WO2009148549A2

WO2009148549A2 - Novel biosensors for live cell imaging of intracellular proteins

Info

Publication number: WO2009148549A2
Application number: PCT/US2009/003297
Authority: WO
Inventors: Peter Drain
Original assignee: University of Pittsburgh
Current assignee: University of Pittsburgh
Priority date: 2008-05-29
Filing date: 2009-05-29
Publication date: 2009-12-10
Anticipated expiration: 2010-11-29
Also published as: WO2009148549A3

Abstract

This invention is related to the field of imaging intracellular protein processing. Fluorescent probes capable of changing color over time are used to identify and track the synthesis, trafficking, and secretion of intracellular proteins. One such probe useful in this invention is a monomeric multifluorescent protein, specifically a mcTimer probe as a biosensor for secretory proteins. After expression, mcTimer sequentially changes from green to red over a twenty- four hour period. In particular, a monomeric multifluorescent protein is inserted into the C-linker portion of the insulin prepropeptide as a marker for normal insulin responses to glucose in cultured donor beta cells.

Description

Novel Biosensors For Live Cell Imaging Of Intracellular Proteins

This invention was made in part with government support under (Grant Nos. DK064383-02) awarded by (National Institutes of Health). The government has certain rights in the invention.

Field Of Invention

This invention is related to the field of imaging intracellular protein processing. Fluorescent probes capable of changing color over time are used to identify and track the synthesis, trafficking, and secretion of intracellular proteins. One such probe useful in this invention is a monomelic multifluorescent protein, specifically a mcTimer probe as a biosensor for secretory proteins. After expression, mcTimer sequentially changes from green to red over a twenty- four hour period. In particular, a monomelic multifluorescent protein is inserted into the C-linker portion of the insulin prepropeptide as a marker for normal insulin responses to glucose in cultured donor beta cells.

Background

Diabetes is a complex set of diseases characterized by chronically high levels of blood glucose (hyperglycemia) Auto immune Type 1 diabetes mellitus (TlDM; IDDM) is a complex disease that still presents many puzzles relative to its etiology and more importantly to effective therapies. Bottino et al., "Islet/pancreas transplantation: challenges for pediatrics" Pediatric Diabetes 3:210-223 (2002). Insulin-dependent diabetic patients have few options for treatment. While the most common therapy is daily multiple injections of insulin, one alternative that has most recently gained favor is the replacement of the islets of Langerhans by allotransplantation, following the Edmonton protocol. Shapiro et al., "Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen" N. Engl. J. Med. 343:230-238 (2000). Thus insulin-producing cells (i.e., the β cells) already lost at the time of the clinical onset of the disease can be replaced in this way. However, one of the major problems with islet transplantation is that two or three human donor organs are necessary to successfully treat one diabetic patient because of the damage these cells suffer during isolation and subsequent manipulation. Ryan et al., "Successful islet transplantation" Diabetes 51 :2148-2157 (2002). In particular, it is difficult to access insulin production when islets are held for extended time periods in culture prior to transplantation, especially when insulin is added to the cell culture medium. Thus raising the question of how functional these cells are at the time of transplantation since the standard method is to access the quality of the cells under the microscope prior to transplantation. Further, it is not known what happens to the islets after transplantation or why and when they stop producing insulin; the percentage of patients who did not require exogenously administered insulin in the Edmonton protocol, close to 80% the first year, apparently reaches the 50% mark at the end of the second year. Ryan et al., "Clinical follow-up after islet transplantation" Diabetes 5 l(Suppl 2):A32-33 (2002).

Non-auto-immune Type II diabetes (T2D) is characterized by insufficient insulin secretion and, resistance to insulin actions in the target cells to take up glucose from the blood, or a combination of both. In T2D, there is no auto-immuno destruction of insulin producing cells. Thus T2D and TlD both exhibit hyperglycemia but are distinguished by the absence or presence of auto-immune destruction of insulin-producing cells. Diagnostically, the two types of diabetes are distinguished by the absence or presence of auto-antibodies. T2D thus results at least in part from a functional defect in the synthesis, folding, trafficking or secretion of insulin but the cells are still present, together with resistance in the target cells to what insulin, however insufficient, is secreted.

Thus compositions and methods are needed for determining the insulin producing capability of β-cells before and after transplantation.

Summary This invention is related to the field of imaging intracellular protein processing.

Fluorescent probes capable of changing color over time are used to identify and track the synthesis, trafficking, and secretion of intracellular proteins. One such probe useful in this invention is a monomeric multifluorescent protein, specifically a mcTimer probe as a biosensor for secretory proteins. After expression, mcTimer sequentially changes from green to red over a twenty-four hour period. In particular, a monomeric multifluorescent protein is inserted into the C-linker portion of the insulin prepropeptide as a marker for normal insulin responses to glucose in cultured donor beta cells.

In general, the present invention provides biosensors for proteins comprising and derived from prepropep tides. Specifically, the present inventions provide biosensors as markers inserted into and thus comprising C linker or a C peptide of a prepropeptide (in some embodiments, a propeptide). In particular, the present inventions provide biosensors for processing and trafficking of insulin (Ins). In one embodiment, the biosensor is a fluorescent molecule whose coding sequence is inserted into a C linker (C) of an insulin molecule. In another embodiment, the biosensor is a fluorescent molecule whose coding sequence is inserted into a C linker (C) of a somatostatin gene. Thus a variety of biosensors are provided as described herein.

In general, the present inventions provide biosensors as monomelic single and multifluorescent proteins.

In some embodiments, the present invention provides, a monomeric single fluorescent protein. Wherein said single fluorescent protein is an emerald green fluorescent protein. In one embodiment, the monomeric fluorescent protein comprises a mutation. In one embodiment, the mutation causes an increase in brightness of the fluorescent protein. In one embodiment, the mutation decreases dimerization of the fluorescent protein. In some embodiments, the present invention provides, a multifluorescent protein.

In one embodiment, over time said multifluorescent protein fluoresces a first color followed by a second color. In one embodiment, said first color is green. In one embodiment, said second color is red. It is not meant to limit the color of the timer molecule, such that molecules of other molecules comprising a fluorescent color coding sequence with 3' and 5' GFP coding sequences are contemplated, such as described in Shaner et al., Nature Biotechnology, 12, 2004, Figure 2a, herein incorporated by reference in its entirety. Further, other molecules which do not have GFP encoding sequences are also contemplated as Timer probe markers, for example, see Shaner et al., Nature Biotechnology, 12, 2004, Figure 2a, herein incorporated by reference in its entirety. However these examples are not meant to limit the fluorescent molecule contemplated for Timer mutations. In addition, Timer molecules whose first color is colorless or a version of their primary color that would "time" to become their second color. In one embodiment, said color is bright. In one embodiment, said red is bright. In one embodiment, said green is bright. Li one embodiment, said monomelic multifluorescent protein comprises SEQ ID NO.l. In one embodiment, said monomelic multifluorescent protein comprises SEQ ID NO:2. In one embodiment, said multifluorescent protein comprises a proteolytic fragment of a prepropeptide. In one embodiment, said monomelic protein comprises a peptide C. In one embodiment, said monomelic protein comprises a prepropeptide. It is not meant to limit the type of prepropeptide. Indeed, any protein translated into a prepropeptide is contemplated, including but are not limited to a mouse insulin prepropeptide, such as mouse insulin II, a human insulin prepropeptide, and the like. In one embodiment, said prepropeptide further comprises an A peptide, a B peptide, and a C peptide. In one embodiment, said C peptide is located in between the A peptide and the B peptide. In one embodiment, said monomelic protein comprises a Timer mutation. In one embodiment, said Timer mutation is a 197T mutation.

In some embodiments, the present invention provides, a protein, comprising SEQ ID NOr Ol.

In some embodiments, the present invention provides, a protein, comprising SEQ ID NO: 02. In some embodiments, the present invention provides methods of using biosensors as platforms for drug screening.

In some embodiments, the present invention provides methods of using biosensors as platforms for treatments, including but not limited to clinical treatment. In some embodiments, the present invention provides, a method, comprising: a) providing; i) a vector encoding a monomelic multifluorescent protein, wherein said protein expresses over time a first color fluorescence and a second color fluorescence; ii) an isolated tissue sample; and b) administering the vector to the tissue under conditions such that the protein expresses a fluorescence. In one embodiment, said monomelic multifluorescent protein comprises SEQ ID NO:01. In one embodiment, said monomelic multifluorescent protein comprises SEQ ID NO:02. hi one embodiment, said method further comprises a step of measuring the first color fluorescence intensity and the second color fluorescence intensity. In one embodiment, method further comprises a step of determining a ratio of a first color fluorescence intensity to a second fluorescence intensity. In one embodiment, first color fluorescence co-localizes in vesicles comprising insulin. In one embodiment, tissue comprises pancreatic beta cells. In one embodiment, said method further comprises, provides, a test compound, hi one embodiment, said method further comprises contacting said compound with said tissue.

In some embodiments, the present invention provides kits comprising monomelic biosensors of the present inventions.

In some embodiments, the present invention provides kits comprising multifluorescent biosensors of the present inventions.

In some embodiments, the present invention provides kits comprising insulin reporting molecules of the present inventions. In some embodiments, the kits further comprise methods of using insulin reporting molecules.

In some embodiments, the present invention provides Ins-C-Fluorescent Protein (Ins-C-FP) reporters (biosensors) comprising a mouse proinsulin II gene and further provided a reporter comprising a human proinsulin gene. There were several advantages of using the human proinsulin gene over reporters using the mouse proinsulin gene. One of the primary advantages of using the human proinsulin gene was for testing mutations in insulin genes, i.e. mutations associated with clinical symptoms of diabetes in humans. These types of studies were not possible using the mouse proinsulin II gene. For example, mutations discovered in genes of diabetic patients were introduced into the human proinsulin gene of the huIns-C-FP in order to study effects of a mutation on insulin processing and trafficking. These studies were not as informative using a mouse proinsulin because when the mouse gene was aligned with human proinsulin it was apparent that these two genes were different, and further the aligned proteins showed major structural differences. Moreover, in exemplary attempts to align these genes and proteins to position the clinically relevant human mutation locations into mouse insulin, it was found that these mutation points were not well aligned and thus would be unlikely to provide the same results in mouse insulin when compared to human insulin. Therefore, in preferred embodiments, methods for studying point mutations in humans comprise human proinsulin reporter molecules. Therefore, human (hu or h) Ins-C-FP's are contemplated as centerpieces of platforms for drug and treatment studies. In a preferred embodiment, the propeptide is a human insulin propeptide.

Thus the inventor further contemplated that by minimizing or eliminating a co- factor associated with or causing ER accumulation or misfolding, would allow more normal folding of either mutant or wild-type pro insulins for restoring normal processing and trafficking, i.e. trafficking of secretory granules to the cell membrane for receptor mediated release of functional insulin. For example, interference of ubiquitylation of human proinsulin would allow the mutant and wild-type proinsulins to eventually fold and traffic to the secretory granules for release. One example of such protein folding treatment is shown in Makino, et al., Journal of Cell Science, 119, 923-932 (2006), herein incorporated by reference, where Elmol inhibits ubiquitylation of DocklδO thus increasing the stability of Dock 180 protein. Thus in one embodiment, the inventor contemplates a strategy for a diabetes therapeutic by mutating a ubiquitin site on human proinsulin for interfering with ubiquitin binding. In another contemplated embodiment a chemical (therapeutic) chaperone is used to increase normal folding of the misfolded mutant proinsulin. hi another comtemplated embodiment a protein chaperone is used to increase normal folding of the misfolded mutant proinsulin.

Further, mutations were introduced into the mouse and human Ins-C-FP's to improve and diversify insulin fluorescent reporter functions. Mutated versions of fluorescent proteins in Ins-C-FP's were contemplated and provided. An example is the A206K mutation in the emerald GFP, designated Ins-C-emGFP206K, which further minimizes dimerization due to the fluorescent protein moiety of Ins-C-FP's, within the emerald fluorescent versions.

Even further, the inventor provided Ins-C-FP's comprising additional fluorescent proteins into the C peptide region for improving and diversifying the fluorescence reporting functions for processing and trafficking of propeptide molecules. Specifically, a Timer sequence was inserted into a human Ins-C-FP, designated huIns-C-Timer, such that a construct of Figure 6 had a human insulin molecule in place of the mouse insulin molecule. In one embodiment the promoter is a mouse insulin promoter. In another embodiment the promoter is a human insulin promoter. However any promoter is contemplated for use such that the encoded protein was expressed. The inventor further contemplates inserting fluorescent proteins in place of emerald, mcTimer, and the like, into huIns-C-FP for providing a biosensor compositions including GCaMP2, RedTag, and Turbo, and various mutant versions of these proteins in a constant effort of evolving the Ins-C-FP family of fluorescent insulin reporters for improved performance or to diverse their reporting functions. For example, with mutant versions of GCaM P2 we should be able to detect the free calcium concentration within secretory granules. The free calcium within secretory vesicles is thought to be a critical parameter affected secretory rates. Mutants of GCaMP2 are contemplated as a fluorescence reporter of the proteolytic maturation of proinsulin to insulin within the secretory vesicle, see, Wang, et al., Structure, Volume 16, Issue 12, 1817-1827, 2008, herein incorporated by reference, for examples of types of reporters). Red fluorescent protein and Timer Ins-C-FP's using RedTag (Mishin, et al., Biochemistry 2008, 47:4666- 4673, herein incorporated by reference, and Turbo fluorescent proteins (for example, including any one of a bright green, yellow, red/orange, true red, and far-red proteins (their recent far-red product is called Katushka, or TurboFP635) U.S. Pat. # 7,417,131, herein incorporated by reference, Evrogen Joint Stock Company, Miklukho-Maklaya str, Moscow Russia) and their mutant variants are contemplated for use as fluorescent markers of propeptides in the present inventions.

There are at least three major categories of compositions and methods of using the Ins-C-FP's of the present applications contemplated for application in the Pharmaceutical and Biotechnology industries: (i) stable cell lines, (ii) viral transduction vectors for islet expression, and (iii) transgenic animal models. Contemplated compositions (platforms) and methods using the Ins-C-FP's of the present inventions are for discovery of antidiabetic drugs using the stable cell lines, viral transduction vectors for islet expression, and transgenic animal models. These core platforms are contemplated for screening drug libraries, profiling toxicity, and developing systems biology for insulin secretion and diabetes.

The inventions further provide compositions based upon a clever strategy of tagging, in one embodiment flurophore tagging, a propeptide, such as proinsulin. In preferred embodiments the mature processed peptide is not tagged. Additional contemplated embodiments include tagging propeptides of peptide hormones, such as proglucagon, and peptide neurotransmitters, such as somatostatin, substance P, enkephalins, endorphins, neuropeptide Y, and substance P. In one embodiment, the inventor constructed a fluorescently tagged propeptide of somatostatin, hi a further embodiment, the inventor contemplate methods of using the somatostatin propeptide construct as live-cell neuropeptide reporters of brain function for allowing the screening and development of neuroactive drugs. Thus in another embodiment, the tagged neurotransmitter peptides are contemplated for use in the field of psychiatry.

Definitions As used herein, the term "diabetes" or "diabetic" or "diabetes mellitus" or

"human diabetes mellitus" generally refers to a physiological disorder that develops when glucose regulation is altered, i.e. compared to a nondiabetic, such that a patient is hyperglycemia corresponding to abnormally high levels of circulating glucose, respectively, correlating with altered circulating insulin levels, for examples, due to diminished amounts in circulating insulin, (i.e. lack of release from beta cells, loss of beta cells, etc.), due to resistance to insulin, typically resulting in increased amounts of circulating insulin, (i.e. lack of insulin binding to it's receptor, lack of insulin bound receptor mediated enocytosis, lack of glucagon release or lack of glucagon production, etc.), and the like. As used herein, the term "Type 1 diabetes" or "human type 1 diabetes" or "type 1 diabetes mellitus" or "insulin dependent diabetes" or "TlD¹Or "TlDM¹Or "E)DM" refers to a disorder developing from diminished secretion of insulin, for example, when insulin-producing cells of the pancreas have reduced capability to secrete insulin from malfunction or cell death. In other words, type 1 diabetic patients are typically hyperglycemic, specifically because they lack insulin-producing cells due to autoimmune destruction. One major characteristic of Type I diabetes is the presence of serum autoantibodies that bind to pancreatic beta cells, including internal molecules such as insulin, Glutamic Acid Decarboxylase, et cetera.

As used herein, the term "human Type 2 Diabetes" or "T2D" or "Type II diabetes" refers to a disorder developing from diminished function of insulin, for example, when circulating insulin fails to regulate glucose. Type II diabetic patients are hyperglycemic, specifically due to insufficient insulin secretion and/or resistance to its action on target cells to take up glucose from the blood, or both, in the absence of autoimmune destruction of the insulin-producing cells.

As used herein, the term "clinically" or "medically" in reference to an observation, a symptom, and a treatment refers to association with diagnosing diabetes and reversing the symptoms of diabestes, such that physicians primarily depend upon the results of specific glucose tests for determing diabetic stage. However, test results are just part of the information that goes into the diagnosis of diabetes. Doctors also take into account a physical exam, presence or absence of symptoms, and medical history. Some people who are significantly ill will have transient problems with elevated blood sugars which will then return to normal after the illness has resolved. Also, some medications may alter your blood glucose levels (most commonly steroids and certain diuretics, such as water pills). Two main tests used to measure the presence of blood sugar problems are the direct measurement of glucose levels in the blood during an overnight fast and measurement of the body's ability to appropriately handle the excess sugar presented after drinking a high glucose drink. Current tests include: Fasting Blood Glucose (Blood Sugar) Level and The Oral Glucose Tolerance Test. "Clinical symptoms" or "medically relevant" or "medically relevant symptoms" include but are not limited to hypoglycemia, including symptoms such as fatigue, weight gain, and hyperglycemia, including symptoms such as weight loss, excessive thirst (polydipsia), excessive urination

(polyuria), poor wound healing, increased risk of infections, microvascular complications (eg, retinopathy, nephropathy), neuropathic complications, macrovascular disease, et cetera.

As used herein, the term "altered glucose" refers to circulating (serum) glucose levels showing a physiological alteration, for example, glucose levels that rise higher than normal after drinking a glucose drink and come down to normal levels much slower (insulin is either not produced, or it is produced but the cells of the body do not respond to it).

As used herein, the term "subject having diabetes" or "subject displaying signs or symptoms or pathology indicative of diabetes" or "subjects suspected of displaying signs or symptoms or pathology indicative of diabetes" refer to a subject that is identified as having or likely to have diabetes based on known diabetes signs, symptoms and pathology.

As used herein, the term "subject at risk of displaying pathology indicative of diabetes" and "subject at risk of diabetes" refer to a subject identified as being at risk for developing diabetes (e.g., due to age, weight, race, or familial inheritance pattern of diabetes in the subject's family).

As used herein, the term "diabetes therapeutic" refers to an agent used to treat or prevent diabetes. Such agents include, but are not limited to, small molecules, drugs, antibodies, pharmaceuticals, and the like. For example, therapeutics used to treat diabetes include, but are not limited to, oral medication to increase insulin sensitivity (e.g., metformin, a thiazolidinedione (TZD)), intermediate-acting insulin (eg, neutral protamine Hagedorn (NPH)), a long-acting insulin (eg, glargine (Lantus) insulin, insulin detemir (Levemir)), Incretin mimetics (e.g., Exenatide (Byetta)), Sulfonylurea agents (e.g., chlorpropamide, tolbutamide, tolazamide, acetohexamide, glyburide, glipizide, and glimepiride), Meglitinides (e.g., Repaglinide (Prandin)), Biguanides (e.g., Metformin (Glucophage)), Alpha-glucosidase inhibitors (AGIs) (e.g., Acarbose (Precose), Miglitol (Glyset)), thiazolidinediones (e.g., Pioglitazone (Actos), Rosiglitazone (Avandia)), and Amylin analogs (e.g., Pramlintide acetate (Symlin)). In one embodiment, a therapeutic is a molecule for inhibiting ubiquanylation of an insulin protein. As used herein, the terms "chaperone" and "molecular chaperone" refer to a molecule whose function is to assist another protein in achieving proper folding, for increasing exportation of the protein from a cell. A chaperone may include but is not limited to a "high temperature protein" or "htp" and "heat shock protein" or Hsp," "chaperonin," SecB," "Syc," and the like. As used herein, the term "chaperonin" refers to a protein or a protein complex that assists in the folding of nascent, non-native (mutant) polypeptides into their native, functional state, and for altering exportation of protein or a protein complex from a cell. Examples include molecular chaperones or Group I chaperonins or Group II chaperonins. As used herein, the terms "host," "subject" and "patient" refer to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.) that is studied, analyzed, tested, diagnosed or treated. As used herein, the terms "host," "subject" and "patient" are used interchangeably, unless indicated otherwise.

As used herein, the terms "prevent" and "preventing" include the prevention of the recurrence, spread or onset of type I diabetes. It is not intended that the present invention be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

As used herein, the term "prophylactic agent" includes any agent that can be used in the prevention of a disease.

As used herein in connection with the term "therapeutic agent", "therapeutically effective amount" includes the amount of the therapeutic agent sufficient to delay, reduce or minimize symptoms associated with type I diabetes. A therapeutically effective amount also includes the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of diabetes.

As used herein, the term "therapeutic agent" includes any agent(s) that can be used in the treatment of a disease.

As used herein, the terms "treat" and "treating" are not limited to the case where the subject (e.g. patient) is cured and the disease is eradicated. Rather, the present invention also contemplates treatment that merely reduces symptoms, and/or delays disease progression. Thus, in certain embodiments, treatment aids in the management or control of type I diabetes.

As used herein, the terms "peptide," "polypeptide" and "protein" all refer to a primary sequence of amino acids that are joined by covalent "peptide linkages." In general, a peptide consists of a few amino acids, typically from 2-50 amino acids, and is shorter than a protein. The term "polypeptide" encompasses peptides and proteins. In some embodiments, the peptide, polypeptide or protein is synthetic, while in other embodiments, the peptide, polypeptide or protein are recombinant or naturally occurring. A synthetic peptide is a peptide that is produced by artificial means in vitro (i.e., was not produced in vivo).

As used herein, the terms "co-administration" and "co-administering" refer to the administration of at least two agent(s) (e.g., composition comprising SEL-PLEX and one or more other agents— e.g., an Alzheimer's disease therapeutic, or, a second form of selenium) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and/or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent.

As used herein, the term "at risk for disease" refers to a subject (e.g., a human) that is predisposed to experiencing a particular disease. This predisposition may be genetic (e.g., a particular genetic tendency to experience the disease, such as heritable disorders), or due to other factors (e.g., age, weight, environmental conditions, exposures to detrimental compounds present in the environment, etc.). Thus, it is not intended that the present invention be limited to any particular risk, nor is it intended that the present invention be limited to any particular disease. As used herein, the term "suffering from disease" refers to a subject (e.g., a human) that is experiencing a particular disease. It is not intended that the present invention be limited to any particular signs or symptoms, nor disease. Thus, it is intended that the present invention encompass subjects that are experiencing any range of disease (e.g., from sub-clinical manifestation to full-blown disease) wherein the subject exhibits at least some of the indicia (e.g., signs and symptoms) associated with the particular disease.

As used herein, the terms "disease" and "pathological condition" are used interchangeably to describe a state, signs, and/or symptoms that are associated with any impairment of the normal state of a living animal or of any of its organs or tissues that interrupts or modifies the performance of normal functions, and may be a response to environmental factors (such as malnutrition, industrial hazards, or climate), to specific infective agents (such as worms, bacteria, or viruses), to inherent defect of the organism (such as various genetic anomalies, or to combinations of these and other factors.

As used herein, the term "biosensor" in general refers to a molecularly engineered construct, such as an Ins-C-FP molecule of the present inventions. Examples of constructs include but are not limited to constructs comprising human proinsulin, mouse proinsulin, and the like, further comprising a fluorescent protein sequence within the C peptide region, see, Figure 6C and 6D for an exemplary biosensor construct wherein the nucleotide sequences of a florescent protein are ligated into the C peptide region of proinsulin. A biosensor construct may further comprise a promoter sequence, an expression vector sequence and the like, such as in another embodiment, wherein the biosensor of Figure 6C and D is ligated to a promoter region of Figure 6B, and further wherein the expression vector sequences are ligated in operable combination to a promoter, an insulin propeptide comprising the fluorophore within peptide C, to form a biosensor of the present inventions, for exemple, Figure 6 sequences ligated in operable combination.

