WO2021119639A1 - Compositions, systems, and methods for measuring protein secretion at a single-cell level - Google Patents

Abstract

Compositions, systems and methods for measuring secretion of a protein-of- interest that include trap proteins and reporter proteins for secretion by a cell. Each trap protein comprises a binding partner, a first fluorescent label, and an outer leaflet anchor to anchor the trap protein to the outer leaflet of the cell to form a secretory trap. Each reporter protein comprises a protein-of-interest, a second fluorescent label, and an optogenetic tool to, in a dark conformation, reversibly heterodimerize with the binding partner, and, in a lit conformation, photo-release the binding partner. Upon secretion of the trap and reporter proteins by the cell, optogenetic tools in the dark conformation, reversibly heterodimerizes with binding partners to reversibly sequester secreting reporter proteins at a secretory trap, and in the lit conformation photo-release the binding-partner of sequestered reporter proteins to facilitate measurement of the secretion of protein-of interest comprised by the reporter proteins.

Description

COMPOSITIONS, SYSTEMS, AND METHODS FOR MEASURING PROTEIN SECRETION AT A SINGLE-CELL LEVEL

Copyright Notice

[0001] © 2020 Oregon Health & Science University. A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. 37 CFR § 1.71(d).

Technical Field

[0002] Generally, the field relates to systems, compositions, and methods for measuring peptide or protein secretion. More specifically, the field relates to systems, compositions, and methods for measuring peptide hormone secretion, such as insulin, glucagon, leptin, or neuropeptides.

Background Information

[0003] The secretion of protein, including peptide hormones regulates many of the metabolic and behavioral functions of higher organisms. Dysregulation or impairment of these physiological regulatory mechanisms governing, for example, metabolism, immune response, or organ function, often leads to significant pathology. Therefore, characterizing the secretion of peptides and proteins as well as the regulation of their secretion would allow for the development of treatments to ameliorate such pathology. And measuring secretion of these peptides or proteins would be valuable because it is one of a few physiological events that can be followed experimentally as the secretagogue is released into the surrounding tissue or the bloodstream. [0004] There are numerous assays available to measure secretion, however, there are very few techniques available that monitor secretion at a single cell level or continuously over time. For example, an array of antibody-based assays (ELISAs) are commercially available and are used routinely in research labs and the clinic to measure the secretion of peptide hormones. ELISAs permit quantifying the hormone concentration in cell supernatant or the blood circulation at a given point in time. But secretion measured by an ELISA or other assays only allows for bulk measurement of hormone in tissue culture supernatants or blood samples and cannot measure secretion at a single-cell level. This greatly limits investigations of secretion dynamics such as the interplay between a- and b-cells within the islet of Langerhans. In the islet, it is currently difficult to study the effect of insulin on glucagon secretion or vice versa as pharmacologically, as both cell types are equipped with similar receptors. Thus it is to date difficult to monitor hormone secretion at the single cell level and in a continuous fashion.

[0005] Accordingly, a light-driven technique that is capable of continuous measurement of a single cell secreting protein would allow for better characterization of these secreted proteins and their regulatory schema and development of treatments that ameliorate secretion-protein-related pathology. Optogenetic systems, such as the LOVTRAP system exist in the art that provide for reversible heterodimerization and photo-induced peptide and protein dissociation (Wang et al., Nat Methods, (2017)). However, optogenetic systems such as LOVTRAP and others provide for measuring peptide or protein subcellular localization and activity, but do not provide for measuring peptide or protein localization and activity at the plasma membrane of a cell, such as secretion. Rapid dissolution of secreted peptide or proteins after cellular release poses a barrier to providing meaningful measurement of the secreted peptides or proteins, which prevents measurement at a single-cell level. Moreover, unregulated release of labeled secreted peptides or proteins can lead to saturation of signal.

[0006] Thus, there remains a need for systems, compositions, and methods to carry out measuring peptide or proteins by such a light-driven technique. For example, there remains a need for fluorescent or luminescent reporter molecules that are concomitantly secreted with the endogenous hormones and that are applicable to experiments in cells, organoids, organs, or entire animals.

Measurement of rapidly dissolving secreting peptides after cellular release would ideally measure a signal that is locally defined at the surface of a cell by a light- driven technique.