As used herein, the term "probe" or "marker" in reference to a biosensor protein refers to a fluorescent protein molecule, such as GFP, any of the fruit molecules, and derivatives thereof, that "identify or "mark" a particular molecule or location. For the purposes of the present inventions, a probe may also mark time. A probe in reference to an amino acid sequence refers to a sequence encoding the probe. A probe in reference to nucleic acid refers to a sequence encoding the amino acid sequence. A sequence for a probe may occur naturally as in a purified restriction digest of genomic DNA, produced synthetically, produced recombinantly, by PCR amplification and the like. A fluorescent probe may be a monomer, dimer, trimer, tetramer and the like. A nucleic acid probe may be single-stranded or double-stranded. Nucleic acid probes are useful in the detection, identification and isolation of particular gene sequences. It is contemplated that any probe used in the present invention will be labeled with any "reporter molecule," so that is detectable in any detection system, including, but not limited to a fluorescent molecule, an enzyme (e.g., ELISA, as well as enzyme-based histochemical assays), radioactive, an antibody, and the like. It is not intended that the present invention be limited to any particular detection system or label or probe. As used herein, "mc-Timer peptide" or "mcTimer probe" refers to a peptide comprising a monomelic (m) form of Cherry (c) with a 197T mutation as described herein which caused the stable red monomelic cherry fluorescent molecule to express initially as a first color, i.e. as a green fluorescent molecule, which shifts to a second fluorescence, i.e. red fluorescence, over time to function as a multifluorescent timer molecule.

As used herein, "T197 point-mutation" or T197" or "197T" or "Timer mutation" in reference to a multifluorescent protein refers to a homologous mutation of a nucleic acid resulting in an amino acid change to a T amino acid at an equivalent position to 197 in dsRedl, which in dsRedl was a S to T substitution, also termed "S197T" which in dsRedl resulted in a timer function as the original timer molecule.

As used herein, "multifluorescent protein" refers to a molecule that is capable of emitting fluorescence at more than one fluorescent peak, such as molecules described in Terskikh, et al., 2000, Science 290:1585-1588, United States Patent No. 7,230,080 and WO 2001/096373 20011220; all of which are herein incorporated in their entirety.

As used herein, "monomelic" refers to a molecule that is capable of emitting fluorescence as a single molecule. As opposed to fluorescent molecules that must form dimers or trimers or tetramers in order to be capable of emitting fluorescence.

As used herein, "green" refers to the fluorescent intensity emission directly from a "green fluorescent molecule" or "GFP." As used herein, "red" in reference to a fluorescent emission refers to a fluorescent emission from a range of molecules, such as cherry, strawberry, tomato, and the like (see, Clonetech). In terms of the present inventions described herein, unless otherwise specified, red refers to the fluorescence emission from a cherry molecule (see, Clonetech). As used herein, "color" refers to a color assigned to a designated emission wavelength peak (of an emitting range), for example, see Shaner et. al, 2004, Table 1 and Figure 1 , herein incorporated by reference in its entirety. A color, for example, red, green, and the like, as used herein reference emission specta and combinations of emission spectra not colors artificially imposed by microscope users. As used herein, "yellow" in reference to a type of emission directly from a fluorescent molecule refers to emissions from any one of banana, melan, orange, tangerine, and the like (see, Clonetech). In contrast, yellow may also refer to overlapping emissions of green and red, due to single green and single red fluorescent protein molecules mixed in approximately equal amounts within a given volume, such as in the secretory vesicles, for example, shown in Figure 4 herein.

As used herein, "bright" in reference to a sequence or fluorescent emission refers to increasing the fluoresence intensity at least two-fold, for example, an amino acid mutation that allows at least a 2 fold stronger fluorescent emission of the fluorescent "color" encoded by the molecule, such as a V105A mutation or equivalent in a coding sequence of a fluorescent molecule as shown in dsReD-E5.

As used herein, "voxel" refers to a "3-dimensional" or "3D" or "3-D" equivalent of a 2-dimensional pixel with an additional volume measurement.

The term "label" or "detectable label" are used herein, to refer to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Such labels include gold particles conjugated to antibodies for identifying proteins for electron microscopy, biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabeads^®), fluorescent dyes (e.g., fluorescein, texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels include, but are not limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241 (all herein incorporated by reference). The labels contemplated in the present invention may be detected by many methods. For example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using confocal microscopes or any device capable of detecting a fluorescent molecule, a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting, the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the colored label.

As used herein, "vector" refers to a molecule for moving a nucleic acid molecule into a cell, such as a nucleic acid molecule encoding a fusion protein into a cell. As used herein, "Adenoviral vector" refers to a vector based upon an adenovirus, such as an adlox vector of the present invention.

As used herein, "protease" refers to an enzyme that promotes proteolysis (a splitting of amino acid sequences by hydrolysis of a peptide bond resulting in formation of smaller "polypeptides or "fragments." As used herein, "proteolytic" in reference to enzymes, refers to enzymes that cleave peptide bonds, for example, prohormone convertases (PCl and PC2), exoprotease carboxypeptidase E, serine proteases, such as trypsin, chymotrypsin, carboxypeptidase and the like.

As used herein, "proteolytic" in reference to cleavage refers to producing fragments of proteins by proteases.

As used herein, "proteolytic fragment" refers to a peptide derived from the action of a protease (i.e. cleavage of a peptide bond).

As used herein, "a peptide" in reference to a prepropeptide merely refers to one of the peptides comprising the cleaved peptide. As used herein, "B peptide" in reference to a prepropeptide merely refers to one of the peptides comprising the cleaved peptide that is not A.

As used herein, "C peptide" in reference to a prepropeptide merely refers to the coding sequence between the A peptide and the B peptide prior to cleavage. A c peptide may refer to a cleaved portion of the amino acid sequence and may refer to the "linker" or "linking" amino acids in between the A peptide and the B peptide..

As used herein, "disulfide bond" refers to a "SS-bond" or "disulfide bridge" linkages formed from the oxidation of sulfhydryl (-SH) groups thiol groups of cysteine residues. The term "transfection" or "transfected" refers to the introduction of foreign DNA into a cell, such as transfecting with an adenoviral vector of the present invention for introducing a sequence encoding a fluorescent molecule. Examples of methods of transfection include but are not limited to calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, biolistics and the like.

The term "co-transfection" or "co-transfected" refers to the introduction of 2 types of foreign DNA into a cell, such as transfecting a construct of the present invention comprising a wild-type insulin gene and transfecting a construct of the present invention comprising a mutant insulin gene, wherein the transfecting may be simultaneous or transfecting sequentially in time.

As used herein, "fusion protein" refers to a protein sequence resulting from joining at least two separate sequences, such as an insulin molecule comprising a peptide bonded fluorescent molecule, a c peptide-fluorescent molecule construct, and the like, as described herein.

As used herein, "tissue" refers to any group of cells in or from a subject.

As used herein, "eukaryotic cell" or "cell" refers to a biological cell with internal membrane-bound compartments such as a nucleus. As used herein, a "bonafide" beta cell, such as a cell derived from a stem cell, refers to a cell that secretes insulin in response to glucose.

The term "beta cell" or "β cell" refers to a cell that should be capable of insulin secretion.

As used herein, "plasmalemma" or "plasma membrane" refers to a membrane that encloses the cytoplasm of a cell.

As used herein, "intracellular compartments" refers to any intracellular organelle, i.e. any intracellular structure surrounded by a membrane. The term "endoplasmic reticulum" or "ER" refers to a complex system of flattened sacs, and it is the site of many important syntheses, including the production of new surface membrane and the intracellular transport of various biochemical entities.

The term "Golgi apparatus" or "Golgi complex" or "Golgi" refers to a collection of organelles or "Golgi bodies" in eukaryotic cells that essentially function as a collecting and packaging center for substances that a cell manufactures for export.

The term "trans-Golgi network" or "TGN" refers to a network of interconnected tubular and cisternal structures located at the side of the Golgi apparatus distal to the endoplasmic reticulum, from which secretory vesicles and lysosomes emerge. The trans- Golgi network is important in the later stages of protein secretion where it is thought to play a key role in the sorting and targeting of secreted proteins and substances to the correct destination, such as a vesicle destined for the plasma membrane, a vesicle specialized for hydrolytic enzymes, et cetera.

As used herein, "vesicle" refers to any intracellular membrane bound space and is a specialized organelle for moving molecules around inside of a cell in "intracellular transport," for example, to and from the nucleus, to and from the cell membrane, between other organelles, for holding molecules, moving molecules to the surface (exocytotic vesicles), moving molecules from the surface (endocytotic vesicles), and to the cell membrane. In general a particular designation is provided based upon a general characteristic (such as a general size range, a general location, pH and the like) or having a specific identifying characteristic, such as a protein marker functioning as a zip code, a secretory vesicle containing a molecule known to be secreted, a lysosomal vesicle of a particular stage identified by an antibody marker, such as LAMP-I. However these designations are not absolute such that vesicles may also fuse together and thus show characteristics of more than one vesicle (mixed characteristics), such as when an early endosome fuses with a lysosome, or when a secretory vesicle fuses with a lysosome (vesicle) instead of the plasma membrane.

As used herein, "granule" refers specifically to a secretory vesicle that contains condensed cargo, such as crystalline insulin and zinc, which appears as a dark dense core of the secretory granule. Such vesicles are designated either secretory granules or granules, interchangeably. However, a secretory vesicle that does not appear to include such a dense core is not designated a granule where merely the term vesicle is used.

As used herein, "secretory" refers to relating to or performing secretion, for example, a "secretory" pathway, "exocytotic pathway" a process, an associated molecule, et cetera.

As used herein, "secretory" in reference to a secretory vesicle, refers to a vesicle related to or undergoing secretion, such as a vesicle derived from the Golgi containing secretory molecules, for example, insulin. The type of secretion of a vesicle may be constitutive, glucose-regulated, for example insulin, or receptor mediated. An act of secretion is referred to as "exocytosis."

The term "test compound" or "test agent" or "compound" or "agent" as used herein, refers to any compound or molecule suspected or known of having a capability for altering the insulin processing or secretion pathway. For example, a "test compound" includes, but is not limited to, protein translation inhibitors, metabolic inhibitors, polypeptides, small molecular weight organic molecules, hormones, and the like. Test compounds are contemplated for therapeutic drugs. As used herein, "insulin pathway" in reference to processing or secretion and the like, refers to any cellular or extracellular interaction with insulin, such as insulin folding, insulin packaging, insulin secretion and the like. Compounds suitable for assay in the methods of this invention include, but are not limited to, proteins, glycoproteins, antibodies, saccharides, lipids, nucleotides, nucleotide analogues, nucleic acids (e.g., DNA, RNA, peptide nucleic acids, etc.), and organic molecules, particularly small organic molecules. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

As used herein, "omega structure" refers to a snap-shot (for example, an electron micrograph) of a secretory vesicle undergoing fusion with the plasma membrane for actual secretion by releasing contents of the secretory vesicle.

As used herein, "secretagogue" or " secretagogues" refers to a substance or substances which causes another substance to be secreted, for example, extracellular glucose causes insulin secretion from functional pancreatic beta cells.

As used herein, "puncta" or "punctate" refers to a pattern of a marker, such as the appearance of a fluorescent marker that shows relatively large dots or spotty marking (such as produced by spots of high fluorescent intensity) within a cell. As opposed to "diffuse" which shows a more even distribution (fluorescence intensity) of a marker within a cell. hi general, the term "endocytosis" refers to the uptake of external materials by cells by means of phagocytosis (uptake of particulate material) or pinocytosis (uptake of liquid material). In both cases, the cell surface membrane literally folds completely around the entity to be taken up, and the membrane-bound is in effect pulled into the cell.

The term, "lysosome" refers to a membrane-bound lytic organelle that contains hydrolases active at acid pH within cells. Lysosomes digest foreign material (for example when fused to endocytotic vesicles) or defective proteins or other cellular organelles.

The term, "secretory lysosomes" refer to modified lysosomes that can undergo regulated secretion in response to external stimuli.

The term, "peroxisome" refers to an organelle rich in enzymes that act on or generate hydrogen peroxide. As used herein, "human cadaver" refers to live human tissue from a dead body where that tissue was donated to research.

The term, "signal peptide" in reference to an insulin peptide fragment refers to a sequence that is removed in the cisternae of the endoplasmic reticulum as the remainder of the protein is taken up into the lumen of the er and via the secretory pathway packaged into secretory vesicles and shipped to the golgi.

As used herein, the term "insulin" refers to a molecule, i.e. hormone, which has extensive effects on metabolism and other body functions, such as blood glucose level regulation, vascular function, et cetera. For examples, insulin causes cells in the liver, muscle, and fat tissue to take up glucose from the blood, storing it as glycogen in the liver and muscle, while reducing use of fat as an energy source. As insulin levels are reduced, glucose take-up by body cells is also reduced, thus increasing circulating glucose levels, especially notable (measured) following meals. When control of insulin levels fails, diabetes mellitus typically is a result.

The term "normal insulin" in reference to secretion refers to an insulin molecule that is folded into its native structure, and locked in this conformation by the formation of 3 disulfide bonds. Specific protease activity then cleaves the center third of the molecule, which dissociates as a C peptide, leaving the amino terminal Beta peptide disulfide bonded to the carboxy terminal Alpha peptide by two disulfide bonds. A third disulfide bond is located intrachain, between two residues of the alpha peptide. Normal insulin is capable of being transported to a cell membrane, released outside of the cell and functions to control blood glucose levels.

The term "mutated" in reference to an insulin molecule refers to a change in nucleic acid that causes a change in an amino acid of an insulin molecule.

The term "prepropeptide" refers to an amino acid sequence that undergoes post- translation modification which cleaves apart biologically active fragments from relatively nonbiologically active prepropep tides. Pre refers to the signal peptide. Pro refers to the entire uncleaved beta peptide-c peptide-alpha peptide. The term "protein" as used herein, refers to any of numerous naturally occurring extremely complex substances (as an enzyme or antibody) that consist of amino acid residues joined by peptide bonds, contain the elements carbon, hydrogen, nitrogen, oxygen, usually sulfur. In general, a protein comprises amino acids having an order of magnitude within the hundreds. The term "peptide" as used herein, refers to any amino acid sequence that derived from at least two or more amino acids bound together when a covalent bond formed as the carbon atom from the carboxyl group of a first amino acid began sharing electrons with a nitrogen atom from the amino group of a second amino acid forming a "peptide bond."

The terms "amino acid sequence" and "polypeptide sequence" and "peptide" as used herein, are interchangeable and to refer to a sequence of amino acids.

The term, "purified" or "isolated", as used herein, may refer to a peptide composition that has been subjected to treatment (i.e., for example, fractionation) to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the composition (i.e., for example, weight/weight and/or weight/volume). The term "purified to homogeneity" is used to include compositions that have been purified to 'apparent homogeneity" such that there is single protein species (i.e., for example, based upon SDS-PAGE or HPLC analysis). A purified composition is not intended to mean that some trace impurities may remain.

As used herein, the term "substantially purified" refers to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and more preferably 90% free from other components with which they are naturally associated. An "isolated polynucleotide" is therefore a substantially purified polynucleotide.

The terms "amino acid sequence" and "polypeptide sequence" as used herein, are interchangeable and to refer to a sequence of amino acids.

As used herein the term "portion" when in reference to a protein (as in "a portion of a given protein") refers to fragments of that protein. The fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.

The term "portion" when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue. The term "antibody" refers to immunoglobulin evoked in animals by an immunogen (antigen). It is desired that the antibody demonstrates specificity to epitopes contained in the immunogen. The term "polyclonal antibody" refers to immunoglobulin produced from more than a single clone of plasma cells; in contrast "monoclonal antibody" refers to immunoglobulin produced from a single clone of plasma cells.

The terms "specific binding" or "specifically binding" when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., for example, an antigenic determinant or epitope) on a protein; in other words an antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

The term "small organic molecule" as used herein, refers to any molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size from approximately 10 Da up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

A "variant" of a protein is defined as an amino acid sequence which differs by one or more amino acids from a polypeptide sequence or any homolog of the polypeptide sequence. The variant may have "conservative" or "silent" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. A variant, such as an engineered molecule of the present inventions may have a "nonconservative" changes, e.g., replacement of a valine with an alanine at a position equivalent to 105 of the original Timer molecule (V 105A), which causes an increase fluorescent intensity, or the replacement of an amino acid with a Threonine (T) at position equivalent to S197T in the original timer molecule, which creates a multifluorescent molecule such as those described herein. Similar minor variations may also include amino acid deletions or insertions (i.e., additions), or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity may be found using computer programs including, but not limited to, DNAStar^® software. The term "Nucleic acid sequence" and "nucleotide sequence" as used herein refer to an oligonucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.

The term "functionally equivalent codon", as used herein, refers to different codons that encode the same amino acid. This phenomenon is often referred to as "degeneracy" of the genetic code. For example, six different codons encode the amino acid arginine. Degenerate codons may be used in point mutations for inducing silent changes may also be induced in nucleic acid codons that do not change the amino acid sequence, such as those of the present inventions, for removing or inserting endonuclease restriction sites, such as in removing at least one restriction site in the sequences of Figure 7 for providing an Ins-C-mcTimer sequence of the present inventions.

A "variant" of a nucleotide is defined as a novel nucleotide sequence which differs from a reference oligonucleotide by having deletions, insertions and substitutions. These may be detected using a variety of methods (e.g., sequencing, hybridization assays etc.). A "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.

An "insertion" or "addition" is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues.

A "substitution" results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.

The term "isolated nucleic acid", as used herein, refers to any nucleic acid molecule that has been removed from its natural state (e.g., removed from a cell and is, in a preferred embodiment, free of other genomic nucleic acid).

The term "derivative" or "derived from" as used herein, refers to any chemical modification of a nucleic acid or an amino acid. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. For example, a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics.

The term "portion" when used in reference to a nucleotide sequence refers to fragments of that nucleotide sequence. The fragments may range in size from 5 nucleotide residues to the entire nucleotide sequence minus one nucleic acid residue.

The term "biologically active" refers to any molecule having structural, regulatory or biochemical functions.

The term "immunologically active" defines the capability of a natural, recombinant or synthetic peptide, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and/or to bind with specific antibodies.

The term "antigenic determinant" as used herein refers to that portion of a molecule that is recognized by a particular antibody (i.e., an epitope). When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants. An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.

The terms "immunogen," "antigen," "immunogenic" and "antigenic" refer to any substance capable of generating antibodies when introduced into an animal. By definition, an immunogen must contain at least one epitope (the specific biochemical unit capable of causing an immune response), and generally contains many more. Proteins are most frequently used as immunogens, but lipid and nucleic acid moieties complexed with proteins may also act as immunogens. The latter complexes are often useful when smaller molecules with few epitopes do not stimulate a satisfactory immune response by themselves.

The term "antibody" refers to immunoglobulin evoked in animals by an immunogen (antigen). It is desired that the antibody demonstrates specificity to epitopes contained in the immunogen. The term "polyclonal antibody" refers to immunoglobulin produced from more than a single clone of plasma cells; in contrast "monoclonal antibody" refers to immunoglobulin produced from a single clone of plasma cells. The terms "specific binding" or "specifically binding" when used in reference to the interaction of an antibody and a protein or peptide means that the interaction is dependent upon the presence of a particular structure (i.e., for example, an antigenic determinant or epitope) on a protein; in other words an antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A", the presence of a protein containing epitope A (or free, unlabelled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

As used herein, the terms "complementary" or "complementarity" are used in reference to "polynucleotides" and "oligonucleotides" (which are interchangeable terms that refer to a sequence of nucleotides) related by the base-pairing rules. For example, the sequence "C-A-G-T," is complementary to the sequence "G-T-C-A." Complementarity can be "partial" or "total." "Partial" complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. "Total" or "complete" complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods which depend upon binding between nucleic acids.

As used herein, the term "an oligonucleotide having a nucleotide sequence encoding a gene" means a nucleic acid sequence comprising the coding region of a gene, i.e. the nucleic acid sequence which encodes a gene product. The coding region may be present in a cDNA, genomic DNA or RNA form. When present in a DNA form, the oligonucleotide may be single-stranded (i.e., the sense strand) or double-stranded. Examples of an oligonucleotide are constructs of the present inventions combined together to form fusion sequences of the present inventions.

Suitable control elements for coding sequences such as enhancers/promoters, splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding region of the gene if needed to permit proper initiation of transcription and/or correct processing of the primary RNA transcript, for example, a preproinsulin of the present inventions. Alternatively, the coding region utilized in the expression vectors of the present invention may contain endogenous enhancers/promoters, splice junctions, intervening sequences, polyadenylation signals, etc. or a combination of both endogenous and exogenous control elements. As used herein, the term "regulatory element" refers to a genetic element which controls some aspect of the expression of nucleic acid sequences. For example, a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. Transcriptional control signals in eukaryotes comprise "promoter" and "enhancer" elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription. Maniatis, T. et al., Science 236:1237 (1987), herein incorporated by reference. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in plant, yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest.

The presence of "splicing signals" on an expression vector often results in higher levels of expression of the recombinant transcript. Splicing signals mediate the removal of introns from the primary RNA transcript and consist of a splice donor and acceptor site. Sambrook, J. et al., In: Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor laboratory Press, New York (1989) pp. 16.7-16.8, herein incorporated by reference. A commonly used splice donor and acceptor site is the splice junction from the l6S RNA ofSV40. The term "poly A site" or "poly A sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3¹ end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene. Efficient expression of recombinant DNA sequences in eukaryotic cells involves expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.

The terms "homology" and "homologous" as used herein in reference to nucleotide sequences refer to a degree of complementarity with other nucleotide sequences. There may be partial homology or complete homology (i.e., identity). A nucleotide sequence which is partially complementary, i.e., "substantially homologous," to a nucleic acid sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence. The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence to a target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding the probe will not hybridize to the second non- complementary target. An oligonucleotide sequence which is a "homolog" is defined herein as an oligonucleotide sequence which exhibits greater than or equal to 50% identity to a sequence of interest when sequences having a length of 100 bp or larger are compared.

The terms "homology" and "homologous" as used herein in reference to amino acid sequences refer to the degree of identity of the primary structure between two amino acid sequences. Such a degree of identity may be directed a portion of each amino acid sequence, or to the entire length of the amino acid sequence. Two or more amino acid sequences that are "substantially homologous" may have at least 50% identity, preferably at least 75% identity, more preferably at least 85% identity, most preferably at least 95%, or 100% identity.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.

The term "poly A site" or "poly A sequence" as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a poly A tail are unstable and are rapidly degraded. The poly A signal utilized in an expression vector may be "heterologous" or "endogenous." An endogenous poly A signal is one that is found naturally at the 3' end of the coding region of a given gene in the genome. A heterologous poly A signal is one which is isolated from one gene and placed 3' of another gene. Efficient expression of recombinant DNA sequences in eukaryotic cells involves expression of signals directing the efficient termination and polyadenylation of the resulting transcript. Transcription termination signals are generally found downstream of the polyadenylation signal and are a few hundred nucleotides in length.

As used herein, "A" or "linker A" in reference to a linker amino acid refers to a string of at least 2 A's (i.e. AA, AAA, and the like) synthetically inserted as codons for alanine into areas of sequence in between separate coding sequences, such as those inserted in between a c peptide sequence and a coding sequence for a fluorescent molecule as described herein.

The term "DNA" or "deoxyribonucleic acid" refers to molecules of hereditary material comprising a string of 4 heterocyclic bases, adenine (A), guanine (G), cytosine (C) and thymine (T) forming either coding and noncoding regions. DNA molecules are said to have "5' ends" and "3' ends" because mononucleotides are reacted to make oligonucleotides in a manner such that the 5' phosphate of one mononucleotide pentose ring is attached to the 3' oxygen of its neighbor in one direction via a phosphodiester linkage. Therefore, an end of an oligonucleotide is referred to as the "5' end" if its 5' phosphate is not linked to the 3' oxygen of a mononucleotide pentose ring. An end of an oligonucleotide is referred to as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of another mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5' and 3* ends. In either a linear or circular DNA molecule, discrete elements are referred to as being "upstream" or 5' of the "downstream" or 3' elements. This terminology reflects the fact that transcription proceeds in a 5¹ to 3' fashion along the DNA strand. The promoter and enhancer elements which direct transcription of a linked gene are generally located 5¹ or upstream of the coding region. However, enhancer elements can exert their effect even when located 3' of the promoter element and the coding region. Transcription termination and polyadenylation signals are located 3' or downstream of the coding region. As used herein, the terms "nucleic acid molecule encoding", "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence. As used herein, the terms "nucleic acid molecule encoding", "DNA sequence encoding," and "DNA encoding" refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the amino acid sequence. As used herein the term "coding region" when used in reference to a gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is typically bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3¹ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

The term "Southern blot" refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists. J. Sambrook et al. (1989) In: Molecular Cloning: A Laboratory Manual Cold Spring Harbor Press, NY, pp 9.31-9.58, herein incorporated by reference. The term "Northern blot" as used herein refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists. J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52, herein incorporated by reference.

The term "reverse Northern blot" as used herein refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligoribonuclotide probe or RNA probe to detect DNA species complementary to the ribo probe used.

As used herein the term "coding region" when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of a mRNA molecule. The coding region is bounded, in eukaryotes, on the 5' side by the nucleotide triplet "ATG" which encodes the initiator methionine and on the 3' side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).

As used herein, the term "gene" means the deoxyribonucleotide sequences comprising the coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the "coding region" or "exon" interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into heterogeneous nuclear RNA (hnRNA); introns may contain regulatory elements such as enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA comprises exons and functions during translation to specify the sequence or order of amino acids in a nascent polypeptide.

In addition to containing introns, genomic forms of a gene may also include sequences located on both the 5' and 3' end of the sequences which are present on the

RNA transcript. These sequences are referred to as "flanking" sequences or regions (these flanking sequences are located 5 ' or 3' to the non-translated sequences present on the mRNA transcript). The 5' flanking region may contain regulatory sequences such as promoters and enhancers which control or influence the transcription of the gene. The 3' flanking region may contain sequences which direct the termination of transcription, posttranscriptional cleavage and polyadenylation.

As used herein, the term "structural gene" refers to a DNA sequence coding for RNA or a protein. In contrast, "regulatory genes" are structural genes which encode products which control the expression of other genes (e.g., transcription factors). The term "sample" as used herein is used in its broadest sense and includes environmental and biological samples. Environmental samples include material from the environment such as soil and water. Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables). A biological sample suspected of containing nucleic acid encoding a collagen-like family protein may comprise a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell, genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like. As used herein, the term "polymerase chain reaction" ("PCR") refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195 and 4,683,202, herein incorporated by reference, which describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. The length of the amplified segment of the desired target sequence is determined by the relative positions of two oligonucleotide primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the "polymerase chain reaction" (hereinafter "PCR"). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be "PCR amplified". With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of ³²P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.

As used herein, the term "amplifiable nucleic acid" is used in reference to nucleic acids which may be amplified by any amplification method. It is contemplated that "amplifiable nucleic acid" will usually comprise "sample template." As used herein, the term "sample template" refers to nucleic acid originating from a sample which is analyzed for the presence of a target sequence of interest. In contrast, "background template" is used in reference to nucleic acid other than sample template which may or may not be present in a sample. Background template is most often inadvertent. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.

The term "Amplification" is defined as the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction. Dieffenbach C. W. and G. S. Dveksler (1995) In: PCR Primer, a Laboratory Manual. Cold Spring Harbor Press, Plainview, New York. As used herein, the term "primer" refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent. The exact lengths of the primers will depend on many factors, including temperature, source of primer and the use of the method.

The term "Southern blot" refers to the analysis of DNA on agarose or acrylamide gels to fractionate the DNA according to size, followed by transfer and immobilization of the DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect DNA species complementary to the probe used. The DNA may be cleaved with restriction enzymes prior to electrophoresis. Following electrophoresis, the DNA may be partially depurinated and denatured prior to or during transfer to the solid support. Southern blots are a standard tool of molecular biologists. J. Sambrook et al. (1989) In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, pp 9.31-9.58.

The term "Northern blot" as used herein refers to the analysis of RNA by electrophoresis of RNA on agarose gels to fractionate the RNA according to size followed by transfer of the RNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized RNA is then probed with a labeled oligodeoxyribonucleotide probe or DNA probe to detect RNA species complementary to the probe used. Northern blots are a standard tool of molecular biologists. J. Sambrook, J. et al. (1989) supra, pp 7.39-7.52, herein incorporated by reference. The term "reverse Northern blot" as used herein refers to the analysis of DNA by electrophoresis of DNA on agarose gels to fractionate the DNA on the basis of size followed by transfer of the fractionated DNA from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized DNA is then probed with a labeled oligoribonuclotide probe or RNA probe to detect DNA species complementary to the ribo probe used.

Low stringency conditions comprise conditions equivalent to binding or hybridization at 42°C in a solution consisting of 5 x SSPE (43.8 g/1 NaCl, 6.9 g/1 NaH₂PO₄-H₂O and 1.85 g/1 EDTA, pH adjusted to 7.4 with NaOH), 0.1% SDS, 5x Denhardt's reagent {50x Denhardt's contains per 500 ml: 5 g Ficoll (Type 400,

Pharmacia), 5 g BSA (Fraction V; Sigma)} and 100 μg/ml denatured salmon sperm DNA followed by washing in a solution comprising 5x SSPE, 0.1% SDS at 42°C when a probe of about 500 nucleotides in length. Numerous equivalent conditions may also be employed to comprise low stringency conditions; factors such as the length and nature (DNA, RNA, base composition) of the probe and nature of the target (DNA, RNA, base composition, present in solution or immobilized, etc.) and the concentration of the salts and other components (e.g., the presence or absence of formamide, dextran sulfate, polyethylene glycol), as well as components of the hybridization solution may be varied to generate conditions of low stringency hybridization different from, but equivalent to, the above listed conditions. In addition, conditions which promote hybridization under conditions of high stringency (e.g., increasing the temperature of the hybridization and/or wash steps, the use of formamide in the hybridization solution, etc.) may also be used.

As used herein, the term "hybridization" is used in reference to the pairing of complementary nucleic acids using any process by which a strand of nucleic acid joins with a complementary strand through base pairing to form a hybridization complex. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is impacted by such factors as the degree of complementarity between the nucleic acids, stringency of the conditions involved, the T_m of the formed hybrid, and the G:C ratio within the nucleic acids. As used herein the term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bounds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. The two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration. A hybridization complex may be formed in solution (e.g., Co t or Ro t analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized to a solid support (e.g., a nylon membrane or a nitrocellulose filter as employed in Southern and Northern blotting, dot blotting or a glass slide as employed in in situ hybridization, including FISH (fluorescent in situ hybridization)).

As used herein, the term "T_m " is used in reference to the "melting temperature." The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. As indicated by standard references, a simple estimate of the T_m value may be calculated by the equation: T_m = 81.5 + 0.41 (% G+C), when a nucleic acid is in aqueous solution at IM NaCl. Anderson et al., "Quantitative Filter Hybridization" In: Nucleic Acid Hybridization (1985), herein incorporated by reference. More sophisticated computations take structural, as well as sequence characteristics, into account for the calculation of T_m. As used herein the term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds such as organic solvents, under which nucleic acid hybridizations are conducted. "Stringency" typically occurs in a range from about T_m to about 20⁰C to 25°C below T_m. A "stringent hybridization" can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. Alternatively, when conditions of "weak" or "low" stringency are used hybridization may occur with nucleic acids that are derived from organisms that are genetically diverse (i.e., for example, the frequency of complementary sequences is usually low between such organisms).

The term "wild-type" refers to a gene or gene product isolated from, or copied from, a naturally occurring source. A wild-type gene is that which is most frequently observed in a healthy population and is thus arbitrarily designed the "normal" or "wild- type" form of the gene. In contrast, the term "modified" or "mutant" refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences, altered trafficking, altered folding, etc.) when compared to the wild-type gene or gene product.

The terms "Western blot," "Western immunoblot" "immunoblot" and "Western" refer to the immunological analysis of protein(s), polypeptides or peptides that have been immobilized onto a membrane support. The proteins are first resolved by polyacrylamide gel electrophoresis (i.e., SDS-PAGE) to separate the proteins, followed by transfer of the protein from the gel to a solid support, such as nitrocellulose or a nylon membrane. The immobilized proteins are then exposed to an antibody having reactivity towards an antigen of interest. The binding of the antibody (i.e., the primary antibody) is detected by use of a secondary antibody that specifically binds the primary antibody. The secondary antibody is typically conjugated to an enzyme that permits visualization of the antigen- antibody complex by the production of a colored reaction product or catalyzes a luminescent enzymatic reaction (e.g., the ECL reagent, Amersham). The term "sample" as used herein is used in its broadest sense and includes environmental and biological samples. Environmental samples include material from the environment such as soil and water. Biological samples may be animal, including, human, fluid (e.g., blood, plasma and serum), solid (e.g., stool), tissue, liquid foods (e.g., milk), and solid foods (e.g., vegetables). A biological sample suspected of containing nucleic acid encoding a collagen-like family protein may comprise a cell, tissue extract, body fluid, chromosomes or extrachromosomal elements isolated from a cell, genomic DNA (in solution or bound to a solid support such as for Southern blot analysis), RNA (in solution or bound to a solid support such as for Northern blot analysis), cDNA (in solution or bound to a solid support) and the like. As used herein, the term "kit" is used in reference to a combination of reagents and other materials. It is contemplated that the kit may include reagents such as nutrients and drugs as well as administration means. It is not intended that the term "kit" be limited to a particular combination of reagents and/or other materials.

Brief Description of The Figures Figure 1 shows exemplary data of Ins-C-Timer transgenic pancreatic islet exposure to interleukin lβ. Lack of vesicular secretion of insulin was shown by comparison of Figures IA to IB.

Figure IA: shows an exemplary confocal micrograph of an Ins-C-Timer transgenic pancreatic islet without exposure to IL-I β. Overall vesicular fluorescence shows a green- yellow color with punctate staining indicating the presence of green containing vesicles comprising recently synthesized insulin and yellow vesicles indicating several types of contents, such as the presence of recently synthesized green molecules and older red fluorescent insulin, intermediate aged insulin vesicles, and red vesicles indicating the presence of relatively very old insulin.

Figure IB: shows an exemplary confocal micrograph of the same islet in Figure IA after a 24-h exposure to interleukin lβ (IL-I β; 50 LVmL). The Timer probe fluorescence shows recently synthesized green containing vesicles, yellow vesicles reflecting intermediate aged insulin, and a marked increase in red vesicles containing old insulin. Original magnification was 40 x.

Figure 2 presents exemplary data of insulin measurements in vitro and in vivo demonstrating the delay in reduction of blood glucose levels in Ins-C-Timer transgenic mice.

Figure 2A: shows an exemplary insulin responsiveness to high glucose measured by an in vitro perfusion assay. A 30-min. low glucose (2.8 mM) incubation was followed by the introduction of high glucose (20 mM) for 30 min, followed by low glucose. The insulin levels were measured by ELISA. The chart on the left shows the insulin-release profile of nontransgenic control islets, while the chart on the right is from Ins-C-Timer transgenic islets. Figure 2B: shows an exemplary insulin responsiveness to high glucose measured in vivo by a intraperitoneal glucose tolerance test (IPGTT). Animals were fasted overnight and then given an intraperitoneal injection of glucose (100 mg/mL) at 2 g/kg body weight. Blood glucose was measured at time 0 (before injection), 15, 30, 60, 90, and 120 min. The chart on the right shows the results of Ins-C-Timer mice from three different litters, and the chart on the left shows the corresponding littermates. Comparing blood glucose levels demonstrated that Ins-C-Timer animals have a noticeable impairment in normalizing their blood glucose compared to controls.

Figure 3 represents one embodiment of an Ins-C-GFP (monomer) reporter construction and subsequent expression in isolated mouse islets showing targeting to an insulin secretory granule.

Figure 3 A: illustrates one embodiment of an Ins-C-GFP construct created by inserting emerald-green GFP in- frame within the middle of a C-peptide coding region of a genomically derived mouse insulin II gene under the control of a mouse insulin II promoter region. The figure depicts a beta cell showing transcription from the 2408 nucleic acid sequence containing the GFP coding region, splicing of the hnRNA sequence into an mRNA, translation and targeting to the insulin granule.

An electron-dense core is shown as a gray circle within the secretory granule, depicted as an open oval. S 1-24 represents 24 amino acids of a mouse signal peptide; B 1-31 represents 31 amino acids of the mouse peptide; C 1-31 represents 31 amino acids of the cleaved C peptide wherein amino acids 1-238 of emerald-GFP flanked by 3 A's on b peptide side and 2 A's on a peptide side were inserted which created a 274 amino acid C-emeraldGFP peptide coding region; and A 1-21 represents 21 amino acids of an alpha peptide.

Figure 3B: presents exemplary data of confocal microscopy of uninfected islets for GFP showing no detectable fluorescence. PlanApo 6OX photomicrograph taken in oil; NA 1.4 objective lens. The scale bar in the confocal images is 2mm.

Figure 3C: presents exemplary data of confocal microscopy of expression of a control construct in which the mouse insulin II promoter of the sequence in Figure 3 A was fused directly to a GFP sequence, without any proinsulin coding DNA in the construct. The uniform green fluorescence is spread throughout the cytoplasm of β cells with increased uniform fluorescence within the nucleus. PlanApo 6OX photomicrograph taken in oil; NA 1.4 objective lens. The scale bar in the confocal images is 2mm.

Figure 3D: presents exemplary data of confocal microscopy of expression of the Ins-C-GFP reporter results in intense punctate staining within β cells. PlanApo 6OX photomicrograph taken in oil; NA 1.4 objective lens. The scale bar in the confocal images is 2mm. Figure 3E: presents exemplary data of immuno-electron microscopy of Ins-C- GFP expressing β cells of islets in 70-100 ran thin sections showing colocalization of antibodies for GFP labeled with large gold particles and insulin labeled with small gold particles. The scale bar in the electron micrograph is 200nm. The circular dark objects are the electron dense cores of the insulin secretory granules. The oblong dark object is a mitochondrion, which does not label.

Figure 4. shows an exemplary color assay of insulin secretory competence provided by an Ins-C-mcTimer of the present inventions. A C(peptide)-m(monomeric )c(cherry)-Timer was expressed in mouse islets in Mat-Tek optical chambers and image with filters for green and red fluorescence. Yellow represents vesicles comprising red and green fluorescent molecules.

Figure 4A: shows exemplary green- red distribution 14 hours post-expression, and overall appears mostly green with some yellow.

Figure 4B: shows exemplary green- red distribution 28 hours post-expression without diazoxide at 28 hours, and overall appears mostly yellow with some green.

Figure 4C: shows exemplary parallel experiments with the potassium channel opener diazoxide to block secretion beginning after the 14 hour time point which showed red β cells at the 28 hour time point. With insulin secretion blocked by diazoxide, overall the islets show mostly yellow with red. Images are representative of nine islets in the presence of diazoxide and nine islets in the absence of diazoxide (B).

Figure 4D: shows exemplary representative quantification data of the change in relative fluorescence intensity ratios (λ) of green/(green+red), where λ is the ratio of green cells to total fluorescent cells in an islet over time from the images in Figures 4A- 4C. Black lines/boxes: Pancreatic islets incubated in absence of diazoxide. Red lines/boxes: Pancreatic islets incubated in the presence of diazoxide between 14-28 hours post-expression. Olympus 1X70 widefield epifluorescence with 6OX NA 1.4 oil objective, secretion-dependent green/red fluorescence ratio.

Figure 5 presents exemplary data showing that glucose stimulus cycling restores insulin secretory competency to compromised human donor islets having a λ < 0.30. Ins-C-mcTimer fluorescence was measured with 15 mM glucose stimulus cycling (red lines and blocks) versus 4 mM glucose stimulus cycling (black lines and blocks).

Figure 6 presents one embodiment of a double stranded nucleic acid sequence encoding an Adlox-Ins-C-mCherry-BstEII-stop-Timer+Hind III vector (wherein the sequences shown in this figure were ligated together in operable combination to form this exemplary expression vector construct, SEQ ID NO:03, for a biosensor of the present inventions) illustrating exemplary modular regions for inserting sequences for a promoter region, expressed peptide regions (for this example, genomic sequences for expressing insulin), wherein an exemplary fluorescent molecule coding sequences were inserted into peptide region C of genomic insulin sequences, exemplary endonuclease restriction sites, et cetera. An isolated exemplary Ins-C-mcTimer coding sequence which was inserted into this vector is shown in Figure 7. The numbered nucleic acids for the plus (encoding strand), SEQ ID NO:03, are shown on top of the minus strand.

Figure 6A: adenoviral shuttle vector, Adlox sequence, Figure 6B: a mouse insulin II promoter (mRNA starts at nucleic acid number

1512 within shaded region),

Figure 6C: additional promoter sequences, proinsulin sequences including intron 1 and beta (B) sequence (shaded region shows signal sequence beginning with MAL to TQA amino acids followed by beta peptide amino acid translation FVK to PMS), RR shows the protyolytic cleavage site between B peptide and C peptide, C peptide nucleic acid sequence extends from EVE through intron 2 (nucleic acids numbers 1879-2366) to the AAA fusion point near the BstEII site with mcTimer (Figure 6D) and introns;

Figure 6D: fluorescent molecule insertion into peptide C, and

Figure 6E: remainder of peptide C, cleavage site KK separating peptide C from peptide A, Alpha (A peptide) sequence (shaded region showing alpha peptide amino acid translation) and remainder of vector.

Figure 7 shows an exemplary coding sequences for Timing fluorescent proteins, for example, sCherry showing an "I" to "T" amino acid substitution at position 197 ("I197T") turning monomelic Cherry into a multifluorescent protein m(monomeric) c(cherry)Timer comprising an mcTimer coding sequence of the present invention (in conjunction with exemplary sequences shown in Figure 6) and showing an optional V105A mutation (at amino acid 110) for "bright" which when present increases fluorescence of GFP and Cherry (red) approximately 2 fold. The mcTimer as used herein was inserted in- frame within the middle of c peptide of proinsulin, similar to Ins-C- dsRedl (Michael, et al., Diabetes, 2007, herein incorporated by reference.), GFP (Watkins, et al, Traffic 2002; 3: 461-471, herein incorporated by reference.) and the original Timer (Bertera, et al BioTechniques 35:718-722 (October 2003); Michael, et al., Biophys J. BioFAST on October 29, 2004), all of which are herein incorporated by reference in their entirety.

Figure 8 shows exemplary differences between trafficking and secretory patterns of Ins-C-Timer (a tetrameric aggregating probe) versus Ins-C-mcTimer (a monomelic probe) expression in beta cells.

Figure 8A: Original mouse Ins-C-Timer and

Figure 8B: Mouse Ins-C-mcTimer of the present inventions. In each panel three to four beta cells are shown. The central non-fluorescent disc within a cell shows the nucleus of the cell surrounded by fluorescent puncta in the cytoplasmic area of the cell.

Figure 9: shows an exemplary alignment of mouse and human pro-insulin(insulin) for use in providing a human Ins-C-mcTimer of the present inventions.

Figure 10: presents one embodiment of a human "emerald" expressing vector. The inventor inserted a mutation preventing dimerization for providing a monomelic C- peptide tagged GFP construct named: Adlox-M-promoter-Human-insulin-C-emGFP- A206K, a human insulin protein is shown in relation to the fluorescent protein.

Figure 11 shows exemplary expression of Ins-C-emGFP in human islets targets green fluorescence to punctate organelles.

Figure HA: Column shows confocal images of human islets from three donors, representative of islets from 22 donor organs studied.

Figure HB: DIC images.

Figure 11C: Merged images shows cells identified by green fluorescent puncta within cytoplasm circumscribed by unlabeled nucleus and plasma membrane. The fluorescent images are from confocal optical sections immediately above the coverslip, approximately 0.5 μm thick. Fluorescence was not found in uninfected human islets under the experimental conditions (n=22). Bar, 2 mm. Figure 12 shows exemplary Ins-C-emGFP fluorescent puncta co-localize with insulin secretory granules.

Figure 12 A: 3D image reconstructions showing a beta cell within a human islet expressing Ins-C-emGFP, representative of islets from 10 donor organs studied. Images within column show a beta cell at various perspectives.

Figure 12B: Same reconstructions showing anti-insulin staining.

Figure 12C: Merged reconstructions with yellow puncta. Green and red channel imaged by sequential excitation and simultaneous detection (n=10). Bar, 2 mm.

Figure 13 shows exemplary human islets in perfusion fluids that demonstrated glucose-stimulated insulin secretion. Isolated non-diabetic islets used in this study were tested for secretory responses to steps in glucose from 5.6 to 20 mM. Results of separately assayed islets from three non-diabetic donors are shown. R/Rmax is the relative rate of insulin release per min at the indicated time points normalized to the maximal rate in an experiment. Figure 14 shows exemplary Ins-C-emGFP labeled human beta cells that exhibited stimulated fluorescent decay in response to insulin secretagogues and insulin secretory granule mobility. Mouse Ins-C-emGFP labeled cell before (A) and after (B) stimulation by 20 mM glucose. Note changing granule positions and modest decay in fluorescence in response to step from 5.6 to 20 mM glucose. C. Time course of Ins-C- emGFP fluorescence decay.

Figure 15 shows exemplary fluorescent glibenclamide co-localizes with Ins-C- dsRedl Bright in human islets.

Figure 15A-C: Three islet beta cells, one in each row, representative of islets from 12 donor organs. Within a row from left to right, a confocal z section from the green channel detecting BODIPY FL glibenclamide, from the red channel detecting Ins-C- dsRedl Bright, and merge. Puncta are yellow demonstrating co-localized green glibenclamide and red Ins-C-dsRedl Bright markers. Images taken by sequential excitation and simultaneous detection, with no channel cross talk (i.e. . Bar, 2 mm.

Figure 16 shows exemplary Ins-C-emGFP labeled insulin in Human Type 2 Diabetes (T2D) islets shows non-punctate diffuse cytoplasmic staining. Figure 16A: Non-diabetic human islet cell expressing Ins-C-emGFP co-stained with ER marker anti-calnexin exhibited punctate green GFP fluorescence and net-like red calnexin staining.

Figure 16B: T2D human islet cell expressing Ins-C-emGFP and with ER marker anti-calnexin.

Figure 16C: Two T2D human islet cells expressing Ins-C-emGFP and co-stained with anti-calnexin. The T2D islets (n=23) showed Ins-C-emGFP with calnexin localized to the ER. Approximately 0.4 micron thick z sections were taken 2 mm apart from each other. Figure 17 shows exemplary The ER marker Grp78 co-localizes with Ins-C- emGFP in the Human Type 2 Diabetes (T2D) islets.

Figure 17A: As in previous Figure except Ins-C-emGFP expressing cells co- stained with anti-Grp78 antibody.

Figure 17B: As in previous Figure except Ins-C-emGFP expressing cells co- stained with anti-Grp78 antibody in reticulated structures.

Figure 17C: Cells from additional islets showing same co-localization of insulin with the ER marker. The T2D islets (n=l 8) from this donor showed Ins-C-emGFP with Grp78 localized to ER.

Figure 18 shows exemplary Human Type 2 Diabetes (T2D) islet preparation showing accumulated traffic of Ins-C-emGFP and KATP channels at ER.

Figure 18 A: Reticulate expression of Ins-C-emGFP in islet cells from a second diabetic donor. The images are perspectives of a 3D reconstruction of optical section from a representative single beta cell within an islet. The nucleus appears as a non- fluorescent disc near top. The surrounding reticulate green fluorescence within the cytoplasmic compartment is similar to that in Figures 15 and 16.

Figure 18B: Islet with beta cells showing green Ins-C-emGFP fluorescence.

Figure 18C: Same islet as in Figure 18A stained with anti-Grp78 with red fluorescent secondary antibody.

Figure 18D: Merged image of Figure 18B and Figure 18C with anti-Grp 78 and Ins-C-emGFP labeling appearing as yellow fluorescence in the ER. Figure 18E: Islet from same donor stained with green glibenclamide-BOD IPY-FL similar to the reticulate staining in the ER, with minor asymmetrical staining with respect to the nucleus.