Summary of the Disclosure

[0007] The present disclosure provides multiple aspects and forms, including novel compositions, systems, and methods to measure the secretion of a proteins of interest at a single-cell level. In some embodiments, the compositions, systems, and methods herein are useful in studying hormone secretion from endocrine cells. In other embodiments, they are useful for studying release of neuropeptides and methods of neuromodulation. In other embodiments, they are useful for collecting information about individual cell behavior rather than a population of cells, which is an improvement on traditional bulk detection methods, such as quantification of hormone release by ELISA techniques.

[0008] A first embodiment herein provides a set of one or more trap proteins, each trap protein in the set comprising, a first fluorescent-label (FL1) configured to, upon excitation, convey a set of one or more fluorescent reference F_ref signals; and an outer-leaflet anchor to anchor the trap protein to an outer leaflet of a plasma membrane of a cell; whereby, upon secretion of the set of trap proteins by the cell, secreted trap proteins accumulate at the outer leaflet of the plasma membrane to a form a set of one or more secretory traps.

[0009] A second embodiment herein provides a set of one or more reporter proteins for measuring cellular secretion of a protein-of-interest from a plasma membrane of a cell, each reporter protein in the set comprising: a protein-of-interest (POI) component comprising the protein-of-interest, a second fluorescent-label (FL2) configured to, upon excitation, convey a set of one or more fluorescent quantification (Fq_nt) signals, and an optogenetic tool configured to, in a dark conformation, reversibly heterodimerize with a binding partner, and, in a lit conformation, photo release the binding partner.

[0010] A third embodiment herein provides a secretion trap (secretrap) system for measuring cellular secretion of a protein-of-interest, the system comprising: a set of trap proteins and a set of reporter proteins; a set of one or more cells, each cell in the set of cells configured to secrete at least one protein selected from the set of trap proteins and the set of reporter proteins, whereby, upon secretion: the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membranes to form a set of one or more secretory traps, and the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps, and in the lit conformation photo-release the binding-partner of sequestered reporter proteins (or optionally sequestered trap proteins) to facilitate release of the sequestered reporter proteins from the set of secretory traps; an illumination system configured to: (a) selectively convey light to the set of secretory traps to selectively switch optogenetic tools between the dark and lit conformations, and (b) selectively excite FL1s in the set of trap proteins and FL2s in the set of reporter proteins; an optical detection system for detecting the sets of F_ref signals and the sets of F_qnt signals conveyed, respectively, from the set of secretory-trap proteins and the set of the reporter proteins; and a processor coupled to the optical detection system and configured to analyze the sets of F_ref signals and the sets of F_qnt signals to determine the quantity of the POI component secreted by the cell.

[0011 ] A fourth embodiment herein provides a method for measuring cellular secretion of a protein of interest, the method comprising: (a) expressing in a set of one or more cells, each cell in the set of cells having a plasma membrane with an outer leaflet, a set of one or more trap proteins of any of claims 1 to 7 whereby, upon secretion by the set of cells, the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membrane to form a set of one or more secretory traps; (b) expressing in the set of cells, a set of one or more reporter proteins of any of claims 8 to 18 whereby, upon secretion, the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps; (c) selectively conveying, during a time period, light to the cell to excite the FL1 s (or optionally the FL3s) of the set of secretory traps to convey a set of time-resolvable F_ref signals; (d) detecting the set of time-resolvable F_ref signals; (e) selectively conveying light to the set of secretory traps to selectively switch the optogenetic tools of the secreting trap proteins (or optionally the secreting reporter proteins) to the lit conformation to facilitate release of the sequestered reporter proteins from the set of secretory traps; (f) selectively conveying, during the time period, light to the set of secretory traps to excite the FL2s (or optionally the FL3) of the set of secretory traps to convey a set of time- resolvable Fq_nt signals; (g) detecting the set of time-resolvable F_qnt signals; (g) (optionally) repeating steps (c) through (g); (optionally) after step (c), selectively conveying, light to the set of secretory traps to excite the FL1s (or optionally the FL3s) in the set of molecular traps to convey a set of one or more F_ref baseline signals, detecting the set of F_ref baseline signals, and calculating, from the set of F_ref signals, a F_ref baseline; (optionally) after step (c), selectively conveying, light to the cell to excite the FL2s (or optionally the FL3s) of the set of molecular traps to convey a set of one or more F_qnt baseline signals, detecting the set of F_qnt baseline signals, and calculating, from the set of F_qnt signals, a F_qnt baseline; and (optionally) stimulating the cell to secrete one or more trap proteins or one or more reporter proteins.