Figure 18F: Islet from same donor showing Golgi-EYFP around nucleus. Bar, 5 mm. Figure 19 Figure 2 shows an exemplary β cell fluorescence phenotype of the diabetes mutants L30P and C96Y (hAkita) compared to wild-type.

Figure 19A: Expression of hIns-C-emGFP-L30P in rat INSl cells. A background of diffuse, near uniform green fluorescence was observed, never seen for hlns-C-emGFP- WT (Fig 1; n = 28), consistent with ER accumulation. Green punctate structures the size of insulin granules were also evident (white arrows).

Figure 19B: Expression of hIns-C-emGFP-C96Y (hAkita) shows a diffuse green fluorescence pattern, consistent with ER accumulation, consistent with results from the homologous mouse mAkita mutant.

Figure 19C: Figure 1 shows exemplary hlns-C-GFP-WT expressed in INSl cells demonstrating and strong signal and corresponding strong expression of insulin within insulin secretory granules with relatively little labeling of Golgi or ER. A single cell shown here was typical of the wild-type hlns-C-GFP biosensor expression (n = 28). The optical section shown was approximately 400 run thick, comparable to the diameter of a single insulin secretory granule. The number of granules visualized (labeled) were in the order of one hundred granules, reflecting highly efficient expression and trafficking of the wild-type hlns-C-emGFP biosensor.

Figure 20 shows exemplary trafficking of insulin using a biosensor of the present inventions.

Figure 2OA: Figure 3 shows an exemplary ratio of hlns-C-emGFP to C-emGFP that quantified trafficking from the ER and proteolytic cleavage in the insulin secretory granule. hlns-C-emGFP-WT, -L30, and -C96Y were expressed in parallel in the rat INS 1-832/13 cell line for two days. Equal total protein was added per lane and the Western blots were probed with anti-GFP specific for the human peptides. L30P was a B chain mutation and C96Y (hAkita) was an A chain mutation. Figure 2OB: Figure 4 shows an exemplary western blot of hlns-C-GFPs transfected into INS1-832/13 cells without (-) or with (+) co-expressed Derlin-1. In each mutant case, Derlin-1 resulted in decreased levels of the proinsulin and C peptide bands. The blots were probed with anti-GFP. Actin was separately probed on the same blots, and confirmed equal amounts of loading per lane.

Figure 21 Figure 5 shows an exemplary evidence for poly-ubiquitinylation of secretory pathway proteins.

Figure 21 A: INSl cell membrane fractions were split into two aliquots, immunoprecipitated with anti-GFP or control IgG, then the immunoprocipitates analyzed as in 2 IB,

Figure 2 IB: Western blots with anti-GFP. Figure 22: Figure 7 shows exemplary β cell fluorescence evidence that human

Akita mutant blocks ER exit of wild-type proinsulin.

Figure 22A: Punctate red fluorescence from wild-type Ins-C-mCherry co- transfected with wild-type human Ins-C-emGFP in INS 1-832/13 β cells.

Figure 22B: Mostly uniform red fluorescence from wild-type Ins-C-mCherry co- transfected with human Akita mutant hIns-C-emGFP-C96Y. The uniform red fluorescence reflects ER accumulation of the wild-type Ins-C-mCherry reporter due to the presence of the green Akita mutant.

Figure 22C: The red fluorescence channel output, alone, from Panel B emphasizes the mostly uniform β cell fluorescence (n = 7). Compare to punctate fluorescence of wild- type alone in Figure 22.

Figure 22D: Figure 6 shows exemplary secretion of endogenous wild-type C peptides blocked by expression of human mutant but not wild-type proinsulin. hlns-C- GFP-WT and hlns- C-GFP-C96Y (hAkita) were expressed in parallel in rat INSl cells and rat C peptide secretion assayed by perifusion. C peptide secretion was shown normalized to the peak ng/min/mg protein of the total cell extract of the hlns-C-GFP-WT sample, for easy comparison of the time course. The assay method does not detect significant human protein or rat proinsulin.

Figure 22E: Figure 8 shows exemplary diabetes mutations L30P and C96Y that enhance the rate of apoptosis of β cells. hlns-C-GFP-Wild-type, hIns-C-GFP-L30P, and hIns-C-GFP-C96Y were expressed in INS1-832/13 cells for three days. The cell cultures were assayed for apoptosis by using an ELISA kit (Roche). The data shown was from n > 3 experiments for each sample.

Deailed Description

This invention is related to the field of imaging intracellular protein processing. Fluorescent probes capable of changing color over time are used to identify and track the synthesis, trafficking, and secretion of intracellular proteins. One such probe useful in this invention is the Timer probe (a multifluorescent protein) which, after expression, sequentially changes from green to red over a twenty- four hour period. In one embodiment, the invention comprises a monomeric version of a Timer probe. In another embodiment, a monomeric multifluorescent protein is inserted into the C-linker portion of the insulin prepropeptide as a marker for normal insulin responses to glucose in cultured donor beta cells.

In particular, the inventions provided herein provide multifluorescent proteins as probes for marking insulin trafficking and responses to glucose in beta cells of the pancreas.

One such probe useful in this invention is a new Timer probe based upon a Timer mutation used in a DsI Red E5 multidimer Timer probe (referred to as the "original" Timer (Terskikh et al., 2000, SCIENCE 290:1585-1588, herein incorporated by reference), where a DslRed sequence was shown inShaner, et al., 2004, Nature Biotechnology, 22:1567-1572, without the Timer mutation, called T197 (S197T) or a Bright mutation of V105A as described in the E5 Timer probe. However, unlike the original E5 Timer which aggregates into multimers, after a 197T equivalent insertion was made in a nontiming mCherry (Clonetech) fluorescent gene it provided a monomeric version of a Timer probe referred to as "rncCherry" or a "new Timer" of the present inventions, SEQ ID NO:01,

MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG VVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGA LKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNgKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYK, where the boxed BOLD T is the timer mutation at amino acid 202 of mCherry, and SEQ ID NO:02, mcCherry-Bright, MVSKGEEDNMAIDCEFMRFKVHMEGSVNGHEFEΓEGEGEGRPYEGTQTAKLKVT KGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGG V@TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGA LKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNgKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYK, where the boxed BOLD A is a bright mutation equivalent at amino acid 110 of mCherry. Like the original Timer, expression of mcCherry fluorescence in a cell sequentially changes from green fluorescence to the red fluorescence of cherry (see, Figure Ib and Id of Shaner, et al., 2004, Nature Biotechnology, 22:1567-1572, herein incorporated by reference) over a twenty-four hour period. Such "timing" provides a time-dependent marker contemplated for use in determining when and where any fusion or linked protein in located. In one embodiment, the new Timer probe marks (is fused to) a specific molecule, such as fused with an insulin molecule. In one embodiment, the Timer probe co-trafficks with a molecule of interest (where the markers is not fused to the molecule of interest, such as the proteolyically cleaved and disulfide linked insulin alpha and beta chain. Previous attempts at fluorescently labeling insulin for trafficking studies showed that inserting a fluorescent molecule at the 5' end of the beta chain, or the 3' end of the alpha chain caused the fusion insulin protein to have altered retention times and to translocate in patterns that did not match insulin trafficking in beta cells that were considered healthy due to glucose induced insulin secretion (for example, see Pouli et al., 1998 Biochem. J. 331 :669-675); herein incorporated by reference).

Therefore, the inventor created a mouse Ins-C-Timer molecule for providing a time-dependent marking of an insulin molecule and insulin containing vesicle. However, this Timer molecule caused apparent mistrafficking and/or altered retention times of the original Timer probe (as shown in Figures 1 and 2) were contemplated as the result of dimer and trimer formation and larger aggregates of Timer, it was contemplated that a monomelic form of Timer would provide a probe which would not interfere with insulin trafficking, thus allowing normal and mutated forms of insulin to traffϊck as if no marker were attached, such that normal insulin would be secreted and mutations that interfered with normal insulin trafficking would continue to traffick abnormally. In order to begin testing a monomelic fluorescent molecule, the inventor created a mouse Ins-C-Emerald molecule wherein they inserted a GFP molecule into the C-peptide, as shown in Figure 3. Although this green molecule showed great improvement as a tracking molecule over the original Timer probe for insulin secretory vesicles and over previous molecules used for trafficking, this molecule lacked the capability to show a time-dependent marking of the vesicles and showed low levels of dimerization. Further, without extreme biochemical, cell biological, immunological, etc., methods, it was difficult to determine the age of the vesicle comprising the Ins-C-Emerald (green fluorescent) molecule.

Thus, in one embodiment, a monomelic (m) version of a Timer probe was contemplated for use. In one embodiment, a fruit monomelic probe was contemplated as a Timer molecule due to residual GFP coding regions on the 3' and 5' ends of the nongreen encoding regions. Therefore, in one embodiment, cherry (Clontetech) was contemplated as a recipient of a Timer mutation, wherein an amino acid homologous to the original T 197 mutation was mutated to a T. In a further embodiment, the "bright" mutation of a V 105 A was made at a homologous amino acid of cherry for a two-fold brighter fluorescent molecule. A bright mutation in addition to being brighter, provides a longer lasting fluorescent color over time when undergoing excitation. Further, a bright mutation allows visualization of a cell within a millisec rather than longer periods of time for allowing live cell fluorescent trafficking with minimal laser/heat damage to the living cell. In a preferred embodiment, the mutated cherry, comprising T 197 was inserted into the C-linker portion of the insulin propeptide. In one embodiment, the mCherry of the present invention was inserted into an adenoviral vector for expression in a mammalian cell. In one embodiment, the mcCherry of the present invention was inserted into an adenoviral vector for expression in a mammalian cell.

In one embodiment, the insulin molecule, alpha, beta and c chain is derived from a mouse genomic sequence. In another embodiment, the insulin molecule, alpha, beta and c chain is derived from a human cDNA sequence. In one embodiment, the human insulin molecule is expressed under a mouse insulin II promoter of the present inventions.

Various embodiments of the present invention contemplate the development of anti-diabetic drug discovery that may effect insulin metabolism. For example, live-cell fluorescent biosensors faithfully report insulin biology and have increased our understanding of insulin action in health and disease. The present invention contemplates various improvements to these biosensors (monomeric Timer probes of the present inventions), including but not limited to, the ability of the fluorescent insulin biosensor to time its own biology. Developing these features without disrupting a faithful reporting of insulin biology has proven difficult. One problem that was overcome during the development of the present inventions was the unexpected behavior of such biosensors arising from a combination of the aggregating nature of the fluorescent protein moiety and the folding and trafficking of insulin beginning in the ER. While routinely constructing and testing numerous variants of these fluorescent biosensors for advantageous properties year after year, yet encountering problems with each new construct, a new construct of described herein in the present inventions surprisingly appeared capable to time insulin biology without affecting normal folding or trafficking of the insulin molecule. In one embodiment, this construct comprises an Ins-C-mcTimer gene.

In one embodiment, the present invention contemplates a method for assessing protein secretory capacity of human donor tissues. In one embodiment, the donor tissues are used in transplantation surgeries. In one embodiment, the donor tissue comprises pancreatic β cells. Although it is not necessary to understand the mechanism of an invention, it is believed that the transplantation of pancreatic β cells capable of insulin secretion should be capable of curing Type 1 diabetes. In one embodiment, the method further comprises a fluorescent probe capable of quantifying the age of a secretory protein. In one embodiment, the secretory protein is insulin. In one embodiment, the fluorescent probe comprises a mcTimer construct. Although it is not necessary to understand the mechanism of an invention, it is believed that a mcTimer construct encodes a protein that exhibits a green fluorescence for a few hours after its biosynthesis, and then changes to red fluorescence between about 14-24 hours after its biosynthesis. By taking the ratio red:(red+green) fluorescence the average age of a secretory protein can be determined using standard microscopy. For example, an mcTimer-tagged secretory protein in healthy cells remain green and yellow (a comparable mix of green and red fluorescence) and only when protein secretion is impaired, such as unhealthy donor tissue, damage resulting from isolation or transport of donor tissue, exposure to secretory inhibitors or mutations in insulin that inhibit insulin release at the plasma membrane, would the mcTimer age sufficiently to present a substantially red fluorescence within a cell or tissue comprising islet beta cells.

The experimental results shown herein demonstrated wild-type insulin trafficking using human Ins-C-FP's. Specifically, human Ins-C-FP's target (were found in) insulin secretory granules and tracked their behavior (i.e. vesicle movement through the cell, such as from the endoplasmic reticulum (ER) to the Golgi, to the cell membrane, etc.). Further, application of the human Ins-C-FP's demonstrated that the human Ins-C-FP's allowed easy use of these biosensors (i.e. using routine methods) for following vesicle trafficking and insulin processing by live-cell imaging and biochemically (i.e. Western blots, etc.) such that wild-type insulin trafficking and processing was directly compared to that of specific mutant proinsulins associated with clinical diabetes.

The majority of the human mutant Ins-C-FP's tested showed marked accumulation in the ER and further provided evidence that insulin was misfolding in the ER. These abnormal cellular phenotypes were consistent with additional evidence generated by the inventor during the course of development of the present inventions for degradation of the proinsulin via the unfolded protein response pathway. This pathway includes degradation of unfolded proteins tagged by ubiquitylation (otherwise known as ubiquitination) which refers to a post-translational modification of a protein by the attachment of one or more ubiquitin monomers. A major function of ubiquitin attachment is labeling proteins for proteasomal degradation.

Further, the inventor found that when mutant proinsulins were co- expressed with wild-type proinsulin, the wild-type proinsulin was then abnormally degraded. This observation shows the strong potential for a mutant proinsulin gene causing phenotypic dominance over a wild-type proinsulin gene.

I. Fusion Protein Fluorescent Probes as Biosensors of the present inventions.

A. Cherry Fluorescent Protein. In general, m(monomeric) Cherry refers to a mutant fluorescent protein derived from the tetrameric Discosoma sp. red fluorescent protein, DsRed (Shaner, et al. (2004) Nature Biotech. 22(12): 1567- 1572, herein incorporated by reference.). The excitation and emission maxima for both molecules are 587 nm and 610 nm, respectively. However, unlike DsRed, the mCherry coding sequence was human codon-optimized for high-level expression in mammalian cells (Haas, J., et al. (1996) Curr. Biol. 6(3):315-324, herein incorporated by reference).

Numerous fruit fluorescent proteins including multimeric and monomer versions of Cherry, were well characterized and recognized in the literature as developed in Dr. Roger Tsien's lab (Shaner, et al. (2004) Nature Biotechnol. 22(12): 1567-1572; Wang, L. et al. (2004) Proc. Nat. Acad. ScL 101(48):16745-16749; and Shu, X. et al. (2006) Biochemistry 45(32):9639-9647; and Clonetech published technical literature (at clonetech.com; all of which are herein incorporated by reference). These fruit fluorescent molecules collectively have a wide emission range, at least two antibodies for detection of the expressed protein, repeatable stable expression, and performed successfully in numerous fusion applications.

The Fruit Fluorescent Proteins are mutants derived from mRFPl, a monomelic mutant of DsRed, by directed mutagenesis (Campbell, R. E. et al. (2002) Proc. Nat. Acad. ScL 99(12):7877-7882, herein incorporated by reference.), briefly described below. Monomeric Cherry expressing cell lines were established, for example, three stably-transfected HEK 293 cell lines with different levels of mCherry expression (as measured by flow cytometry). Transfected cells were observed to grow at a rate similar to nontransfected control cells, without increased cell death, as determined by visual inspection. See, Clonetech. Further, effective mCherry Fusion Constructs were successfully created by fusing mCherry to other proteins, including actin and tubulin (see, Clonetech). Other fusion proteins containing mCherry have been reported in Arabidopsis ((Song, et al. (2007) Proc. Nat. Acad. Sci. 104(13):5437-5442), zebrafish (Pisharath, et al. (2007) Mech. Dev. 124(3)'218-229), E. coli (Pradel, et al. (2007) Biochem. Biophys. Res. 353(2):493-500), HIV virions (Campbell, et al. (2007) Virology 360(2):286-293), and yeast (Snaith, et al. (2005) EMBO J. 24(21):3690-3699). These fusions were used for quantitative imaging techniques including fluorescence resonance energy transfer (FRET; Picard, et al. (2006) Exp. Cell Res. 312(19)3949-3958), fluorescence recovery after photobleaching (FRAP; Picard, D. et al. (2006) Exp. Cell Res. 312(19)3949-3958), and fluorescence lifetime imaging microscopy (FLEvI; Tramier, et al. (2006) Microsc. Res. Tech. 69(11)933-939, all of which are herein incorporated by reference). Simple Detection is available using DsRed Antibodies, such as Clontech's Living Colors® DsRed Monoclonal and Polyclonal Antibodies (Cat. Nos. 632392, 632393, & 632496) which are contemplated for use to detect the Fruit Fluorescent Proteins, including mcCherry, by Western blot analysis and histology (Fruit Fluorescent Proteins (2007) Clontechniques XXII(3)8). However, in contrast to fluorescent markers of the present inventions, the monomelic mCherry expresses few green fluorescent molecules while expressing a majority of red molecules that remains a stable "cherry" or "red" color over time.

B. Multimeric and Monomelic versions of Fruit fluorescent molecules were developed as briefly shown in Figure 2b Shaner, et al. (2004) Nature Biotechnol. 22(12): 1567-1572, herein incorporated by reference; from Dansyl Red Protein (DsRed).

1. DsRed fluorescent proteins refer to coelenterate fluorescent proteins that were cloned to display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP did not impede its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. Thus a stepwise evolution of DsRed was undertaken to create a smaller dimer aggregate and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFPl (red fluorescent protein 1) (s monomeric red fluorescent protein). Briefly, to reduce the aggregates of DsRed molecules within cells, each subunit interface of DsRed was disrupted by insertion of arginines, which initially crippled the resulting protein by interfering with aggregate formation, but red fluorescence was then rescued in the mutant molcecule by random and directed mutagenesis totaling 17 substitutions in the dimer molecule and 33 in mRFPl. Tests showed that smaller sized fusion molecules were more functional for example, wherein fusions of the gap junction protein connexin43 to mRFPl were created that formed fully functional junctions in cells, whereas analogous fusions to the tetramer and dimer failed. Although mRFPl has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFPl matured >10 times faster, so that it shows similar brightness as DsRed in living cells. In addition, the excitation and emission peaks of mRFPl, 584 and 607 nm, are 25 nm red-shifted from DsRed, which was contemplated to confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins. See, Clonetech at clonetech.com.

2. Formation of fruit multimeric and monomelic fluorescent proteins derived from the mutated Ds Red, see above, are briefly summarized in a schematic diagram in Figure 2b of Shaner et al., 2004, Nature Biotechnology,

22:1567-1572, herein incorporated by reference.

An amino acid sequence comparison between DsRed, mRFPl, Cherry and other fruit proteins are shown in Figure 2a of Shaner, et al., 2004, Nature Biotechnology, 22: 1567- 1572, herein incorporated by reference. . Further, this alignment demonstrates that for comparison purposes, the Timer 197 "T" mutation is generally at position 217 of the aligned molecules as shown in Figure 2a of Shaner, et al., 2004, Nature Biotechnology, 22:1567-1572. . Thus the inventor further contemplate mutating other fruit fluorescent molecules to express a T amino acid at that that homologous location (generally referred to as amino acid position 197 by custom) for creating additional timer molecules, in particular those that retain gfp (green) coding sequences, however other timer molecules without the gfp sequences are contemplated for a time dependent location for creating additional Timer molecules, in particular those that retain GFP (green) coding sequences, however other Timer molecules without the GFP sequences or any sequences capable of providing a first fluorescence are contemplated for a time dependant initiation of "color" for example, a multifluorescent molecule wherein the first color is silent or a very low or no fluorescent intensity version of the second color.

C. Original Timer, pTimer (plasmid Timer) is a prokaryotic expression vector that encodes DsRed 1-E5, a mutant of the red fluorescent protein, DsRed 1 (Living Colors® Red Fluorescent Protein (October 1999) Clontechniques XIV(4):2-6). (The cDNA for the wild-type protein, DsRed, was originally isolated by Matz et al., who refer to the protein as drFP583 (Matz, et al. (1999) Nat. Biotechnol. 17:969-973). DsRedl-E5 contains two amino acid substitutions (V(valine)105A(alanine) and S(serine)197T(Threonine), respectively), which increase its fluorescence intensity and endows it with a distinct spectral property: As the protein ages, it changes color (Terskikh, A., et al. (2000) Science 290:1585-1588). When first synthesized, DsRedl-E5 is bright green (excitation & emission maxima = 483 nm & 500 ran). As time passes, the green fluorophore undergoes additional changes that cause its fluorescence to shift to longer wavelengths — when fully matured, the protein is bright red (excitation & emission maxima = 558 nm & 583 nm). In mammalian cells transfected with a Tet-inducible DsRed 1-E5 expression vector, the green-to-red transition starts about 3 hours after the protein first becomes fluorescent (Terskikh, A., et al. (2000) Science 290:1585-1588, herein incorporated by reference). In addition to the amino acid replacements, DsRedl- E5's coding sequence contains a series of silent base-pair changes, which correspond to human codon-usage preferences, for high expression in mammalian cells (Haas, et al. (1996) Curr. Biol. 6:315-324, herein incorporated by reference). The DsRedl-E5 coding sequence is flanked by separate and distinct multiple cloning sites at the 5' and 3' ends so that the gene can easily be excised for use in other expression systems. Alternatively, the DsRed 1-E5 coding sequence can be amplified by PCR. The DsRed 1-E5 gene was inserted in frame with the lacZ initiation codon from pUC19 so that DsRed 1-E5 is expressed from the lac promoter (Plac) in E. coli host cells. A Kozak consensus sequence is located immediately upstream of DsRed 1-E5 to enhance translational efficiency should you wish to express the gene in eukaryotic systems (Kozak, (1987) Nucleic Acids Res. 15:8125-8148, herein incorporated by reference). The entire DsRedl-E5 expression cassette in pTimer (Clonetech) is supported by a pUC backbone, which contains a high- copy number origin of replication and an ampicillin resistance gene for propagation and selection in E. coli.

D. Emerald (em) Green Fluorescent Protein constructs as biosensors. The inventor further contemplated constructs comprising a monomelic Green

Fluorescent Protein (GFP). In one embodiment, mutations were inserted for interrupting dimerization of GFP while retaining its fluorescent properties. GFP is a standard fluorescent molecule described in Tsien RY., "The green fluorescent protein" Annu Rev Biochem 67:509-544 (1998).

Unlike previous GFP molecules used for trafficking, monomelic GFP molecules of the present inventions provided a smaller molecule for allowing more accurate trafficking of the protein of interest, for example, proteins of interest in the present inventions include but are not limited to preproinsulin, proinsulin, and processed insulin. Specifically, any intracellular processed propeptide, including all propeptide hormones and all propeptide neurotransmitters are laid out with a molecular framework and biologic of proinsulin and therefore could be made into such fluorescent biosensors.

π. Preproprotein Probes as Biosensors.

Inserting fluorescent labels into prepropeptides provides numerous advantages for studying complex molecular and cellular mechanisms in live cells (i.e., for example, regulated secretory peptide vesicle trafficking and exocytosis). First, prepropeptide labeling takes advantage of the crystalline concentration of propeptide is targeted to large dense-core secretory vesicles that results in an increase in signal intensity (i.e., for example, vesicle trapping). For example, a propeptide fluorescent label would be expected to define subcellular structures and dynamics at the confocal optical resolution limit of 100's of nm. Second, fluorescent protein (FP) technology affords real-time durations of millisecond-to-days for imaging live cells that identify subcellular and molecular landmarks defining underlying spatial and temporal cellular mechanisms. Third, a live-cell assay incorporating spatial and temporal dynamics involving time-lapse photography and direct intracellular measurements provide more accurate and relevant data when compared to traditionally used multiple endpoint assays of regulated secretory peptide vesicle trafficking and exocytosis using crude extracts, fixed cells, and perifusion samples.