[0012] Additional aspects and advantages will be apparent from the following detailed description of preferred embodiments, which proceeds with reference to the accompanying drawings.

Brief Description of the Drawings

[0013] FIGS. 1A and 1 B are schematics showing, respectively, a proglucagon and a fluorescent-protein (FP)-tagged proglucagon construct.

[0014] FIG. 2 provides an illustration showing a generalized and non-limiting representation of a reporter protein self-cleaving prior to secretion and subsequent sequestration by an optogenetic tool that switches to a lit conformation to dissociate the dissociate the reporter protein and thereby release it from the secretory trap. [0015] FIGS. 3 provides an illustration showing: (a) a trap protein reversibly heterodimerized to a reporter protein when the optogenetic tool is in a dark conformation to form a trap protein/reporter protein complex, and (b) a reporter protein dissociating from the complex when the optogenetic tool is in a lit conformation.

[0016] FIGS 4A and 4B are illustrations showing generalized and non-limiting examples of a set of reporter proteins being selectively released from a secretory trap; Fig. 4A shows (i) a set of reporter proteins accumulating in the secretory trap, creating a saturation of signal, which are then (ii) released by selectively conveying 490 nm light to the secretory trap, and Fig. 4B shows (iii) an substantially empty secretory trap and (iv) the set of reporter protein reaccumulating in the trap.

[0017] FIG. 5 provides a graph representing a time-course experiment showing first, a saturation of fluorescent signal that is reduced after release of reporter protein from a secretory trap and the re-accumulation of fluorescent signal after application of a stimulus.

[0018] FIG. 6 shows exocytosing FP-gcg-Lov2 fusion protein bound to Zdk1 at the plasma membrane of aTC1 c9 cells that to form a secretory trap.

[0019] FIG. 7 shows glucagon-bearing reporter proteins secreting at the plasma membrane of aTC1c9 cells that are quantifiable by measuring F_min and F_ref signal to calculating secretion.

[0020] FIG. 8 shows GPI-GFP-Zdk1 trap proteins accumulating at the surface of HeLa cells to form a secretory trap.

[0021] FIGS. 9A and 9B are each graphs of normalized fluorescence overtime showing a drop of fluorescence after illumination of Zdk1-LOV2 complexed to the GPI-GPF-Zdk1 trap protein forming the secretory trap.

Detailed Description of Preferred Embodiments [0022] The present disclosure provides novel compositions, systems, and methods to measure the secretion of a peptide or protein of interest at a single-cell level. In a preferred embodiment, a secretory-trap (secretrap) system measures hormone release of a hormone-fluorescent protein (HFP) fusion and its subsequent sequestration to a secretory trap on the surface of a secreting cell by providing a locally defined fluorescent signal that accumulates at the secretory trap overtime to facilitate measurement of the locally defined fluorescent signal. After sequestration, accumulated HPP signal may be selectively released from the secretory trap through use of an optogenetic switching tool that provides for selectively dissociating the HPF from the secretory trap by selectively conveying light to the optogenetic tool. In some embodiments, the compositions, systems, and methods herein are useful in studying hormone secretion from endocrine cells. In other embodiments, they are useful for studying release of neuropeptides and methods of neuromodulation. In other embodiments, they are useful for collecting information about individual cell behavior rather than a population of cells, which is an improvement over traditional bulk detection methods, such as quantification of hormone release by ELISA techniques.

[0023] A first aspect provides a set of one or more trap proteins. In a preferred embodiment, each trap protein in the set of trap proteins comprises, a binding- partner having a conformation-dependent affinity for an optogenetic tool; a first fluorescent-label (FL1) configured to, upon excitation, convey a set of one more fluorescent reference F_ref signals; and an outer-leaflet anchor to anchor the trap protein to an outer leaflet of a plasma membrane of a cell, whereby, upon secretion of the set of trap proteins by the cell, secreted trap proteins accumulate at the outer leaflet of the plasma membrane to form a set of one or more secretory traps. In another embodiment, the present disclosure provides an isolated polynucleotide encoding a trap protein comprising a sequence at least 90% identical to SEQ ID NO: 1.