A. C-Fluorescent Proteins Ins-C-GFP & Ins-C-emGFP.

A prepropeptide marker was created using a green fluorescent protein (GFP) coding segment fused in-frame within a part of an insulin fragment that is cleaved from a mature secreted peptide. Watkins et al., "Imaging Secretory Vesicles By Fluorescent Protein Insertion In Propetotide Rather Than Mature Secreted Peptide" Traffic 3:461-471 (2002); herein incorporated by reference. Maturation and cleavage of the nascent insulin protein occurs in response to a reduction in intravesicular pH that activates convertases Cl and C2, as well as carboxypeptidase H and proteolytically cleaves C-peptide from the A and B chains within proinsulin, (see, Tager, et al., 1973, J Biological chemistry,

248" 10:3476-3182). The mature folded insulin condenses to form the electron dense core of the secretory granule. Thus, by insertion into the C peptide of proinsulin, the FP(fluorescent protein) is trapped as a stoichiometric reporter within the vesicle, without being part of mature insulin. The 'vesicle trapping' strategy thereby obviates problems such as improper secretory regulation that might result from modification of the mature peptide hormone or neuropeptide by the fusion of the fluorescent protein marker. It was observed that a reporter may be optimized by using a native gene including a promoter and regulatory signals for physiologically relevant expression, targeting, and trafficking. Highly efficient targeting by Ins-C-GFP to insulin vesicles was supported both by confocal fluorescence imaging and by immuno-electron microscopy. Confocal imaging verified that mistargeting was absent. In over three-quarters of approximately 20,000 mouse islets infected by adenovirus expressing the Ins-C-GFP, a punctate pattern of fluorescence was found, with punctal diameter consistent with that of insulin vesicles (i.e., for example, up to 1 mm). See, Figures 3A-3D. Immuno- electron microscopy showed that anti-GFP staining was markedly above background only at dense cores and always accompanied by anti-insulin staining. See, Figure 3E. The reverse, anti-GFP staining without anti-insulin staining, was not found, for example, mitochondria were not stained with anti-GFP antibodies. These observations are consistent with a mouse insulin II promoter driving β-cell-type specific expression of Ins-C-GFP. Immuno-electron microscopy also showed that about 80% of the dense cores had no significant anti-GFP staining, consistent with fluorescence observations indicating that Ins-C-GFP expression by adenovirus in infected islets was present in approximately 30% of the cell volume, localized at the perimeter.

These results demonstrate that that C-prepropeptide targeting is efficient. The success likely results from using the native mouse insulin II gene, including a 952-bp native promoter, and/or by inserting GFP in the middle of the C-peptide. Other attempting this type of approach failed as their constructs fused GFP to or within a human pre-proinsulin sequences outside the C peptide. Pouli et al., "Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase" Biochem J331 :669-675 (1998). An alternative construct was developed to monitor insulin secretion at the cellular level using fluorescent protein technologies using the mouse based Ins-C-emeraldGFP (Ins-C-emGFP) probe. Watkins et al., "Imaging secretory vesicles by fluorescent protein insertion in propetide not mature secreted peptide" Traffic 3:461-471 (2002). The mouse based Ins-C-emGFP probe provided extraordinary bright florescent insulin granules and numerous physiological attributes, featuring proper proteolytical processing, insulin folding, and secretion. Michael et al., "Pancreatic β-cells secrete insulin in fast- and slow-release forms" Diabetes 55:600-607 (2006).

However, while the Ins-C-emGFP was efficiently processed and showed fast exocytic release kinetics indistinguishable from the vast majority of wild-type insulin release events, the probe revealed numerous disadvantages. The probe was inefficient because it did not enable easy normalization of the fluorescence to control samples showing variable expression from cell to cell, its use for optically assay insulin secretion required tedious analysis of high numbers of images showing small changes in fluorescent intensity following stimulation by secretagogues, and finally, low levels (weak) but detectable dimerization were found that would impair its use for studying the effects of medically/physiologically relevant mutations in human insulin.

Therefore the inventor contemplated a complete monomelic GFP molecule for an Ins-C-emerald construct and found a location for a point mutation that provided a monomelic GFP of the present invention. An exemplary monomelic GFP inserted and expressed within a human insulin probe is provided herein as a human Ins-C-GFP-A206. In one embodiment, a pH sensivitive monomelic probe is contemplated as a sensor of secretory granule pH.

As used herein, the terms "Ins-C-GFP" and "Ins-C-emeraldGFP" and "Ins-C- emGFP" are interchangeable and refer to a similar construct where a GFP molecule coding sequence was inserted into an insulin C peptide coding region of an insulin prepropeptide coding region, these terms refer to a construct comprising mouse insulin II unless specifically designated as a human Ins-C-GFP where the GFP molecule is inserted into a an insulin C peptide coding region of an human insulin prepropeptide coding sequence.

B. Tetrameric Timer Biosensor Probes (C-Timer Constructs). In order to overcome the above cited disadvantages of the Ins-C-emGFP probe, a fluorescent color ratio method was developed to control for variable reporter expression from cell to cell and islet to islet that also indicates to what extent each cell was secreting insulin. The Ins-C-Timer construct replaces the emGFP gene of the Ins-C-emGFP probe with a gene encoding a Timer protein. Terskikh et al., "Fluorescent timer": Protein that changes color with time" Science 290:1585-1588 (2000).

The C-Timer probe was derived from a dsRedl fluorescent protein, a coral protein that has to assemble in a homo-tetramer to fluoresce at practical levels. Timer when expressed alone, shows green fluorescence for the first few hours and then over time, by undergoing a slow conformational transition, with a characteristic time constant of about 14 hours, turns red (16). The time-dependent change from green to red fluorescence is independent of cell-type, level of expression, or protein to which C-Timer is fused, which can be used to determine how old C-Timer is at any given time.

It was observed, however, that C-Timer has many disadvantages. C-Timer is known to aggregate, and when administered as an Ins-C-Timer probe: i) disrupts processing of Proinsulin; ii) aggregates to form large fluorescent puncta in lysosomes, thereby showing delayed secretion from beta cells (Michael et al., "Fluorescent cargo proteins in pancreatic beta-cells: design determines secretion kinetics at exocytosis" Biophys J. 87: 3-5 (2004), and iii) induces glucose intolerance in Ins-C-Timer transgenic mice result in glucose intolerance (Bertera et al., "Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein" Biotechniques 35:718-722 (2003). Although it is not necessary to understand the mechanism of an invention, it is believed that these problems arose from the tetramerization and aggregation of C-Timer, thereby greatly limiting the use of Ins-C- Timer (2) as compared to Ins-C-emGFP. Fluorescent Timer is a mutated version of a red fluorescent protein obtained from the Anthozoa coral. Matz et al., "Fluorescent proteins from nonbioluminescent Anthozoa species" Nature 17:969-973 (1999). These mutations permit its fluorescence to change from green when it is first synthesized to red over approximately 24 h.

One such fluorescent Timer probe protein and gene was previously reported. Terskikh et al., "Fluorescent Timer": protein that changes color with time" Science 290:1585-1588 (2000). The development of fluorescent proteins included constructs wherein the probe was inserted into a prepropeptide such that cleavage resulted in a functional mature peptide. This advance allowed live cell monitoring in transgenic animals of intracellular protein synthesis, trafficking and secretion. Bertera et al., "Body Window-Enabled In Vivo Multicolor Imaging Of Transplanted Mouse Islets Expressing An Insulin-Timer Fusion Protein" BioTechniques 35:718-722 (2003). This probe was then inserted into a sequence encoding the C-peptide of murine insulin II to create Ins-C- Timer. Watkins et al., "Imaging Secretory Vesicles By Fluorescent Protein Insertion In Propeptide Rather Than Mature Secreted Peptide" Traffic 3:461-471 (2002). Although it is not necessary to understand the mechanism of an invention, it is believed that the C- peptide is normally cleaved when the A and B chains fold to generate the mature protein, the insulin molecule in the transgenic animal may still fold properly and the hormone may function physiologically.

C. Monomelic Multicolor Timer Biosensors (C-mcTimer Constructs).

In one embodiment, the present invention contemplates a multicolor monomelic fluorescent biosensor that uniquely tags the C peptide segment which is a peptide segment of the propeptide that, only after arrival in the secretory granule is proteoltically cleaved from the propeptide resulting in the tagged peptide and the mature peptide hormone or peptide neurotransmitter of an intracellular preproprotein (i.e., for example, preproinsulin). Because they are from the same precursor propeptide and obligatorily contained ("trapped") within the vesicle membrane, the fluorescent peptide sensor and either mature peptide hormone or mature peptide neurotransmitter are necessarily in 1"1 stoichiometry. In one embodiment, the tagged preproprotein is localized in the endoplasmic reticulum (i.e., for example, newly synthesized). In one embodiment, the preproprotein is localized in the intracellular space (i.e., for example, undergoing trafficking). In one embodiment, the preproprotein is localized at or near the inner cell membrane (i.e., for example, undergoing secretion). In one embodiment, the preproprotein is cleaved, thereby releasing the fluorescent biosensor. In one embodiment, the biosensor comprises an mcTimer biosensor. In one embodiment, the mcTimer biosensor is incorporated into the C peptide segment of the insulin prepropeptide (i.e., for example, Ins-C-mcTimer).

D. Schematic of the design of Ins-C-mcTimer.

Ins-C-mcTimer biosensors were developed based on a mouse emerald green fluorescent protein construct (Ins-C-emGFP). Watkins et al., "Imaging Secretory Vesicles By Fluorescent Protein Insertion In Propetotide Rather Than Mature Secreted Peptide" Traffic 3:461-471 (2002). As discussed above, a tetrameric version (Ins-C- Timer) was first developed but was discovered to have several disadvantages including but not limited to the development of diabetes. For example, Ins-C-Timer transgenic mice were shown to have symptoms of glucose intolerance. Bertera et al., "Body Window-Enabled In Vivo Multicolor Imaging Of Transplanted Mouse Islets Expressing An Insulin-Timer Fusion Protein" BioTechniques 35:718-722 (2003). Follow-up studies presented herein show that this glucose intolerance was indeed a precursor to diabetes and pancreatic tissue damage.

In one embodiment, the present invention contemplates a monomelic C-Timer probe based, in part, on a monomelic form of dsRedl, designated mCherry. Shaner, et al., "A guide to choosing fluorescent proteins" Nat Methods 2:905-909 (2005). In one embodiment, a monomeric C-Timer probe (i.e., for example, C-mcTimer) comprises a point mutated mCherry Timer probe, wherein the C-mcTimer probe changes color with time but does not aggregate.

Although it is not necessary to understand the mechanism of an invention, it is believed that, unlike a C-Timer probe, a C-mcTimer probe comprises a fluorescent coding region that does not require aggregation to initiate fluorescence. In one embodiment, the C-mcTimer probe comprises a monomeric fluorescent protein that changes color (i.e., for example, from green to red) subsequent to protein expression. In one embodiment, the C-mcTimer probe converts from green to red in approximately 24 hours. Although it is not necessary to understand the mechanism of an invention, it is believed that because intracelluar protein (i.e., for example, insulin) lifetimes are about one day (i.e., for example, twenty-four hours), healthy cells fluoresce green and yellow; wherein the yellow is a result of a mixing of mostly green and some red C-mcTimer probes. It is further believed that if there if a defect in protein folding, trafficking, or secretion then C-mcTimer probe intracellular lifetimes are increased and the cells fluoresce more yellow and red than green. In one embodiment, an intracellular measurement is taken by calculating a ratio of red fluorescence to total fluorescence (i.e., for example, red + yellow + green fluorescence). In one embodiment, an intracellular measurement may be selected from the group comprising post-protein expression time (i.e., for example, protein age), cell secretory capability (i.e., for example, vesicle release kinetics), or identifying intracellular locations responsible for deficiencies in intracellular protein processing including, but not limited to, synthesis, trafficking, and/or secretion. Although it is not necessary to understand the mechanism of an invention, it is believed that such processing deficiencies would be expected to underlie causation, development, and/or expression of disease states (i.e., for example, deficiencies in insulin secretion being responsible for diabetes).

As outlined above, previous studies have introduced the construction and application of Ins-C-emGFP to create Ins-C-Timer via a point mutation resulting in a color change from green to red. Unfortunately, the Ins-C-Timer proprotein is not processed, aggregates and therefore the reporter was not widely usable or disseminated. This problem was overcome by mutating the fluorescent mCherry protein (a monomelic derivative of DsRedl). mCherry has been reported to not aggregate (13, 43). As reported herein, the mCherry protein was mutated to create the Ins-C-mcTimer probe. Ins-C-mcTimer expressed in mouse islet β cells reliably identifies insulin secretory competence. See, Figure 4. A metric (λ) was then developed to calculate the fractional islet β cells expressing C-mcTimer that are competent for insulin secretion based on the color of C-mcTimer under standardized fluorescence data acquisition conditions: λ = (# green cells)/(# green cells + # red cells).

The λ metric may be used with high reproducibility. For example, after the first 14 h of mouse pancreatic islets expressing Ins-C-mcTimer, β cells are largely green with some yellow (λ = 0.65). After the first 28 hours, the cells remain largely green and yellow with little or no red (λ = 0.52). In parallel experiments, islets treated from the 14 h time point onward with 100 μM diazoxide (a potassium channel opener blocking insulin secretion) showed dramatic accumulation of red fluorescence (λ = 0.29). See Figure 4D. The results indicate that Ins-C-mcTimer can be used as an easy color assay of insulin secretory competence in mouse islets. Although it is not necessary to understand the mechanism of an invention, it is believed that the present invention improves imaging capabilities of conventional fluorescent microscopes. It is further believed that the mcTimer probes will result in novel modeling of disease pathology (i.e., for example diabetes) and/or the development of highly useful cell and animal systems to understand and cure disease.

III. Methods Of Using C-mcTimer Probes.

In one embodiment, the present invention contemplates using C-mcTimer probes to facilitate identification of basic subcellular signaling mechanisms (i.e., for example, coupling glucose metabolism to insulin granule exocytosis) but to develop disease and metabolic disorder drugs and/or therapies (i.e., for example, for diabetes). In one embodiment, a C-mcTimer is used in transplant protocols. In one embodiment, the tissue mass and secretory capacity of donor tissue expressing a C-mcTimer probe can be quantitatively distinguished from host tissue. For example, a determination of a C- mcTimertotal C peptide ratio may be measured in the circulation over the life-time of the transplant. In another embodiment, genetically engineered cells comprise C-mcTimer probes and/or derivatives thereof stably inserted into stem cells.

In one embodiment, the present invention contemplates fluorescent live cell monitoring of C-mcTimer stem cells for screening compounds that inhibit and/or stimulate secretory peptide processing, trafficking, and/or vesicular secretion. In one embodiment, the monitoring is performed by fluorescent microscopy. Although it is not necessary to understand the mechanism of an invention, it is believed that C-mcTimer probes are better than the simple secretory peptide promoter- GFP fusion proteins that have been previously employed because these simple fusion proteins only reflect any cell state that turns on a secretory peptide promoter. It is believed that such a disadvantage would provide signals of transient gene activation that is not related to overall regulation of the secretory peptide. In one embodiment, the present invention contemplates, a method comprising administering a secretagogue to a subject under conditions such that a genetically engineered therapeutic peptide is secreted into the circulation or synaptic junctions. In one embodiment, the administering comprises oral, intranasal, injection (i.e., for example, intraperitoneal, intravenous, intramuscular, etc.), or transdermal.

The C-mcTimer probes are advantageous because they allow study of differentiation protocols that follow events well beyond promoter activation including, but not limited to, physiologically relevant sustained vesicle biogenesis, vesicle trafficking, and exocytosis. Such events have been illustrated herein using a completely differentiated pancreatic β cell. C-mcTimer assays are easy to perform, and can be quantified en masse using engineered cell populations.

Specifically, the time course of normal cellular insulin synthesis, trafficking, and secretion was highly similar to the time course of the green to red color change of the timer molecules. Thus, pancreatic cells that normally perform these functions were found to exhibit an approximately equal mix of green and red mctimer appearing green to yellow in fluorescence color, whereas any disruption of these functions delaying or otherwise rendering the cell incompetent for insulin secretion exhibited greater amounts of red than green mctimer appearing yellow to red in fluorescence color, see Examples.

A. Regulated Peptide Secretion.

Regulated peptide secretion is believed to involve coupling of signal flow between stimulus and response. Although it is not necessary to understand the mechanism of an invention, it is believed that identification and characterization of the multiple subcellular sites, molecular interactions, and kinetics of the signal flow involved are modulate the coupling strength between cell stimulus and vesicle exocytosis. Further, in relation to peptide hormones (i.e., for example, insulin) the sites of regulation extend from the nucleus to the plasma membrane, with regulated resupply being as important as exocytosis of the secretory vesicle. Rhodes et al., "Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway" J Cell Biol 105:145-153 (1987); Wollheim et al., "The exocytotic process of insulin secretion and its regulation by Ca²⁺ and G-proteins" Diabetes Rev 4:277-297 (1996); Lang J., "Molecular mechanisms and regulation of insulin exocytosis as a paradigm of endocrine secretion' Eur J Biochem 259:3- 17 (1999); and Straley et al., "Rapid transport of internalized P-selectin to late endosomes and the TGN: Roles in regulating cell surface expression and recycling to secretory granules" J Cell Biol 15:107-116 (2000).

Regulated secretory peptide granule trafficking and exocytosis are typically measured by using: i) insulin antibodies labeled by radioactivity or enzymes (Albano, et al., "A sensitive, precise radioimmunoassay of serum insulin relying on charcoal separation of bound and free hormone moieties" A eta Endocrinol 70:487-509 (1972); ii) capacitance changes (Eliasson, et al., "PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic b cells. Science 271:813-815 (1996); or iii) amperometry (Finnegan, et al., "Vesicular quantal size measured by amperometry at chromaffin, mast, pheochromocytoma and pancreatic β cells" J Neurochem 66:1914-1923 (1996). One commonly used measure of insulin vesicle trafficking and secretion in the basic research lab or clinic is by immunoassays. Disadvantages of immunoassays include, but is not limited to, dependence upon the use of a relatively large sized biopsy or perfusate tissue and costly I¹²⁵ radioisotopes or enzymes conjugated to a secondary antibody. Further, these methods reveal little about the underlying molecular and cellular mechanisms at work within the cell, in a live-cell, real-time format with dynamic spatial (subcellular, one-cell, multi-cell, islet) and temporal (millisecond, second, min, to hour) monitoring.

1. Diabetes.

During the last 20 years the total number of people with diabetes worldwide rose from 30 million to 230 million, according to the International Diabetes Federation (2006). China and India now have the most diabetes sufferers in the world. Diabetes in general is a chronic disease that is devastating to the lives of at least 240 million people in the world today, with a diabetes- related death coming every ten seconds, or 3.8 million every year. This death rate is similar to those from Human immunodeficiency vires (HIV) and Acquired immune deficiency syndrome or acquired immunodeficiency syndrome (AIDS), according to the International Diabetes Federation. Diabetes is on track to double the number of affected subjects by 2030. There are two types of Diabetes, Type 1 and Type 2. Type 1 is IDDM (Insulin dependent diabetes mellitus) while the majority of Type2 is NIDDM (Non- Insulin dependent diabetes mellitus) however a subset of Type II patients are insulin dependent.

Most people who get Type 1 diabetes do so as a child or young adult however there is no none method of prevention. Inevitably, Type I diabetes involves the inability of the human body to produce enough insulin to lower elevated blood sugar levels after meals or due to the disease. In the majority of cases a type 1 diabetic loses beta cells of the pancreas due to cell death, likely through autoimmune mediated cell death. Type 2 Diabetes usually develops in the people older than 40, people who are very over weight, and people taking various medications whose side effect is weight- gain and onset of Type II diabetes. In general, Type 2 can be controlled with exercise, weight loss, a strict diet and proper nutrition and diet however as referred to previously, a subset of these patients are believed to suffer from defective insulin production.

In 1922, Banting and Best were the first to report the lowering of blood sugar in pancreatectomized dogs by pancreatic extracts. Soon after they were able to show that their extracts injected into a 14 year old boy, strikingly improved his diabetes. A year later, in 1923, Banting and MacLeod received the Nobel Prize in Physiology in Medicine. A protein named insulin was discovered to be the extract component responsible for lowering insulin levels. Thereafter, insulin replacement (injections of the insulin protein) was relatively rapidly optimized in the years following these discoveries. Further, islet cell transplants were also used to replace insulin function however as described elsewhere, when this procedure works it provides merely temporary relief. These therapies are no doubt one of the top historical medical breakthroughs but they are in fact not a cure, but rather only delaying the onset of long term complications of diabetes. Diabetes causes more cases of blindness and visual impairments in adults than any other illness in the developed world. One million amputations each year are caused by diabetes. A diabetes sufferer is up to 40 times more likely to need a lower-limb amputation when compared to a person who does not have diabetes. Diabetes raises the sufferer's risk of developing a cardiovascular disease by two to four times. Cardiovascular disease, the number one cause of death in the industrial world, is estimated to soon become the number one cause of death globally. The costs of diabetes in the United States comprises these devastating complications for 20 million people along with a direct annual medical cost of $100 billion. It is estimated that at this time, diabetes accounts for 5% to 10% of most nations' health budgets. Unfortunately, the statistics from the Center for Disease Control indicate that the number of people with diabetes is expanding at an alarming rate and is expected to double to 40 million by 2025, without dramatic advances in the understanding and treatment of the disease. Therefore if more money was now spent on early detection of diabetes and diabetes prevention the economic savings would be massive into the future. For this to reasonably be expected we need innovative tools to better learn how the body properly regulates insulin secretion and fails to do so in diabetic subjects, and use this knowledge for the discovery of far more effective anti-diabetic drugs. As magnificent a medical milestone as insulin replacement therapy is, now is the worst time to rest. Pancreatic islet tissue graft reliability to provide durable insulin independence in pancreatic transplant patients has minimized the success rates for curing type 1 diabetes. Bertuzzi et al., "Prediction of clinical outcome in islet allotransplantation" Diabetes Care 30:410-7 (2007); and Emamaullee et al., "Factors influencing the loss of beta-cell mass in islet transplantation" Cell Transplant. 16" 1-8 (2007). These reports show that while a minority of clinical engraftments provide durable insulin independence, the majority fail. These observations highlighted a problem in the art regarding the damaging role that secretory incompetent beta islet cells and secretory incompetent graft islets play in the failure of pancreatic tissue transplants. Ren et al., "Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation" J Transl Med. 5" 1-8 (2007).

Moreover, treating diabetes and approaches to finding preventative indicators and cures are now known to require far more detailed knowledge of how insulin is dynamically secreted commensurate with ever-changing blood glucose levels after meals, during fasting, and personal medical differences including disease states. The development of more effective drugs and the advance of artificial pancreas projects will require dramatic advances in this knowledge. The data presented herein demonstrates that when imaging pancreatic tissue at a single islet or single cell resolution, cadaveric islets routinely have a widely varying and often substantial fractions of cells impaired in their responsiveness to glucose. Although it is not necessary to understand the mechanism of an invention, it is believed that these impaired cells stress the islets and in turn the entire graft and increase the fraction of impaired cells, leading to graft failure. Disadvantages of presently used assays (i.e., for example, ELISA) for islet cores include an inability to determine what fraction of islet cells, or when an unacceptably low fraction of cells, are secretory competent. An innovative methodology is needed to 50 10 15 20 quantitatively assess and optimize the fraction of clinical islet beta cell cores secreting insulin in a given donor islet preparation. Glucose-stimulated insulin secretion is regulated over a wide range of time scales, including but not limited to" i) day-to-hour (i.e., for example, nuclear transcription: Leibiger et al., "Short-term regulation of insulin gene transcription by glucose" Proc Natl Acad Sci USA 95:9307-9312(1998); ii) hour-to-minute (i.e., for example, cytoplasmic translation (Goodge et al., "Translational regulation of proinsulin biosynthesis and proinsulin conversion in the pancreatic i3 cell" Semin Cell Dev Biol 11:235-243(2000) or vesicular trafficking and/or recycling (Easom RA., "[3 -granule transport and exocytosis" Cell Dev Biol 11:253-266 (2000); or iii) minute-to-millisecond (i.e., for example, exocytosis at the plasma membrane (Steyer et al., "Transport, docking and exocytosis of single secretory granules in live chromaffin cells" Nature 388:474-478(1997). Although it is not necessary to understand the mechanism of an invention, it is believed that live-cell monitoring of the spatial and temporal features of the underlying molecules and mechanisms would be facilitated by a method that avoids modification of the mature bioactive secretory peptide for not only glucose-regulated insulin secretion but also regulated peptide secretion in general. In one embodiment, the present invention contemplates a biosensor that is incorporated into a prepropeptide such that a functional mature secretory peptide is released upon cleavage and exocytosis. A transgenic mouse carrying pancreatic β cell Timer probe demonstrated both insulin production and the time course of the release kinetics. Bertera et al., "Body Window-Enabled In Vivo Multicolor Imaging Of Transplanted Mouse Islets Expressing An Insulin-Timer Fusion Protein" BioTechniques 35:718-722 (2003). 2. Bone Marrow Transplants.