[0024] In a preferred embodiment, the binding partner has a conformation- dependent affinity for an optogenetic tool in which the conformation-dependent affinity ranges between tightly binding (e.g., K_D < 100 nM) and readily releasing (e.g., KD > 1 mM) the binding partner to facilitate, in one conformation, selectively accumulating binding-partner/optogenetic-tool complexes at the set of secretory traps, and in another conformation rapidly releasing the binding partner from the set of secretory traps. One having skill in the art will understand that embodiments of the binding-partner should have a conformation-dependent affinity of sufficient range between functional conformations to facilitate, accordingly, reliable accumulation and release of binding-partner/optogenetic-tool complexes.

[0025] In some embodiments, each binding-partner in the set comprises a Zdark (Zdk1) domain as provided by Wang et al. (2016). In a preferred embodiment, the Zdk1 is a short Zdk1 peptide that reversibly heterodimerizes a light-oxygen-voltage-2 (LOV2). Lov2 is a photosensor domain from Avena sativa phototropin 1.

[0026] In a preferred embodiment, the FL1 is configured to, upon excitation, convey a set of one more fluorescent reference F_ref signals. In a preferred embodiment, the FL1 is configured to, upon excitation, convey light that is about 550 nm or greater to avoid non-specific switching the optogenetic tool to a lit conformation. In some embodiments, the FL1 is configured to convey light that is about 490 nm or greater. In some embodiments, each FL1 in the set of trap proteins comprises a fluorescent protein or a fluorescently labeled protein. In some embodiments, each FL1 in the set of trap proteins comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

[0027] In a preferred embodiment, each outer-leaflet anchor in the set comprises at least one domain selected from a glycosylphosphatidylinositol (GPI) domain or a murine-macrophage-inflammatory-protein-1 (Ml P1 ) domain. GPI-anchored peptides and proteins are known in the art to reside substantially exclusively at the outer leaflet of the plasma membrane and expression of GPI-anchored peptides to the plasma membrane has been successful (Hiscox et. al., Biochem Biophys Res Commun, (2002)). MIP1s are known to bind to a common seven-transmembrane domain receptor.

[0028] In some embodiments, any of the sets of trap proteins of aspects 1 to 12 is a recombinant protein expressed by a cell.

[0029] A thirteenth aspect provides a set of one or more reporter proteins for measuring cellular secretion of a protein-of-interest from a plasma membrane of a cell. In a preferred embodiment, each reporter protein in the set of reporter proteins comprises: a protein-of-interest (POI) component comprising the protein-of-interest, a second fluorescent-label (FL2) configured to, upon excitation, convey a set of one or more fluorescent quantification (F_qnt) signals, and an optogenetic tool configured to, in a dark conformation, reversibly heterodimerize with a binding partner, and, in a lit conformation, photo-release the binding partner. In another embodiment, the present disclosure provides an isolated polynucleotide encoding a reporter protein comprising an sequence at least 90% identical to a sequence selected from SEQ ID NOs: 2-3.

[0030] In some embodiments, each POI component comprises a protein-of- interest having at least one amino acid sequence having at least about 90% sequence identity to at least one sequence selected from an insulin sequence, a glucagon sequence, a glucagon-like sequence, a leptin sequence, a neuropeptide sequence, and a hormone sequence. In some embodiments, the protein of interest is at least one protein selected from an insulin protein, a glucagon protein, a glucagon like protein, a leptin protein, a neuropeptide protein, and a hormone protein.

[0031] In some embodiments, the protein-of-interest comprises at least one protein selected from an insulin protein, a glucagon, a glucagon-like protein, a leptin protein, a neuropeptide protein, or a hormone protein. In some embodiments, the insulin protein may include insulin fusion proteins, insulin analog proteins, insulin (A- and B-peptide), proinsulin, and C-peptide. In some embodiments, the glucagon protein may include glucagon fusion proteins, glucagon-like fusion proteins, and glucagon analogs.