The Ins-C-Timer mouse can also be used as the bone marrow donor in the preparation of hematologically chimeric animals. Zorina et al., "Distinct characteristics and features of allogeneic chimerism in the NOD mouse model of autoimmune diabetes" Cell Transplantation 11:113-123 (2002), herein incorporated by reference. The bone marrow transplant is sufficient to abolish the autoimmune process. Without autoimmunity, the regenerative properties of the endocrine pancreas can replace, over time, sufficient sufficient β cells to guarantee euglycemia for an indefinite period of time, even in mice that are already diabetic. Zorina et al., "Recovery of the endogenous beta cell function in the NOD model of autoimmune diabetes" Stem Cells 21 :377-388 (2003, herein incorporated by reference). The question relative to the source of the regenerative process (i.e., bone marrow vs. pancreas) can be answered using these transgenic mice.

3. Drug Development.

The materials and methods of this invention are particularly useful for analyzing the effects of compounds, including test compounds, on the insulin pathway, including any compound that might alter any aspect of insulin production, processing, trafficking, secretion, break-down, and the like. Compounds may interfere with either or both of first and second phases of insulin secretion. Properties of probes of the present inventions, in particular, Ins-C-mcTimer and Ins-C-emGFP, demonstrated the capability provide a sensitive biosensor for monitoring insulin trafficking in beta cells from normal and diabetic donors.

In particular, Ins-C-mcTimer and Ins-C-emGFP- A206K are contemplated to provide a dynamic diabetic drug discovery tool for screening potential therapeutic compounds for preventing, and reducing or delaying short and long-term effects of diabetes. Such drugs would alter the secretory capacity of the beta cell or any insulin expressing cell, for example, enhancing glucose dependent insulin secretion or inhibiting insulin secretion. In one embodiment provided herein, the test agent alters insulin secretion. In a further embodiment, insulin secretion is increased by an agent. Examples of such agents are Sulfonylureas, in particular Gatifloxacin™, currently used to treat Type II diabetic patients, incretins, and a test compound LY389382 which stimulates insulin secretion over a concentration range of at least two log units in a glucose- dependent manner. Sulfonylureas stimulate insulin secretion independent of the blood glucose concentration. However, this can lead to hypoglycaemia in type 2 diabetic patients. Incretins do not by themselves stimulate insulin secretion, rather they work to augment glucose-stimulated insulin secretion. Compounds include secretagogues, such as an insulin secretagogue efaroxan. Basically any Potassium-channel openers are potential drugs for activating (open) ATP-sensitive K+-channels for inducing insulin release, including but not limited to such as molecules that bind to and act through sulfonylurea receptors (SURs), for example at least 6 chemical families grouped according to their molecular structures: (1) benzopyrans, (2) cyanoguanidines, (3) thioformamides, (4) pyrimidine derivatives, (5) pyridine derivatives, (6) benzothiadiazines, (7) dihydropyridines, (8) nicotinamide derivatives, and (9) aliphatic amines.

Examples of compounds that block insulin secretion, are ATP-sensitive potassium channel agonist compounds, such as diazoxide, Clotrimazole, and the like, that reduce glucose- induced insulin release. It is contemplated that both cell and tissue based assays will be used in drug discovery screenings of test compounds using probes of the present inventions. In one embodiment, a screening method comprises any insulin expressing cell. In another embodiment, the screening method comprises any organism expressing an insulin molecule. Such organisms include animal models wherein real-time fluorescence assays of plasma samples of insulin secretory competency are monitored. In particular, such monitoring is contemplated to comprise a fluorimeter (a device used to measure a parameter of fluorescence, such as intensity, etc.), antibody-based ELISA, etc. In a preferred embodiment, the monitoring of insulin secretion in plasma comprises a fluorimeter. It is contemplated that generation of disease models comprising proinsulin mutations will be combined with methods comprising biosensors of the present inventions. In one embodiment, animals comprising knock-in constructs comprising insulin mutations are contemplated for monitoring using biosensors of the present inventions. Assays for monitoring the rate of insulin degradation in the beta cell are contemplated. Further, because the fluorescent probe is released alongside of the insulin molecule, levels of fluorescence measured in circulation, for example, measuring changes in fluorescent levels in plasma is contemplated to provide a monitoring tool for determining the effects of test compounds on insulin secretion in whole organisms, such as animal models and human tissues. Further, probes of the present inventions are contemplated to provide tools for determining the delivery time and activity of a test compound in a cell, tissue or whole organism.

Furthermore, biosensors of the present inventions are contemplated as vital tools for pre- clinical islet cell transplantation procedures and related stem cell research. Currently stem cell research is aiming to generate a high fraction of bona fide beta cells from stem cells for transplantation. This goal requires a highly differentiated set of cell biosynthetic, trafficking, and regulated secretory processes examples of which were measured by Ins-C-mcTimer. These assays are contemplated to be performed optically in live cells en masse during the differentiation protocols by using live cell imaging. Critically rates of these processes can be determine relative to bona fide beta cell rates of the same process by a simple color ratio process intrinsic to the mcTimer green/red fluorescence which is proportional to time after synthesis of the insulin reporter. These assays are similar to those described herein for using a monomelic fusion protein probe for assaying cadaveric donor islets for viability and insulin secretory competence, see, Examples. 4. Clinical Biopsy/Pathology Applications.

The probes of the present inventions are contemplated for type 1 and type 2 diabetes disease development staging by following expression of the fluorescent molecule in live cadaveric donor islets from diabetic donors and animal models. These types of methods are further contemplated to extend to neonatal diabetes mutation characterization of cellular trafficking defect, neonatal diabetes mutation characterization of a cellular proteolytic cleavage defect required to make mature insulin hormone, and a neonatal diabetes mutation characterization of cellular secretory defect. In other words, any type of mutation, insertion, deletion and the like, within a preproinsulin molecule, in particular those identified in diabetic animals and humans, are contemplated for study over time using monomelic probes of the present inventions. B. Human Clinical Applications

Even though fluorescent proteins are not presently allowed for use in humans, these probes can be used to support human surgical techniques and disease treatments intended for human administration. Islet transplantation, intensely managed insulin dosing, and autoimmune destruction of islet β cells each involves functionality of the insulin secretory granules. How granule proteins work as molecular machines per se, and how they work at the higher levels of insulin secretory granules and islet β cells are new and unexplored areas in identifying novel therapies for diabetes. Such novel concepts and innovative approaches include, but are not limited to, how insulin granules maintain the equilibrium of actively secreting and resting β cells for transplantation therapy, the relationship between 1st and 2nd phase insulin secretion in the body's exquisite, maintaining an enduring glycemic control by proper dynamic dosing for insulin replacement therapy, and establishing a role of ICAs of insulin granules for identifying individuals at risk for TlD. Preclinical pancreatic transplant tissue evaluation including islet transplantation techniques intended to cure type 1 diabetes will require dramatic improvements in the quality of the donor pancreatic islet tissue. Optimized donor pancreatic islets should have beta cells with the highest possible insulin secretory capacity, while at the same time not having beta cells that are secretory-incompetent. Secretory-incompetent cells stress the graft and lead to premature insulin dependence in the engrafted diabetic subjects. What is needed in the art is a simple way to measure the fraction of beta cells that have a healthy secretory capacity in the human donor islet preparations immediately before transplantation.

In one embodiment, the present invention contemplates an C-mcTimer probe providing an optical assay to quantify fractional beta cells from human donor pancreatic islet cells having a healthy secretory capacity. In one embodiment, the islet cells are identified for transplantation. Although it is not necessary to understand the mechanism of an invention, it is believed that this method provides a simple color assay of the quality of donor islets, which can then be optimized to provide permanent insulin independence in the graft recipients. In one embodiment, the method further comprises standardizing the highest secretory quality β cells within the pancreatic islets, thereby providing a cure for type 1 diabetes. In one embodiment, Ins-C-mcTimer may be used to screen cadaveric donor islets for type 1 diabetes islet prior to transplantation. In one embodiment, the results from such screening allows the optimal selection of insulin-producing tissue.

C. Co-Imaging. In one embodiment, the present invention contemplates a method comprising co- imaging secretory vesicles labeled with a plurality of different fluorescently labeled C- mcTimer probes within a spatio-temporal context of at least one cellular landmark. In one embodiment, the landmark comprises a cytoskeleton. In one embodiment, the landmark comprises another vesicle-associated molecule. Although it is not necessary to understand the mechanism of an invention, it is believed that different fluorescent labels would allow the monitoring of vesicle membrane, luminal, or extra- vesicle proteins separately and simultaneously. It is further believed that by changing the microscopic magnification, the spatial interaction context might be intracellular or intercellular, involving different secretory vesicles that underlie intercellular communication, particularly relevant to the regulation of the endocrine pancreas.

In this study, the inventor showed that Ins-C-FPs were robust (easily visualized) reporters of insulin cell biology in live human islets and further were applied to the study of insulin secretory defects contributing to T2D. Further, Ins-C-emGFP colocalized with anti-insulin antibody labeling within islet cells. When the images were subjected to a binary mask analysis, 0.97 of cells with green Ins-CemGFP puncta included red anti- insulin puncta. In no case were green puncta detected in cells without any red puncta.

These findings demonstrated that an Ins- C-emGFP reporter was used routinely to monitor insulin secretory granules in live human islet β cells (27). The Ins-C-FP design for live human islet work was also exploited to investigate aspects of human insulin pathophysiology in T2D (5-14). Unique to this work was the finding of a dramatic change in the trafficking pattern of insulin in the islets from two T2D donors analyzed. Specifically, most of the Ins-C-emGFP was retained in the ER, as shown by co- localization with both anti-calnexin and anti-Grp78 antibodies. These trafficking defects were unlikely to be attributable to agonal causes (such as abnormal trafficking that occurs as a cell dies) or to the islet isolation technique. In fact, no trafficking defects were observed in the 22 non-diabetic and the other 6 diabetic donors' islets prepared and analyzed in parallel in this study.

In these islet preparations robust expression and trafficking of Ins-C-emGFP was shown to be targeted to insulin granules. Thus, the presence of different insulin traffic phenotypes observed in different T2D donors demonstrated heterogeneity of the disease within the β cell. Future investigation of islets from a larger set of T2D donors with our Ins-C-FP live-cell reporters are contemplated.

Another unique finding to this work, was the colocalization at human insulin granules from nondiabetic human islets of the Ins-C-dsRedl Bright reporter with the green fluorescent BODIPY-FL glibenclamide, a marker for KATP channels. There was relatively little fluorescent glibenclamide detectable at other membranes from the ER and Golgi to the plasmalemma.

Thus it was contemplated that plasmalemma sulfonylurea receptors were relatively scarce compared to those at the insulin granule membrane in normal human islets, similar to those in rodent β cells (23-25). These observations using fluorescent glibenclamide on human β cells confirm and extend previous observations showing high affinity sites for radioactive glibenclamide on rodent insulin granules (35-38). Because SURl and Kir6.2 are integral membrane proteins that depend on assembly of one another into KATP channels for ER exit (39-43), these findings also indicated that KATP channels are integral membrane proteins of the human insulin granule. The fluorescent glibenclamide also specifically labeled insulin granules in islets from 7 of the 8 T2D donors. Rather than labeling insulin granules, in the 1 donor diabetic islets were glibenclamide did not specifically label insulin granules, the glibenclamide fluorescence was diffuse and reticulated, consistent with the KATP channels accumulating in the ER membrane. This finding was consistent with defective ER export of insulin cargo and also KATP channels that normally localized to insulin granules. The traffic jams of both insulin cargo and KATP channels at the ER result in abnormal insulin granule phenotypes of the β cell in some forms of T2D.

With the use of Ins-C-GFPs coupled with BODIPY-FL glibenclamide different insulin granule phenotypes in different T2D donor islets were identified herein. Fully differentiated islet β cells used uniquely complex mechanisms for exquisitely tight control of insulin trafficking and secretion,

Thus additional disease phenotypes are contemplated to emerge from application of Ins-C-FPs and related live-cell fluorescence methodologies for studying human islets. In summary, fluorescent reporter technology designed into the Ins- C-FPs of the present inventions was demonstrated to provide a powerful live-cell monitoring tool for studying the physiology of human insulin secretion in health and disease. Moreover, because the biosensors of the present inventions provide live-cell monitoring of fully differentiated islet β cells including pro-insulin processing, trafficking, and glucose regulated insulin secretion (44), the biosensors are further contemplated to provide highly valuable compositions in methods to differentiate stem cells into pancreatic β cells for treatment of diabetes (45).

IV. Kits. In another embodiment, the present invention contemplates kits for the practice of the methods of this invention. The kits preferably include one or more containers containing a C-mcTimer fluorescent assay method of this invention. The kit can optionally include an uncompromised cell culture to be utilized as a control (i.e., for example, a pancreatic islet β cell culture). The kit can optionally include a first fusion prepropeptide comprising a C-mcTimer probe and a second fusion prepropeptide comprising a C-mcTimer probe. The kit can optionally include antibodies capable of binding to the cleaved mature peptide of the first prepropeptide. The kit can optionally include antibodies capable of binding to the cleaved mature peptide of the second prepropeptide. The kit can optionally include fluorescent fusion proteins capable of binding to and identifying intracellular components (i.e., for example, golgi bodies, endoplasmic reticulum, secretory vesicles, cytoplasmic protein, nuclear bodies, plasma lemma etc). The kit can optionally include an incubation solution. The reagents may be provided suspended in the incubation solution or may be provided as a separate component which can be later combined with the incubation solution. The kit can optionally include a secretagogue to induce release of a first or second prepropeptide (i.e., for example, glucose). The kit can optionally include a secretory vesicle release inhibitor (i.e., for example, diazoxide).

The kits may also optionally include appropriate systems (e.g. opaque containers) or stabilizers (e.g. antioxidants) to prevent degradation of the reagents by light or other adverse conditions.

The kits may optionally include instructional materials containing directions (i.e., protocols) providing for the use of the C-mcTimer fluorescent probe in assays to monitor the synthesis, trafficking, and/or release of secretory proteins, hi particular the secretory proteins can include any one or more of the proteins described herein. While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

VI. Detection of Protein.

In other embodiments, secretory vesicle exocytosis and release of prepropeptides in biological tissues may be detected by measuring a mature and/or functional protein or polypeptide. Proteins may be detected by any suitable method, hi some embodiments, proteins are detected by immunohistochemistry. In other embodiments, proteins are detected by their binding to an antibody raised against the protein. The generation of antibodies is described below.

Antibody binding may be detected by many different techniques including, but not limited to, (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays, etc.), complement fixation assays, immunofluorescence assays, protein A assays, and Immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. In some embodiments, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. For example, in some embodiments, software that generates a prognosis based on the presence or absence of a series of proteins corresponding to cancer markers is utilized. In other embodiments, the immunoassay described in U.S. Pat. Nos. 5,599,677 and 5,672,480; each of which is herein incorporated by reference.

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay (e.g., the presence, absence, or amount of a given fluorescent probe or probes) into data of predictive value for a clinician. The clinician can access the predictive data using any suitable means. Thus, in some preferred embodiments, the present invention provides the further benefit that the clinician, who is not likely to be trained in endocrinology or molecular biology, need not understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician is then able to immediately utilize the information in order to optimize the care of the subject.

The present invention contemplates any method capable of receiving, processing, and transmitting the information to and from laboratories conducting the assays, wherein the information is provided to medical personal and/or subjects. For example, in some embodiments of the present invention, a sample (e.g., a biopsy) is obtained from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility, genomic profiling business, etc.), located in any part of the world (e.g., in a country different than the country where the subject resides or where the information is ultimately used) to generate raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to the profiling center, or subjects may collect the sample themselves (e.g., a urine sample) and directly send it to a profiling center. Where the sample comprises previously determined biological information, the information may be directly sent to the profiling service by the subject (e.g., an information card containing the information may be scanned by a computer and the data transmitted to a computer of the profiling center using an electronic communication systems). Once received by the profiling service, the sample is processed and a profile is produced (i.e., expression data), specific for the diagnostic or prognostic information desired for the subject.

The profile data is then prepared in a format suitable for interpretation by a treating clinician. For example, rather than providing raw fluorescence data, the prepared format may represent a diagnosis or risk assessment (e.g., likelihood of compromised tissue), along with recommendations for particular options. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor. In some embodiments, the information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to convert the raw data to information useful for a clinician or patient. The central processing facility provides the advantage of privacy (all data is stored in a central facility with uniform security protocols), speed, and uniformity of data analysis. The central processing facility can then control the fate of the data following treatment of the subject. For example, using an electronic communication system, the central facility can provide data to the clinician, the subject, or researchers.

In some embodiments, the subject is able to directly access the data using the electronic communication system. The subject may chose further intervention or counseling based on the results. In some embodiments, the data is used for research use. For example, the data may be used to further optimize the inclusion or elimination of markers as useful indicators of a particular condition or stage of disease. Experimental

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Example I.

Construction OfAn Ins-C-Green Fluorescent Protein (GFP) Construct This example provides a proof-of-concept experiment showing that a GFP reporter was successfully introduced into a prepropeptide thereby allowing release of a functional mature insulin protein.

An Ins-C-GFP reporter construct was created by placing a green fluorescent protein probe in- frame within the middle of the C peptide connecting the A (herein referred to as alpha) and B (herein referred to as beta) chains of murine proinsulin II. See, Figure 3 A, with the exception that a mouse insulin was used in place of human insulin. The Ins-C-GFP reporter construct was inserted into the El region of an El, E3 deleted adenoviral vector and expressed in wild-type mouse islets by infection. A 2.4 Kbp BamHI genomic fragment of mouse Insulin II (33) was inserted within a Xhol/Hpal fragment into the Adlox shuttle vector. Wentworth et al., "Characterization of the two nonallelic genes encoding mouse preproinsulin" J MolEvol 23:305-312 (1986); and Hardy et al., "Construction of adenovirus vectors through cre-lox recombination" J Virol 71 :1842-1849 (1997). A PCR product of Emerald GFP (26) was cut with Smal and BstEII, then placed into the corresponding sites within the C-peptide coding sequences, such that three alanine codons flank either end of GFP, all in frame with C peptide codons. Tsien RY., "The green fluorescent protein" Annu Rev Biochem 67:509-544 (1998). The entire Ins-C-GFP construct inserted into Adlox was confirmed by DNA sequencing and designated simply 'Adlox.Ins-C-GFP' or Ins-C-emGFP.

Ins-C-GFP expression in mouse islets gave rise to beta cells containing intense punctate fluorescence, as compared to controls. See, Figures 3B-3D. Immuno-electron microscopy was used to further characterize the site of Ins-C-GFP localization. Large gold particles labeling anti-GFP antibody and small gold particles labeling anti-insulin antibody were used to probe 70-100-nm thin sections from islets expressing the Ins-C- GFP reporter. The large and small gold particles were colocalized at dense cores of beta cells, whereas over 100-fold less gold particles localized to mitochondrial structures. Whenever large gold particles were present (GFP), small particles were also detected (insulin), consistent with beta-cell-specific expression. See, Figure 3E. Similarly, a Ins-C-emGFP expression molecule was created and tested wherein live islet insulin granule fluorescent labeling with Ad.Ins-C-emGFP, was used as described (Watkins et al., Traffic 2002;3:461-471) at an MOI of approximately 200. The inventor similarly constructed and used Ad.Ins-C-dsRed IBRIGHT by substituting into the DsRedl (Clontech, CA) of Adlox.Ins-C-dsRedl, the VallO5 and Ala 105 mutations, as originally described (Terskikh et al.,) by site-directed mutagenesis, creating Adlox. Ins- CdsRedlBRIGHT. This was used to make Ad.Ins-CdsRedlBRIGHT. The fluorescence expression levels were representative of those observed within 48 h after infection. Typically, approximately 10% of the cells, mostly located at the perimeter of the islet expressed the Ins-C-FP (fluorescent protein) to intense levels 24 h after infection, and approximately 20% 48 h after infection. The islet cells studied were alive by the LIVE/DEAD fluorescent assay (Molecular Probes, Eugene, OR).

Example II.

Construction OfA Transgenic Mouse Comprising A Tetrameric Ins-C-Timer. This example uses a tetrameric Ins-C-Timer fluorescent probe visualized with a body window device (Fotofab, Chicago, IL, USA) to image insulin-producing cells over time.

The 2.4-kb BamHI genomic fragment of mouse insulin II was inserted within an Xhol/Hpal fragment into an Adlox shuttle vector to form an Adlox. Insulin II vector. Wentworth et al., "Characterization of the two nonallelic genes encoding mouse preproinsulin" J. MoI. Evol. 23:305-312 (1986); and Hardy et al., "Construction of adenovirus vectors through Cre-lox recombination recombination" J. Virol. 71:1842- 1849 (1997).

The tetrameric Timer probe was generated from a dsRedl sequence by introducing the relevant mutations. Terskikh et al., "Fluorescent Timer": protein that changes color with time. Science 290:1585-1588 (2000)(herein incorporated by reference in relevant part). Specifically, the fluorescent Timer probe gene coding sequence was mutated using polymerase chain reaction (PCR) by encoding three flanking alanines at the 5' and 3' ends and adding sufficient C-peptide sequence to span between the Smal and BstEII restriction sites to produce a C-Timer construct. The C-Timer construct was inserted into the Adlox.Insulin II vector at the Smal and BstEII restriction sites having the effect of replacing C-peptide codon 20 (i.e., Ala) with the tri-Ala flanked Timer probe sequence to create the Adlox.Ins-C-Timer construct. The accuracy of the Adlox.Ins-C-Timer construct was then confirmed by the DNA sequencing of a Xhol/Hpal segment. The Ins-C-Timer transgenic founder mice were generated by injecting highly purified Adlox.Ins-C-Timer DNA into fertilized embryos at the University of Cincinnati Transgenic Core Facility (Cincinnati, OH, USA). The transgenic founders and offspring were identified by the PCR amplification of genomic DNA from each animal. Primer sequences were 5'-CCAGTTCCAGTACGGCTCCA-S' (forward primer) and 5'- TGGATCTCGCCCTTCAGCA-3 ' (reverse primer).

Six Ins-C-Timer-positive founder mice were identified out of 50 pups born from injected embryos. Three of the founders did not transmit through the germline. Of the remaining three founders, the one with the best expression (no. 53,128) was bred to produce the Ins-C-Timer transgenic line used in the Examples below.

Example III. Visualization Of Newly Synthesized Insulin Using A Tetrameric Ins-C-Timer Probe.

This example presents preliminary data showing that the tetrameric Ins-C-Timer probe is capable of tracking insulin synthesis, trafficking, and secretion in the transgenic mice made in accordance with Example I.

Pancreatic islets were isolated from Ins-C-Timer transgenic mice, transplanted to recipient mice. Alexander et al., "Indoleamine 2,3-dioxygenase expression in transplanted NOD islets prolongs graft survival after adoptive transfer of diabetogenic splenocytes" Diabetes 51 :356-365 (2002); and Bertera et al., "Gene transfer of manganese superoxide dismutase prolongs transplanted islet function in an autoimmune mouse model of diabetes" Diabetes 52:387-393 (2003). Confocal images were collected using 488 nm excitation with a META 510 Confocal Microscope (Carl Zeiss, Thornwood, NY, USA) using spectral detection. Emission spectra between 490 and 570 nm in 10-nm bands were collected for each image. Using the spectral unmixing capabilities of the instrument, spectra for the green component and red component of Timer were generated and used to unmix the aggregate spectral image. The two separated images were then recombined using green and red channels in a standard two-color image. Geng et al., "The insulin secretory granule is the major site of KATP channels of the endocrine pancreas" Diabetes 52:767-776 (2003). The transplanted pancreatic islets were visualized using a body window sutured into the abdominal wall of the mouse and shown to be responsive to the pancreatic insulin inhibitor, interleukin lβ (IL-I β). Without IL- lβ exposure, the insulin was secreted as it was synthesized, and thus fluorescence did not build up in the β cells, thereby making the islets look green and yellow. See, Figure IA. When insulin secretion was inhibited by IL- lβ, the insulin in the β cells became more abundant and after 24 hours the fluorescence converts from green to red. See, Figure IB. See, Figure IB.