[0032] In some embodiments the neuropeptide protein may include CGRP family neuropetides, such as CGRP (alpha, beta), calcitonin, amylin, and adrenomedullin (AM1 , AM2). In some embodiments the neuropeptide protein may include glucagon/secretin family neuropeptides, such as PACAP, VIP, glucagon, secretin, GHRH, and GIP. In some embodiments the neuropeptide protein may include vasopressin/oxytocin. In some embodiments the neuropeptide protein may include tachykinins, such as Sub P, neurokinin a, neuropeptide K, and neuropeptide gamma. In some embodiments the neuropeptide protein may include Tensins, such as angiotensin, neurotensin, and bradykinin. In some embodiments the neuropeptide protein may include CRH-related neuropeptides, such as CRH, urocortins, urotensins. In some embodiments the neuropeptide protein may include F-amides and Y-amides, such as NPY, PPY, and NPFF. In some embodiments the neuropeptide protein may include adipose neuropeptides, such as leptin, adiponectin, and resistins. In some embodiments the neuropeptide protein may include opiods, such as enkephalins, dynorphin, endorphins, and nociception. In some embodiments the neuropeptide protein may include somatostatin/cortistatin.

In some embodiments the neuropeptide protein may include natriuretic factors, such as ANF, BNF, and CNP. In some embodiments the neuropeptide protein may include GRP and neuromedins. In some embodiments the neuropeptide protein may include endothelins. In some embodiments the neuropeptide protein may include CCG/gastrin. In some embodiments the neuropeptide protein may include insulins, such as insulin (A-and B-peptide), IGFs, and relaxins. In some embodiments the neuropeptide protein may include motelini/ghrelin. In some embodiments the neuropeptide protein may include galanins. In some embodiments the neuropeptide protein may include gonadotropin releasing hormones. In some embodiments the neuropeptide protein may include neuropeptide B/W/S. In some embodiments the neuropeptide protein may include neurexophilins. In some embodiments the neuropeptide protein may include cerebellins. In some embodiments the neuropeptide protein may include granins, such as chromogranins, and secretogranins. In some embodiments the neuropeptide protein may include family less neuropeptides such as orexins, MCH, TRH, PTHrP, CART, AGRP, prolactin, diazepam-binding inhibitor peptide, and kisspeptins.

[0033] In some embodiments, the hormone protein may include hormones of the hypothalamus, including anti-diuretic hormone (ADH), corticotropin-releasing hormone (CRH), Growth hormone-releasing hormone (GHRH) or growth hormone- inhibiting hormone (GHIH), Gonadotropin-releasing hormone (GnRH), oxytocin, thyrotropin releasing hormone (TRH), and prolactin-releasing hormone (PRH). In some embodiments, the hormone protein may include pineal gland hormones, including Melatonin. In some embodiments, the hormone protein may include Pituitary (RFP) Hormones, including Adrenocorticotropic hormone (ACTH), Follicle- stimulating hormone (FSH), Growth hormone (GH), Luteinizing hormone (LH), Prolactin, Thyroid-stimulating hormone (TSH), Anti-diuretic hormone (ADH), and Oxytocin. In some embodiments, the hormone protein may include Thyroid hormones, including triodothyronine (T3) and thyroxine (T4). In some embodiments, the hormone protein may include Parathyroid hormone. In some embodiments, the hormone protein may include Thymosin. In some embodiments, the hormone protein may include Adrenal (RFP) hormones, including hydrocortisone, corticosterone, epinenphrine, and norepinephrine. In some embodiments, the hormone protein may include Pancreatic hormones, including gastrin, glucagon, insulin, somatostatin, and vasoactive intestinal peptide (VIP). In some embodiments, the hormone protein may include estrogen and progesterone. In some embodiments, the hormone protein may include testosterone.

[0034] In some embodiments, each FL2 in the set of reporter proteins comprises a fluorescent protein or a fluorescently labeled protein. In a preferred embodiment, each FL2 in the set of reporter proteins is configured to, upon excitation, convey light that is about 550 nm or greater to avoid non-specific switching the optogenetic tool to a lit conformation. In some embodiments, each FL2 in the set of reporter proteins is configured to convey light that is about 490 nm or greater. In some embodiments, each FL2 in the set of reporter proteins comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label. [0035] In a preferred embodiment, the optogenetic tool is configured to, in the dark conformation, tightly bind (e.g., KD < 100 nM) a corresponding binding partner to facilitate reliable sequestration of optogenetic tool/binding partner complexes and thereby facilitate reliable accumulation of F_qnt signals in a secretory trap at the surface of a cell and thereby provide fluorescent signals that are locally defined to the surface of the cell. In the lit conformation, the optogenetic tool preferably readily releases (e.g., KD > 1 mM) the binding partner to facilitate reliable release of dissociated optogenetic tool/binding partner complexes and provide for selectively removing F_ref signal from the secretory trap. One having skill in the art would understand that the terms “tightly bind” and “readily release” are approximations of the optogenetic tools functional conformations. One having skill in the art would also understand that an effective optogenetic tool should have an affinity for the binding partner of sufficient range between the dark and lit conformations to facilitate, accordingly, reliable accumulation and release of binding-partner/optogenetic-tool complexes through reversible heterodimerization. For example, LOV2 domains, as provided herein, have dark and lit conformation affinities for Zdk1 of, respectively, about 26 nM, and about 4 mM. In some embodiments, an optogenetic tool may have dark and lit conformation affinities for the binding partner that constitute about a 200- fold to about a 100-fold change in KD. In some embodiments, an optogenetic tool may have dark and lit conformation affinities for the binding partner that constitute about a 100-fold to about a 10-fold change in KD. In some embodiments, an optogenetic tool may have dark and lit conformation affinities for the binding partner that constitute about a 10-fold to 2-fold change in KD. In some embodiments, an optogenetic tool may have dark and lit conformation affinities for the binding partner of, respectively, about KD < 100 nM about KD > 1 pM.

[0036] In a preferred embodiment, the optogenetic tool is configured to selectively conformationally switch from the dark conformation to the lit conformation when light is conveyed to the optogenetic tool. In some embodiments, each optogenetic tool in the set of reporter proteins comprises a light-oxygen-voltage (LOV) domain or a light- oxygen-voltage-2 (LOV2) domain. In a preferred embodiment, light having a wavelength of about 490 nm is conveyed to LOV2 domain to switch it to the lit conformation. In some embodiments, the optogenetic tool is configured to selectively conformationally switch when light having a wavelength of about 450 nm to about 550 nm is conveyed to the optogenetic tool. [0037] In a preferred embodiment, the optogenetic tool is switched to the lit conformation by conveying light for about 1 second. In some embodiments, the optogenetic tool is switched to the lit conformation by conveying light for about 1 to about 10 seconds.

[0038] In some embodiments, each LOV domain or LOV2 domain in the set of reporter proteins further comprises a third fluorescent label (FL3) to convey, upon excitation, a set of one or more FL3 signals to facilitate accumulating additional F_ref signals relative to the F_qnt signals to facilitate detection of the F_ref signals. In some embodiments, each FL3 in the set of reporter proteins comprises a fluorescent protein or a fluorescently labeled protein. In some embodiments, each FL3 in the set of reporter proteins comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

[0039] In some embodiments, each reporter protein in the set of reporter proteins comprises and does not comprise an optogenetic tool. In some embodiments, each in the set of reporter proteins comprises a Zdk1 domain. In some embodiments, secretion of reporter proteins comprising Zdkls may be enhanced because the smaller size of the Zdk1 domain, relative to LOV or LOV2 domains may facilitate secretion.

[0040] In some embodiments, each reporter protein in the set of secretion is a recombinant protein expressed by a cell.

[0041] In a preferred embodiment, any of the trap proteins of aspects 1 to 12 and any of the reporter proteins of aspects 13 to 27 may be generated using genetically encoded fluorescent constructs (as provided in the non-limiting examples of Brumbaugh et al., J Am Chem Soc, (2006), Bulusu et al., Dev Cell, (2017), Piljic et al., ACS Chem Biol, (2011), and Bolbat, A. and Shultz, C., Biol Cell, (2017)). In some embodiments, any of the trap proteins of aspects 1 to 12 and any of the reporter proteins of aspects 13 to 27 may be hormone fusion proteins (HFPs). In some embodiments, the HFPs may be generated using genetically encoded fluorescent (HFP) constructs that are transfected to a cell for subsequent expression and secretion the HFPs.

[0042] In a preferred embodiment, HFP constructs for generating any one of the trap proteins of aspects 1 to 12 may include a meGFP-GPI-zdk1 construct, a mlP1- mPlum-zdk1 construct, a mlP1-mPlum-zdk1-LOV2 construct, a GPI-GFP-zdk1 construct, or a zdk1-GFP-GPI, and constructs for generating any one of the reporter proteins of any of aspects 13 to 27 may include a mScarlet-C-peptide-LOV2 construct, a mScarlet-glucagon-LOV2 construct, a mScarlet-insulin-C-peptide-LOV2 construct, an insulin-C-peptide-mScarlet-LOV2 construct, or a mScarlet-C-peptide- Zdk1 construct.