(Figure 1, see, Bertera et al., Biotechniques 35:718-722 (2003); herein incorporated by reference in its entirety).

Example IV. Abnormal Glucose Tolerance Response Of Tetrameric Ins-C-Timer Transgenic Mice.

This example provides data showing that Ins-C-Timer transgenic mice demonstrated an abnormal glucose tolerance response and ultimately developed diabetes.

To compare the insulin-secretory capacity of Ins-C-Timer transgenic pancreatic islets versus non-transgenic controls, a dynamic glucose challenge involving isolated pancreatic islet perfusion was performed with Krebs buffer under conditions of low and high glucose. Pipeleers et al., "Transplantation of purified islet cells in diabetic rats. I. Standardization of islet cell grafts" Diabetes 40:908-919 (1991). The overall basal and stimulated insulin releases were similar between the non-transgenic control pancreatic islets and the Ins-C-Timer transgenic pancreatic islets. A slight but insignificant lower insulin content was measured in the Ins-C-Timer pancreatic islets. See, Table 1. Table 1. In Vitro Functional Testing of Isolated Islets

Data are means (x - ± SEM). Basal and stimulated insulin releases are measured at 2.8 and 20 mM glucose concentrations. The stimulation index expresses the ratio between stimulated and basal insulin releases.

However, the perifusion curve indicates that after stimulation, the Ins-C-Timer transgenic mouse pancreatic islets showed an abnormal fluctuating insulin release. See, Figure 2A.

An in vivo fasting intraperitoneal glucose tolerance test (IPGTT) was then performed. Tatarkiewicz et al., "Reversal of hyperglycemia in mice after subcutaneous transplantation of macroencapsulated islets" Transplantation 67:665-671 (1999). The results show that the Ins-C-Timer transgenic mice showed a higher initial glycemic response and a slight delay in recovery from the glucose challenge compared to non- transgenic mice. See, Figure 2B. These results are also considered to represent an abnormal response to a glucose tolerance test. One consideration for these abnormal glucose tolerance results may be related to a toxic effect of the fluorescence probes on the pancreatic islet cells. Note that fluorescent probes are not approved for use in humans for these very reasons. For example, when comparing in situ insulin synthesis, trafficking, and secretion data generated with either green fluorescent protein fusion proteins (GFP) or Ins-C-Timer probe fusion proteins, the latter seems to be less toxic for the cells of a developing embryo. However, this does not exclude the possibility that a fluorochrome-labeled proinsulin may be processed more slowly than an unlabeled proinsulin or that the presence of the Ins-C-Timer probe protein in the insulin secretory granules has an effect on the secretion of insulin after a hyperglycemic stimulus. Example V.

Construction OfAn Ins-C-mCherry Probe.

This example demonstrates one embodiment for constructing a monomelic Cherry (mCherry) from a dsRedl fluorescent protein for the expression of a very bright red fluorescence in cells.

An Aldox vector comprising Ins-C-mCherry was constructed using the techniques described in accordance with Example II. See, Figure 6. An Ins2 952 bp promoter sequence runs from DNA position 1557 upstream to position 606. The Adlox vector sequence comprises the basepair positions upstream of the Ins2 promoter sequence having an Xhol site between positions 554-559.

The proinsulin sequence begins with its signal amino acid sequence MAL (positions 1693-1701) and ends with the amino acid sequence TQA (positions 1756- 1764). The B insulin chain then begins with the amino acid sequence FVK (positions 1757-1773) and ends with the amino acid sequence PMS (positions 1846-1854). The first portion of the C insulin chain then begins with the amino acid sequence EVE (position 1861), skips across intron 2 (positions 1879-2366) to the first AAA fusion point (positions 2401-2408) where the mcTimer sequence begins and runs to the second AAA fusion point (position 3020-3025) near the BstEII site (positions 3126-3133). The B chain and the C chain are cleaved at the RR proteolytic site (positions 1856-1860). Downstream of the second AAA fusion point, the C peptide continues and ends at the amino acid sequence KR cleavage point (positions 3165-3170) between the C peptide and the A peptide. The A peptide begins at the amino acid sequence GIV (positions 3171- 3179) and ends at position 3233. Although it is not necessary to understand the mechanism of an invention, it is believed that this Ins-C-mcTimer vector incorporates a novel design feature by inserting polyAAA sequences that provide a flexible linker between fused proteins without disturbing the encoded protein's native structure (and therefore function). It is further believed that alanine (A) is the most substituted amino acid residue in proteins of known structure without changing the tertiary and/or quaternary structures. The C-mcTimer protein is point-mutated as the V 105 A mutation (amino acid position 110; nucleic acid position 2737), and is designated as the "Bright" mutation. This mutation is believed to double the quantum efficiency (i.e., for example, fluorescence intensity) of the known mcTimer protein. A further mCherry point-mutation comprises I197T (amino acid position 202; nucleic acid position 3013), which is equivalent to the S197T mutation in dsRedl.

Example VI.

Construction OfAn Ins-C-mcTimer Probe.

The Ins-C-mCherry probe made in accordance to Example V was mutated to make Ins-C-mcTimer (i.e., m = monomelic; c = Cherry). Importantly, mcherry codes for an I at position 197, whereas dsRedl (from which the original timer was derived) codes for an S at position 197. Therefore, the inventor contemplated that the removal of the I from mcherry might have precluded the generation of mctimer with the contemplated substitution of the I by T that instead or providing a timing molecule would instead result in loss of fluorescence or lose the desired rate of timing which matched insulin processing, et cetera. However, when the inventor actually tested the timer substitution at position 197 of mcherry they were surprised that mCherry was fluorescent and were further surprised that mCherry gained the timing function of the original timer.

The data below demonstrates that the Ins-C-mcTimer probe shows a very bright green, yellow, and red fluorescence in mouse islets using live-cell culture in Mat-Tek optical chambers. Secondly, the data shows that the green, yellow, and red fluorescence was the result of the dependence of fluorescent color on the age of C-mcTimer.

Example VII.

Improved Sensitivity OfAn Ins-C-mcTimer Probe. This example demonstrates the improved sensitivity and monitoring capability of

Ins-C-mcTimer probe as compared to the tetrameric Ins-C-Timer probe.

After fourteen (14) hours of Ins-C-mcTimer probe expression all pancreatic islets showed predominantly green fluorescence when imaged with filters simultaneously for green and red fluorescence. Figure 4A. During the next fourteen hours, a first set of pancreatic islets were incubated in the presence of diazoxid and a second set of pancreatic islets were incubated in the absence of diazoxid. Although it is not necessary to understand the mechanism of an invention, it is believed that diazoxid is a potassium channel opener and dramatically blocks insulin secretion by hyperpolarizing pancreatic beta cells thereby preventing calcium influx and insulin secretion. The pancreatic islet set incubated in the absence of diazoxid exhibited roughly equal green and red fluorescence, thereby resulting in largely yellow fluorescence (i.e., 28 hours after Ins-C- mTimer expression. See Figure 4B. The pancreatic islet set cultured in the presence of diazoxide between 14-28 hours after expression exhibited a dramatic combination of yellow and red fluorescent pancreatic beta cells. See, Figure 4C. The results are summarized as fractional green/(green+red) fluorescence ratios observed for both pancreatic islet sets expressing C-mcTimer before and/or after incubation with diazoxide. See, Figure 4 D. At 14 hours post-expression, the two sets of islets were largely green, and the green/(green+red) fluorescence color ratio means were statistically indistinguishable (0.66 v. 0.64; students t test p>0.05). At 28 hours post-expression, however, the pancreatic islet set in the presence of diazoxide were largely yellow-red, and green/(green+red) fluorescence ratio means were dramatically lower as compared to the largely yellow-green pancreatic islets incubated without diazoxide (0.49 v. 0.29; students t test pθ.01). The data demonstrate that the green-to-red color change accurately monitors reductions in insulin secretory capacity at a very high sensitivity.

Example VUI.

Proteolytic Processing Of Ins-C-mcTimer Versus C-mcTimer. This example addresses the question as to whether Ins-C-mcTimer probes are processed as efficiently as the C-mcTimer. Equivalent processing efficiencies were observed between Ins-C-emGFP and C-emGFP. Watkins et al., "Imaging secretory vesicles by fluorescent protein insertion in propetide not mature secreted peptide" Traffic 3:461-471 (2002).

Protein processing techniques were described in Watkins et al. (2002). The data presented in Example VII demonstration a lack of red β cells after 28 hours incubation in pancreatic islets comprising an Ins-C-mcTimer probe, when compared to similar data collected after 24 hours using the tetrameric Ins-C-Timer. Thus, when insulin secretion is blocked, the beta cells shift from green and yellow to yellow and red. In other words, the blockade of secretion causes insulin to be retained by the cell and thereby increases the fraction of old (red) versus yound (green) insulin. In the control islet cells the secreted insulin is in effect infinitely diluted in the extracellular medium and only the young vesicles (green) only intermediate aged vesicles (appearing yellow due to approximately equal mix of green and red insulins) remain in the cell and will be secreted before aging enough to appear red. See, Figure 4.

This example shows that aggregation and lysosomal accumulation is dramatically decreased with a monomelic fluorescent molecule. Consequently, was determined that aggregation and trafficking to lysosomes of the C-mcTimer probe was similar to C- emGFP probe (i.e., low fluorescence intensity); and dissimilar to Ins-C-Timer probe (i.e., high fluorescence intensity), see below. The inventor contemplate a morphometric analysis of the fluorescent puncta in conjunction with co-localization studies with the lysosomal markers lysosensor and LAMPl antibodies, insulin markers and these fluorescent markers for demonstrating whether any of these molecules enter the lysosomal pathway.

Example IX.

Ins-C-mcTimer Does Not Disrupt Insulin Secretion. Islet perifusion and ELISA assays will be used with anti-insulin, anti-C-peptide, and anti-RFP antibodies. (2,6,19). Optically measurements of insulin secretion will be determined using published techniques (6,19).

Cadaveric islet preparations (i.e., for example, at least 12) will be studied both with, and without, Ins-C-mcTimer expression for basal (5.6 mM glucose) and stimulated (22.2 mM glucose) insulin secretory rates. These rates will be compared to determine whether the rates are the same in the presence or absence of Ins-C-mcTimer expression. The data indicates whether Ins-C-mcTimer alters insulin secretory rates and at the same time properly monitors those rates.

Example X. Determination Of Insulin Secretion Capability Of Human Cadaveric Pancreatic Islet

Preparations.

The quality of cadaveric human islets are determined by tabulating statistical parameters including median, mean, and variability of single pancreatic islet C-mc-Timer green/(green+red) fluorescence ratios taken at 14, 28, and 56 hours in accordance with Example XII. The methods are detailed in previously published studies with the exception that mcTimer fluorescent protein replaces the emGFP probe within the Ins-C- emGFP expression module. Balamurugan et al., "Flexible management of enzymatic digestion improves human islet isolation outcome from sub-optimal donor pancreata" Am J Transplantation 3:1135-1142 (2003); Bertera et al., "Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein" Biotechniques 35:718-722 (2003): and Michael et al., "Pancreatic β-cells secrete insulin in fast- and slow-release forms" Diabetes 55:600-607 (2006).

Both individual pancreatic islet β cells and whole pancreatic islet isolate measures will be performed to determine intra-islet cellular variability. Islets expressing Ins-C- mcTimer will be individually placed in microtiter plates designed for wide-field fluorescence (coverslip glass well bottoms). For each islet preparation, statistics of the distribution of the means and variances at the cellular and islet levels are collected. A correlation between the secretory rate from the perifusion assays with the time course of green/(green+red) fluorescence ration will determine how well the fluorescence color ratio averaged over all islets of a preparation measures differences in insulin secretory rates by perifusion.

These data using the Ins-C-mc-Timer reporter will help clinical islet cores correlate fractional islet beta cell tissue with high insulin secretory competency, pre- transplantation, on durability of insulin independence in their engraftment subjects.

Example XI.

Glucose Cycling Restores Isolation-Induced Insulin Release Incompetence. This example demonstrates that the Ins-C-mcTimer probe can measure restoration of insulin secretory competence from human donor pancreatic donor islets expressing abnormally low to normal levels of insulin. The data was collected from rested pancreatic islets from three donor preparations expressing Ins-C-mcTimer in 4 mM glucose after stimulating in 15 mM glucose. A significant recovery of insulin release was seen where previously red fluorescing cells (λ = 0.25 ± 0.03) shifted to green fluorescing cells (λ = 0.63 ± 0.05). See, Figure 5. These findings indicate that pre-transplantation islets injured during isolation protocols can be restored to optimal secretory competence by glucose stimulus cycling. Further, this study shows that an Ins-C-mcTimer probe of the present inventions would be of use as part of donor islet transplantation methods.

This observation has major implications for improving the quality of islets for transplantation.

Thus the inventor contemplate the use of an Ins-C-mcTimer probe of the present invention for identifying functional beta cells prior to transplantation, an example of which is provided below in Example IVX.

' Example XII.

Ins-C-mcTimer Trafficks Without Aggregation Or Abnormal Accumulation. Eliminates aggregation and accumulation in ER and secretory granule, and accelerated degradation via proteosome or lysosome.

Upon side-by-side comparison of Ins-C-Timer (Figure 7A) which forms aggregates with a monomelic marker of the present inventions, Ins-C-mcTimer (Figure 7B), at least two major parameters were markedly different in the organelles that were fluorescently labeled. These were primarily the diameter and frequency of the fluorescent puncta. For example, the original Ins-C-Timer resulted in a dramatically lower frequency of fluorescent puncta with diameters similar to those of insulin secretory granules concurrently with a higher frequency of fluorescent puncta with diameters markedly larger than those of insulin secretory granules. The larger diameter organelles were contemplated to be lysosomes in the process of degrading fused secretory granules and their insulin cargo. Also, the background fluorescence appeared higher with the original Ins-C-Timer, which likely reflected accumulation in the diffuse trafficking network known as the Endoplasmic Reticulum (ER) and transgolgi-network (often appearing as a large dense oval region when staining for secretory proteins. It is contemplated that aggregation of the original Ins-C-Timer via obligate tetramer formation and aggregation of the Timer moiety of the fusion protein in the ER and trans Golgi. Misfolding and aggregation of proteins within the ER is known to trigger increased trafficking of those proteins within the lysosomal pathway. Further, a mutation in insulation is known to lead to increased insulin accumulation in the ER, ER stress, apoptosis of the beta cell, and ultimately diabetes. Because the proteolytic processing of proinsulin into mature insulin and C peptide occurs after trafficking to the insulin secretory granule, the proinsulin accumulating in the ER does not undergoes this proteolytic maturation process. Thus, the cell biological finding of Ins- fluorescence accumulation in the ER, as opposed to the secretory granule, is an easily assayed predictor of the biochemical observation of incomplete proteolytic maturation of insulin.

Further, the use of an Ins-C-mcTimer molecule trafficking in a more physiological normal manner provides an additional biochemical assay for proinsulin processing much easier because the fluorescent protein moiety adds about 240 amino acid residues to the 31 residue long C peptide, making it easily resolvable on standard polyacrylamide protein sizing gels (example, 10-12% polyacrilymide). This is in contrast to labeled C-peptide which are not visible on standard gels and instead require concentrations of polyacrilymide in the range of 20%.

In summary, Ins-C-Timer can provide a marker for time related insulin production and secretion. Such as the average age of the proinsulin accumulated in the ER, and in any other intracellular organelle, such as early lysosomes, late lysosomes, which are contemplated to be of use to compute the trafficking rates of insulin and any of its mutant variants. These considerations underscore major ways Ins-C-mcTimer can report on the intricate and important cell biology of insulin and it's relation to diabetes.

Example XIII.

Ins-C-mcTimer Is Processed To Insulin And C-mcTimer

This example provides an illustrative characterization showing that Ins-C- mcTimer is processed in β cells to insulin and the C-mcTimer biochemical using Western blot analysis. INS 1-832/13 cells and mouse islets will be used to confirm islet proteolytic processing Orci et al., "Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles" Cell 49:865-868 (1987); and Watkins et al. (2002). The Ins-C-mcTimer probe is contemplated for use in a color assay for determining insulin secretory competency of pre-transplantation human islets {supra). In parallel, aliquots of the same islet preparation will be assayed by ELISA insulin assays to validate the color assay. Geng et al., "Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels. Am. J. Physiol. Endocrinol. Metab 293: 293-301 (2007); and Watkins et al., (2002). As shown above in Example XI, human islet preparations may be obtained having a λ of 0.3 or less, that can be assessed with the Ins-C-mcTimer probe.

Example IVX. This example presents a live-cell biology study of human islets, featuring confocal imaging of the fluorescent reporter mouse Ins-C-emGFP labeling insulin secretory granules.

This example further demonstrates that the fluorescently-tagged insulin granules maintain their mobility and secretory response to insulin secretagogues in vitro. Li addition, Ins-C-emGFP reporter co-localized with BODIPY-FL glibenclamide, a high affinity fluorescent ligand of the KATP (potassium ATP) channels. Analysis of human islets obtained from type 2 diabetic donors showed presence of a dramatic retardation in the trafficking of Ins-C-emGFP labeled insulin granules out of the endoplasmic reticulum, a feature not observed in islets from 6 other diabetic and 22 non-diabetic donors. These findings demonstrate that the use of fluorescent reporters can be successfully applied to the study of insulin granule function and differentiated secretory phenotype, which can be disrupted by a variety of unknown causes in cases of type 2 diabetes.

Expression of Ins-C-emGFP in human islets fluorescently labels subcellular puncta. For every human islet preparation; Ad.Ins-C-emGFP infection resulted in intense green fluorescence in up to 20% of the cells of an islet (n=22 donor organs). Figure 11 shows in each row fluorescent, Differential Interference Contrast (DIC), and merged images from different human islet isolations. In panel A (top to bottom), each image shows tens of labeled cells within an islet, where each cell is distinguished by the absence of green fluorescence within the nucleus and the presence of numerous fluorescent puncta within the cytoplasm. Individual puncta appeared to be equivalent in size to large dense- core secretory granules.

The diameter of the fluorescent puncta were quantitated and found to average 375+143 ran in diameter and ranged from the detection limit up to 1.2 mm, although very few were one micron or greater (n=2523 from five donor organs). These results are consistent with measurements from electron micrographs of large dense core insulin secretory granules (Orci, et al., Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987:49:865-868; and Dean, Ultrastructural morphometry of the pancreatic b cell. Diabetologia 1973;9:115— 119; ). In the absence of Ad.Ins-C-emGFP under the same confocal detection conditions no fluorescent puncta were observed. The results showed that Ins-C-emGFP fluorescence was highly localized in islet secretory granules.

Human islets co-localize Ins-C-emGFP to insulin secretory granules within β cells. To demonstrate that the green fluorescent puncta, observed after Ins-C-emGFP expression, were indeed labeling insulin secretory granules, human, non-diabetic, islets were co-stained with an anti-insulin antibody. Figure 12 shows the results representative of experiments performed on non-diabetic, human islet preparations (n=7). To maximize the number of puncta viewable in a single cell, 3D projections of confocal optical sections of cells expressing green Ins-C-emGFP fluorescence (panel A) and red anti- insulin fluorescence (panel B) were constructed.

As shown in panel C, the vast majority of puncta were both green and red as indicated by the high fraction of yellow puncta. This result demonstrated that Ins-C- emGFP expression co-localized to insulin granules. The few red puncta without green voxels (green fluorescence above background) was reasonably contemplated to be from low expression of Ins-C-emGFP or from a granule having been formed prior to expression of Ins-C-emGFP. Green puncta without red voxels (red fluorescence above background) were rare, and when present were contemplated to be due to incomplete permeabilization of the cell or accessibility of the antibody. The green fluorescence intensity was at least an order or magnitude greater at the granule compared to any other subcellular compartment including the endoplasmic reticulum (ER), Golgi, and plasma membrane where typically relatively little fluorescence was observed.

Ins-C-emGFP labeled insulin granules respond to secretagogues and were mobile. Before performing any imaging experiments, each of the islet preparations used in the experiments were tested for being capable of proper secretory function as measured by glucose containing perfusion fluids. Figure 13 shows the typical insulin secretory response to 2OmM glucose of the nondiabetic human islets (n=7) used in this study. As shown in the Figure 13, the islets consistently exhibited substantial secretory responses to high glucose stimulation for 30 min. When whole-cell fluorescence of Ins-C-emGFP labeled granules was measured by live-cell imaging of the fluorescent puncta, a significantly greater time-dependent decay was observed in response to steps in glucose stimulation from 5.6 (Figure 14, panel A) to 2OmM glucose (Figure 14, panel B), compared to mock stimulation by 5.6mM glucose. Panel C (Figure 14) shows the time course of fluorescence over 800 images of the experiment with glucose stimulation (black trace) and with mock stimulation (gray trace). In addition, the fluorescently labeled granules exhibited different mobilities. Most granules were stationary or locally mobile, as if they were contained in approximately 1 mm diameter cages or similarly tethered. Some granules were globally mobile, moving multiple microns at about 1 mm per sec (as observed in a movie made during the course of this experiment, specifically a Time-lapse imaging of Ins-C-emGFP expressed in a non-diabtic human islet b cell. The movie shows stationary, local and long-distance mobility behaviors representative of fluorescently labeled human insulin granules; herein incorporated by reference). These results demonstrate that the Ins-C-emGFP labeled insulin granules retain their functional and biological properties.

Ins-C-FPs help identify additional markers at the insulin secretory granule. In live rodent β cells, fluorescently tagged sulfonylureas were previously used to identify their high affinity receptor SURl on insulin granules (Geng, et al., Diabetes 2003;52:767-776; Zϋnkler, et al., Biochemical Pharmacology 2004;67: 1437-1444; and Varadi, et al., Diabetologia 2006;49: 1567-1577.). Similarly live human islets were herein tested for the presence of SURl on red fluorescent Ins-CdsRedl Bright vesicles by using a green fluorescent sulfonylurea (BODIPY FL glibenclamide) for marking SURl and the red fluorescent Ins-CdsRedl Bright for a live-cell marker of insulin secretory granules. Figure 15 shows the striking intracellular punctate pattern of green glibenclamide fluorescence, together with the pattern of Ins-C-dsRedl Bright fluorescence in three optical sections of a human b cell. In the merged images, most puncta are yellow demonstrating co- localization of the green glibenclamide and red Ins-C-dsRedlBright (n=10 or more islets from each of 3 donors). Image analysis of z sections showed that 0.94 of green puncta contained a substantial number of voxels with significant red fluorescence above background. Membranes other than those of the insulin secretory granules, including the ER, Golgi, and plasma membranes, show comparatively little fluorescence. The results indicate that glibenclamide is highly localized to insulin secretory granules in human islets.

Expression of Ins-C-emGFP reporter in T2D islets identifies novel islet cell phenotypes. Over the past years the inventor studied the expression of the Ins-C-emGFP reporter in islets obtained from T2D cadaveric donors. In two of the eight donors studied, islet expression of the Ins-C-emGFP reporter differed from what was observed in non- diabetic islets (Figure 16). While in the other human islets (n=22 non-diabetic and n=6 diabetic; panel A), the reporter resulted in a discrete, punctate, fluorescence, in two of the T2D donor islet populations' expression of Ins-C-emGFP resulted in a dramatically diffuse net-like green fluorescence throughout the cytoplasm (panels B and C). This phenotype was observed in the islets studied from these two T2D donors, and points to a cellular defect in insulin trafficking in T2D islets.

Defective insulin trafficking out of the ER in the islets from the two T2D donors. As shown in Figure 16 (panel A), the red fluorescence signal of the anti-calnexin antibody at the ER fails to co-localize with the Ins-C-emGFP reporter at the insulin secretory granules in non-diabetic islets. Anticalnexin, however, did co-localize with Ins- C-emGFP in islets from the T2D islets showing diffuse green fluorescence (panel B and C). These T2D islets demonstrate mistargeting or dramatically inefficient trafficking of the Ins-C-emGFP fluorescence resulting in its accumulation in the ER. The results are representative of islet cells studied from the T2D donor (421 β cells from 108 islets). Similar results were obtained by using antibodies against another ER marker, Grp78. As shown in Figure 17, Grp78 failed to co-localize with Ins-C-emGFP labeled secretory granules in non-diabetic islets but did co-localize with Ins-C-emGFP expressed in islet cells from the T2D donor islets (panel B and C), as above for calnexin. Defective trafficking of KATP channels to insulin secretory granules in T2D. The study of islets from another T2D donor showed again presence of non-punctate, diffuse green fluorescence of the Ins-C-emGFP reporter, which colocalized with the ER marker anti-Grp78 (Figure 18; 3 or more cells from each of 52 islets). In addition, fluorescent glibenclamide failed to label punctate structures, but rather mostly labels reticulate structures (5 or more cells from each of 17 islets). This shows that, in the islets from the second T2D donor, the SURl of KATP channels failed to undergo ER export. The results indicated that T2D islets exhibit trafficking phenotypes in which insulin cargo and also resident membrane proteins do not normally traffic to the insulin granule.

Materials and Methods for Example IVX.

Islet isolation. Human islets were isolated from pancreatic organs procured by CORE (Center for Organ Recovery and Education, Pittsburgh) from multi-organ deceased donors using a modification of the semi-automated Ricordi's method on discontinuous density gradients (Ricordi, et al., Automated islet isolation from human pancreas. Diabetes 1989;38(Supplement l):140-142; Liu M, et al., A new method for isolation of murine islets with markedly improved yields. Transplant Proc 1995;27:3208- 3210; and Balamurugan, et al., Flexible management of enzymatic digestion improves human islet isolation outcome from sub-optimal donor pancreata. Am J Transplantation 2003;3:l 135-1142). Eight pancreatic donors had T2D, all of which appeared normal except two whose β cell biology was studied here. The first donor was a 64 year old male treated with metformin for 5 years. Cause of death was a cerebrovascular accident with intracranial hemorrhage with no downtime and no cardiac arrest. Glucose levels were 266 mg/dl on admission. Islets were isolated 12 hours following cross clamp of the aorta during which the pancreas was maintained in HTK (histidine, tryptophan, and ketoglutarate) solution on ice, and exhibited a regular morphology. The second donor was a 57 year old male treated with metformin for 10 years. Cause of death was an intracranial hemorrhage consequent to a stroke with had no downtime or cardiac arrest. Glucose levels were 238 mg/dl on admission. Islets were isolated as above and exhibited a regular morphology. Isolated islets from both non-diabetic and T2D donors were cultured in CMRL- 1066 medium which included glucose (5.6 mM) and was supplemented with Lglutamine (2 mM), Nicotinamide (2 mM), Penicillin (2%),

Streptomycin (2%), heat inactivated fetal calf serum (10%), at 37°C in 95% air, 5% CO2. Viability assays. Non-diabetic islets were cultured in the CMRL medium at 37⁰C overnight prior to viability assay and imaging.

Islet secretory assays. Non-diabetic islets were cultured in the CMRL medium at 37°C overnight before routine dynamic perifusion to assess in vitro glucose responsiveness. Assay of perifusion samples was performed (48) using approximately 100 hand-picked islets which were sandwiched between Bio-Gel P2 plugs in Kreb's Ringer Bicarbonate Buffer (KRBB) with 0.5% BSA and the indicated glucose used to perfuse the islets at 37oC at approximately 0.1 ml/min and fractions taken every minute. Glucose challenge was carried out in a 90-minute protocol where islets were exposed to KRBB buffer containing 2.8 mM glucose during the first and last 30 minutes, and KRBB with 20 mM glucose between minute 31 and 60. The eluates were assayed using an ELISA kit for insulin.

Ins-C-FP expression. Live islet insulin granule fluorescent labeling with Ad.Ins- C-emGFP, was used as described (Watkins, et al., Imaging secretory vesicles by fluorescent protein insertion into propeptide rather than mature secreted peptide. Traffic 2002;3:461-471) at an MOI approximately 200. In another embodiment, a Ad. Ins- C- dsRed IBRIGHT was constructed by substituting into the DsRedl (Clontech, CA) of Adlox.Ins-C-dsRedl, the VaI 105 and Ala 105 mutations, as originally described (Terskikh, et al., Science 2000, 290: 1585-1588) by site-directed mutagenesis, creating Adlox.Ins-CdsRedlBRIGHT. This Adlox.Ins-CdsRedlBRIGHT construct was then used to make Ad.Ins-CdsRed IBRIGHT. The fluorescence expression levels shown herein were representative of those observed within 48 h after infection. Typically, approximately 10% of the cells, mostly located at the perimeter of the islet expressed the Ins-C-FP to intense levels 24 h after infection, and approximately 20% 48 h after infection. The islet cells studied were alive by the LIVE/DEAD fluorescent assay (Molecular Probes, Eugene, OR).

Confocal fluorescence microscopy. Islets were placed into an optical recording chamber (Harvard Apparatus, Holliston, MA) at 37⁰C. Singlephoton confocal microscopy was performed using an Olympus Fluoview 300 Confocal Laser Scanning head with an Olympus 1X70 inverted microscope (Olympus, Melville, NY). Excitation of Ins-C-emGFP and green glibenclamide BODIPY FL was by the 488 nm Argon laser line and emission detected using sharp cutoff 510IF long-pass and BA530RIF shortpass filters. Excitation of red Ins-C-dsRedl BRIGHT glibenclamide was by the 543-nm green HeNe laser line and emission detected using a sharp cutoff B A610IF long-pass filter. Co- imaging was done by sequential excitation, and simultaneous detection of emission, which showed no crosstalk. Guinea pig anti-insulin optimized for detection of human insulin was obtained (Dako, Carpinteria, CA) and used at 5 ug/ml. AlexaFluor 594 goat anti-rabbit IgG and AlexaFluor 594 anti-guinea pig IgG (Molecular Probes) were used at 0.2 ug/ml. Freshly purified islets, naive or infected by Ad.Ins-C-FPs, were fixed with 2% paraformaldehyde in PBS for 20 min, washed in PBS three times, and blocked by incubation in 2% BSA in PBS (pH 7.5) overnight at 4°C. The islets were then incubated with the indicated primary antibodies at 5 mg/ml in blocking buffer overnight at 4°C. The islets were washed three times with PBS, incubated with labeled secondary antibodies in blocking buffer for 2 h at room temperature, and then washed three times with PBS. For imaging responses to secretagogue stimulation, islets expressing Ins-C emGFP were preincubated in 2.8 mM glucose in KRBB secretory medium with 0.5% BSA and stimulated by increasing the glucose to 20 mM using superfusion with a BIOLOGIC RSC- 160 sewer pipe system. Images were recorded from the bottom plasma membrane of a β cell in an intact islet that had attached to the coverslip firmly enough that the superfusion did not move the cell imaged. Fluorescent insulin granules exhibited characteristically dynamic movements and were tracked manually by running the time series using MetaMorph v4.6r3 analysis software from Universal Imaging (West Chester, PA, USA). The integrated whole-cell fluorescence intensity within the z section was determined across the time series.

Co-localization withs Calnexin and Grp78. Anti-calnexin C-terminus and anti- Grp78 polyclonal rabbit antibodies (Stressgen, Victoria BC, Canada) were used separately each at 1 :500 dilution. AlexaFluor-594 goat anti-rabbit (Molecular Probes, Eugene, OR) was used at 1 :10,000 dilution as secondary antibody, as described above for the antiinsulin antibodies.

Expression of Golgi Fluorescent Protein. Overlap PCR was used to amplify the fusion protein coding region of the plasmid EYFP-Golgi (Clontech), which includes the first 81 codons of the human beta- 1 ,4-galactosltransferase fused to EYFP, and insert it downstream from the mouse Ins2 promoter in the plasmid Adlox,Pins2-emGFP, and replacing emGFP. This was then used to make Ad.Pins2-EYFP-Golgi as previously described (Watkins, et al., Imaging secretory vesicles by fluorescent protein insertion into propeptide rather than mature secreted peptide. Traffic 2002;3:461-471).

Fluorescent glibenclamide labeling. The human islets were superfused at 37⁰C with BODIPY-FL glibenclamide (40 nM; Molecular Probes, Eugene, OR) in KRBB secretion buffer (0.05% BSA) for 5 minutes then washed by superfusion with the KRBB buffer without the fluorescent ligand. Control experiments showed that excess unlabeled glibenclamide (400 nM) in the KRBB secretion buffer (0.05% BSA) reversed the granule fluorescent staining. By the fluorescent live-dead cell test (Molecular Probes), >90% of the cells were alive in the islets studied.

The following references are herein incorporated in their entirety: 1. Gembal, et al., Evidence that glucose can control insulin release independently from its action on ATP-sensitive K+ channels in mouse b cells. J Clin Invest 1992;89: 1288-1295;

2. Sato, et al., Dual functional role of membrane depolarization/Ca2+ influx in rat pancreatic b cell. Diabetes 1992;41:438-443;

3. Detimary, et al., Concentration dependence and time course of the effects of glucose: on adenine and guanine nucleotides in mouse pancreatic islets. J Biol Chem

1996;271 :20559-20565; 4. Straub, et al., Glucose-stimulated signaling pathways in triphasic insulin secretion. Diabetes Metab Res Rev 2002; 18:451-463;

5. Cherrington, et al., Physiological consequences of phasic insulin release in the normal animal. Diabetes 2002 ;51 (Supplement 1):S1O3-S1O8; 6. Pørksen, The in vivo regulation of pulsatile insulin secretion. Diabetologia 2002;45:3- 20;

7. Gerich, Is reduced first-phase insulin release the earliest detectable abnormality in individuals destined to develop type 2 diabetes. Diabetes 2002;51 (Supplement 1): S117- S121; 8. O'Rahilly, et al., Impaired pulsatile secretion of insulin in relatives of patients with non-insulin-dependent diabetes. N Engl J Med 1988;318:1225-1230;

9. Lang, et al., Brief, irregular oscillations of basal plasma insulin and glucose concentrations in diabetic man. Diabetes 1981;30:435-439;.

10. Schmitz, et al., Disorderly and nonstationary insulin secretion in relatives of patients with NIDDM. Am J Physiol Endocrinol Metab 1997;272:E218-E226;

11. Del Prato, et al., Phasic release and metabolic regulation in type 2 diabetes. Diabetes 2002;51 (Supplement l):S109-S116;

12. Del Prato, et al., Beta- and alpha-cell dysfunction in type 2 diabetes. Horm Metab Res 2004;36:775-781;. 13. Del Guerra, et al., Functional and molecular defects of pancreatic islets in human type 2 diabetes. Diabetes 2005;54:727-735;

14. Weir, et al., Five stages of evolving β-cell dysfunction during progression to diabetes. Diabetes 2004;53(Supplement 3):S16-21.

15. Aridor, et al., Traffic jam: a compendium of human diseases that affect intracellular transport processes. Traffic 2000;l:836-851;

16. Aridor , et al., Traffic jams II: an update of diseases of intracellular transport. Traffic 2002;3:781- 790;

17. Arvan , et al., Sorting and storage during secretory granule biogenesis: Looking backward and forward. Biochem J 1998;332:593-610; 18. Tsien, The green fluorescent protein. Annu Rev Biochem 1998;67:509-544; 19. Tsien, Building and breeding molecules to spy on cells and tumors. FEBS Lett 2005;579:927-932;

20. Terskikh ,et al., "Fluorescent timer": Protein that changes color with time. Science 2000;290:1585-1588; 21. Lippincott-Schwartz, et al., Development and use of fluorescent protein markers in living cells. Science 2003;300:87-91;

22. Rizzo, et al., A functional link between glucokinase binding to insulin granules and conformational alterations in response to glucose and insulin. J Biol Chem

2002;277:34168-34175; 23. Geng, et al., The insulin secretory granule is the major site of KATP channels of the endocrine pancreas. Diabetes 2003;52:767-776; 24. Zϋnkler, et al., Fluorescent microscopy studies with a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic β-cell ATP-sensitive K+ currents. Biochemical Pharmacology 2004;67:1437-

1444;. 25. Varadi, et al., Intracellular ATP-sensitive K+ channels in mouse pancreatic b cells: against a role in organelle cation homeostasis. Diabetologia 2006;49: 1567-1577;

26. Kasai, et al., A new quantitative (twophoton extracellular polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets. J Physiol 2005;568:891-903; 27. Watkins, et al., Imaging secretory vesicles by fluorescent protein insertion into propeptide rather than mature secreted peptide. Traffic 2002;3:461-471;

28. Bertera, et al., Body window-enabled in vivo multicolor imaging of transplanted mouse islets expressing an insulin-Timer fusion protein. Biotechniques 2003;35: 718-

722; 29. Michael, et al., Pancreatic b-cells secrete insulin in fast- and slowrelease forms.

Diabetes 2006;55:600-607;

30. Tsuboi, et al., Mechanisms of dense core vesicle recapture following "kiss and run"

("cavicapture") exocytosis in insulin-secreting cells. J Biol Chem 2004;279:47115-

47124; 31. Stiernet, et al., Glucose acutely decreases pH of secretory granules in mouse pancreatic islets. Mechanisms and influence on insulin secretion. J Biol Chem 2006;281 :22142- 22151;

32. Takamori, et al., Molecular anatomy of a trafficking organelle. Cell 2006;127:831- 846;

33. Orci, et al., Proteolytic maturation of insulin is a post-Golgi event which occurs in acidifying clathrin-coated secretory vesicles. Cell 1987:49:865-868;

34. Dean, Ultrastructural morphometry of the pancreatic b cell. Diabetologia 1973 ;9:115- 119; 35. Carpentier, et al., Internalization of 3H-glibenclamide in pancreatic islet cells. Diabetologia 1986;29:259-261;

36. Ozanne, et al., Intracellular localization and molecular heterogeneity of the sulphonylurea receptor in insulin-secreting cells. Diabetologia 1995;38:277-282;

37. Gylfe, et al., Interaction of sulfonylurea with the pancreatic b cell. Experientia 1984;40:l 126-1134;

38. Hellman, et al., Glibenclamide is exceptional among hypoglycaemic sulphonylureas in accumulating progressively in β-cell rich pancreatic islets. Acta Endocrinol 1984;105:385-390;.

39. Sharma, et al., Aguilar-Bryan L. The C terminus of SURl is required for trafficking of KATP channels. J Biol Chem 1999;274:20628-20632;

40. Zerangue, et al., A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane KATP channels. Neuron 1999;22:537-548;

41. Schwappach, et al., Molecular basis for KATP assembly: transmembrane interactions mediate association of a K channel with an ABC transporter. Neuron 2000;26: 155-167; 42. Crane, et al., Assembly, maturation, and turnover of KATP channel subunits. J Biol Chem 2004;279:9080-9090;

43. Michelsen, et al., Hide and run. Arginine-based endoplasmic-reticulum-sorting motifs in the assembly of heteromultimeric membrane proteins. EMBO Reports 2005;6:717- 722; 44. Ren, et al., Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation. J Transl Med. 2007; 5:1-8;

45. Bonner-Weir, et al., New sources of pancreatic b cells. Nat Biotechnol 2005;23:857- 861 ; and

46. Ricordi, et al., Automated islet isolation from human pancreas. Diabetes 1989;38(Supplement 1): 140-142.

47. Liu M, et al., A new method for isolation of murine islets with markedly improved yields. Transplant Proc 1995;27:3208-3210. 48. Balamurugan, et al., Flexible management of enzymatic digestion improves human islet isolation outcome from sub-optimal donor pancreata. Am J Transplantation 2003;3:l 135-1142.

Example VX. This example provides biosensors for studying mutations in the human insulin gene that may cause diabetes by causing ER accumulation of insulin due to misfolding and aggregation of variant (mutant) insulin molecules.

ER accumulation of variant insulin might further cause mistrafficking of insulin such as into secretory lysosomal vesicles. However these types of studies are invalid when fluorescent protein is multimerizing and contributing to the misfolding and aggregation of insulin, a limitation of previous fluorescent probes, in particular Timer probes. Thus

Therefore in order to insert and study point mutations representative of human insulin, mcTimer biosensors comprising human insulin were provided using a human version of mcTimer sequences shown in Figure 7, wherein mcCherry-Bright was inserted into a human C peptide sequence. The human version of mcTimer was then inserted into the vector of Figure 6), under the control of a human insulin promoter.

Further, a human version of mouse emerald was developed as described. Wherein emerald-GFP was inserted into a human C peptide sequence (Figure 10B). The human version of mcTimer was then inserted into the vector of (Figure 1OA, C and D), under the control of a human insulin promoter. The vertical lines denote junctions between the insulin peptides B, C, and A, and the dibasic cleavage sites. As in the other constructs, highlighted triple AAA's are the alanine linkers between the fluorescent molecule, in this case emeraldGFP, and the C peptide flanks. The K residue and codon at the bottom of the first page is the 206K mutation to eliminate the weak dimerization of the original human emerald of the present inventions which does not express a K at that position.

The human version of Ins-C-emGFP was successfully expressed in mouse islet cells and a rat INS1-832/13 cell line.

Thus Ins-C-GFP reporters and mcTimer probes of the present inventions are powerful tools contemplated for analyzing specific pathophysiological mechanisms underlying type 2 diabetes.

The following Examples show that fluorescent biosensors of the present inventions demonstrate live-cell imaging of human proinsulin and its clinical mutants and facilitate biochemical measures of their proteasomal degradation and altered proteolytic cleavage.

The observations described herein show that mutations disrupt mutant and wild- type proinsulin in the secretory pathway, including ER exit and proteolytic cleavage, leading to ER stress and ultimately β cell apoptosis. Human Ins-C-emeraldGFP was designed for live-cell imaging the β cell biology of proinsulin and its trafficking to insulin secretory granules.

Example VXI.

Human proinsulin mutants associated with clinical diabetes were tested for ER accumulation and classified based on cell fluorescence phenotype. These results support β cell causation of diabetes by expression of insulin mutants contemplated by the inventor by looking at human insulin trafficking with hlns-C-GFP, as shown in Figure 19. The design of the fluorescent biosensor affords extraordinarily robust fluorescent labeling of secretory granules. Live-cell confocal imaging of hlns-C- emGFP-WT expressed in rat INSl cells is shown in Figure 19C.

Example VXII. Shows exemplary ER misfolding and proteasomal pathway degradation. Insertion of a 238 emGFP moiety within a mutant insulin molecule provides a routine biosensor for trafficking by measuring either unprocessed Ins-C-GFP or processed C-emGFP by Western blot analysis using anti-GFP antibodies. Figure 2OA shows how mutations in proinsulin reverse the ratio of the hlns-C-GFP to cleaved C- emGFP protein bands: For wild-type, the densitometry ratio of proinsulin to C peptide bands detected is 0.34/0.66. For the mutants L30P and C96Y, the same ratios were 0.67/0.33 and 0.75/0.25, respectively. The Westerns provide a population sample result consistent with β cell fluorescence phenotypes. Derlin-1 promotes the efficient degradation of the Cystic Fibrosis Transmembrane

Conductance Regulator (CFTR) and CFTR folding mutants (Sun, et al., J. Biol. Chem., Vol. 281, Issue 48, 36856-36863, 2006). This example shows co-expression of Derlin-1 with certain human proinsulin mutants in huIns-C-emGFP. Figure 2OB shows degradation- enhanced by co-expression of Derlin-1 for five proinsulin mutations. From the Western blot, the fraction of protein degraded by Derlin expression is as follows for the proinsulin form (fraction C peptide degraded given in parantheses): G32S: 0.20 (0.29), C95Y : 0.45 (0.51), C96Y: 0.39 (0.48), L30P: 0.28 (0.26), and Y108Stop 0.22 (0.65). The fractions degraded by Derlin expression for wild-type control were 0.05 (0.02). Therefore, the diabetes mutant peptides result in greater misfolding and proteasomal pathway degradation than wild-type. The total protein added is identical across all lanes yet the wild- type level is markedly higher than the mutant insulin bands, due to expected ER stress and consequent degradation of the mutant secretory protein.

Example VXIII. Proinsulin mutants causing diabetes were tagged for proteasomal degradation by ubiquitin.

This was studied by immunoprecipitating with anti-GFP then using Western blot analysis to test for the presence of ubiquitinylated proinsulin, observed as high molecular weight hlns-C-GFP in Panel A, or as anti-ubiquitin reactivity follow-up Westerns in Panel B. Densitometry of the proinsulin bands shown in Figure 21 A shows a markedly higher ratio of the high molecular weight hlns-C-GFP to the non- ubiquitylated hlns-C- GFP. The ratio flips from <1 for wild-type (0.80), to >1 for C96Y (1.63) and L30P (1.17), consistent with earlier diabetes onset of C96Y (hAkita) compared to L30P. Ubiquitinylation of prosinsulins will be used to explore the β cell pathway of ER stress in diabetes.

Example IVVX.

Mutant proinsulin blocks secretion of non-mutant insulins. One mechanism by which hAkita proinsulin might block secretion of wild-type insulin is by blocking its traffic out of the ER. Figure 22 provides evidence for this effect. In this series of experiments, a wild-type red fluorescent Ins-C-mCherry is co-expressed with either a green fluorescent wild-type human Ins-C-emGFP or the human Ins-C- emGFP-C96Y Akita mutant. The red fluorescence of the wild-type proinsulin is blocked from trafficking to secretory granules by the green Akita mutant compared to the green wild-type proinsulin. The inventor contemplated whether hyperglycemia underlying the diabetes arose from defective secretion of the processed wild-type insulin and C peptide, or normal secretion occur but of unprocessed proinsulin. C96Y (hAkita) mutant proinsulin was distinguished from INSl cells expressing hlns-C-GFP in parallel with those expressing hIns-C-GFP-C96Y (hAkita) by performing ELISA assays of secretion. The ELISA used detects only rat C peptide (100%), with little or no cross-reactivity to rat proinsulin

(<4.6%) or human proinsulin (<0.001%), human insulin (<0.001%) or human C peptide (<0.001%). Secretion of the endogenous processed rat insulin is blocked by the hAkita mutant, then the expression of hAkita proinsulin should result in a decrease in secretory rate of the endogenous rat C peptide and, by deduction, the endogenous rat insulin, as observed in Figure 22D.

One indirect mechanism by which mutant proinsulin might block secretion of wild-type insulin is by destroying β cells. Figure 22E, shows an exemplary significantly enhanced apoptosis of β cells at two diabetes mutations in proinsulin, C96Y and L30P, compared to non-mutant proinsulin. All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described compositions and methods of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in medicine, diagnostics, molecular biology, diabetes, or related fields are intended to be within the scope of the present invention and the following Claims. References provided herein are incorporated in their entirety.

Claims

I Claim:

I . A monomeric multifluorescent protein.

2. The protein of Claim 1, wherein over time said protein fluoresces a first color followed by a second color.

3. The protein of Claim 2, wherein said first color is green.

4. The protein of Claim 2, wherein said second color is red.

5. The protein of Claim 1, wherein said multifluorescent protein comprises a proteolytic fragment of a prepropeptide.

6. The protein of Claim 1, wherein said monomeric protein comprises a peptide C.

7. The protein of Claim 1, wherein said monomeric protein comprises a prepropeptide.

8. The protein of Claim 1, wherein said monomeric protein comprises SEQ ID NO:2.

9. The protein of Claim 7, wherein said prepropeptide further comprises an A peptide, a B peptide, and a C peptide.

10. The protein of Claim 9, wherein said C peptide is located in between the A peptide and the B peptide.

I 1. The protein of Claim 1, wherein said monomeric protein comprises a Timer mutation.

12. The protein of Claim 11, wherein said Timer mutation is a 197T mutation.

13. A protein, comprising SEQ ED NO: 02.

14. A method, comprising: a) providing; i) a vector encoding a monomelic multifluorescent protein, wherein said protein expresses over time a first color fluorescence and a second color fluorescence; ii) an isolated tissue sample; and b) administering the vector to the tissue under conditions such that the protein expresses a fluorescence.

15. The protein of Claim 14, wherein said monomelic multifluorescent protein comprises SEQ ID NO : 02.

16. The method of Claim 14, wherein said method further comprises a step of measuring the first color fluorescence intensity and the second color fluorescence intensity.

17. The method of Claim 14, wherein said method further comprises a step of determining a ratio of a first color fluorescence intensity to a second fluorescence intensity.

18. The method of Claim 14, wherein said first color fluorescence co-localizes in vesicles comprising insulin.

19. The method of Claim 14, wherein said tissue comprises pancreatic beta cells.

20. The method of Claim 19, wherein said tissue is a cell.

21. The method of Claim 20, wherein said cell is a bonafide beta cell.

22. The method of Claim 14, further comprising a test compound.