[0043] In some embodiments, the encoded fluorescent constructs are bicistronic to facilitate simultaneous, or near simultaneous, co-expression of any one of the set of trap proteins of aspects 1 to 12 and any one of the set of reporter proteins of aspects 13 to 27. In some embodiments, the bicistronic constructs may encode a self-cleaving peptide. In some embodiments, the bicistronic constructs may encode a self-cleaving peptide located between a first cistron encoding the trap protein and a second cistron encoding the reporter protein. In some embodiments, the self cleaving peptide is a F2A-T2A peptide. In some embodiments, the self-cleaving peptide is an IRES. In some embodiments, the IRES is a tunable IRES (Koh et al., PLoS One, (2013)).

[0044] It is known in the art from the crystal structure of the LOV2 domain disclosed by Wang et al. that Zdk1 binds to its C-terminal a-helix. Thus, one having skill would understand that an effective trap protein of any of aspects 1 to 12 and an effective reporter protein of any of aspects 13 to 27 would require the LOV2 domain be operatively connected to the C-terminus of a respective construct.

[0045] One having skill in the art will understand that the present disclosure provides systems, compositions, and methods that may be extrapolated to other applications. For example, one having skill in the art will understand how to make HFP constructs comprising proteins-of-interest that are analogous in size and chemical composition the proteins-of-interest disclosed herein.

[0046] A twenty eighth aspect provides a secretion trap (secretrap) system for measuring cellular secretion of a protein-of-interest of any of aspects 1 to 43. In a preferred embodiment, the system comprises a set of trap proteins of any of aspects 1 to 12; a set of reporter proteins of any of aspects 8 to 27; a set of one or more cells, each cell in the set of cells configured to secrete at least one protein selected from the set of trap proteins and the set of reporter proteins, whereby, upon secretion: the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membranes to form a set of one or more secretory traps, and the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps, and in the lit conformation photo-release the of sequestered reporter proteins (or optionally sequestered trap proteins) to facilitate release of the sequestered reporter proteins from the set of secretory traps; an illumination system configured to: (a) selectively convey light to the set of secretory traps to selectively switch optogenetic tools between the dark and lit conformations, and (b) selectively excite: FL1s of the set of trap proteins, FL2s of the set of reporter proteins, (or optionally FL3s in the set of trap proteins or the set of reporter proteins); an optical detection system for detecting the sets of F_ref signals and the sets of F_qnt signals conveyed, respectively, from the set of secretory-trap proteins and the set of the reporter proteins; and a processor coupled to the optical detection system and configured to analyze the sets of F_ref signals and the sets of F_qnt signals to determine the quantity of the POI component secreted by the cell.

[0047] In some embodiments, any of the systems of any of aspect 28 to 37 may provide a secretrap system for measuring cellular secretion of a protein-of-interest of any of aspects 1 to 43.

[0048] A thirty-eighth aspect provides a method for measuring cellular secretion of a protein of interest of any of aspects 1 to 43. In a preferred embodiment, the method comprises: (a) expressing in a set of one or more cells, each cell in the set of cells having a plasma membrane with an outer leaflet, a set of one or more trap proteins of any of claims 1 to 7 whereby, upon secretion by the set of cells, the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membrane to form a set of one or more secretory traps; (b) expressing in the set of cells, a set of one or more reporter proteins of any of claims 8 to 18 whereby, upon secretion, the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps; (c) selectively conveying, during a time period, light to the cell to excite the FL1s (or optionally the FL3s) of the set of secretory traps to convey a set of time-resolvable F_ref signals; (d) detecting the set of time-resolvable F_ref signals; (e) selectively conveying light to the set of secretory traps to selectively switch the optogenetic tools of the secreting trap proteins (or optionally the secreting reporter proteins) to the lit conformation to facilitate release of the sequestered reporter proteins from the set of secretory traps; (f) selectively conveying, during the time period, light to the set of secretory traps to excite the FL2s (or optionally the FL3) of the set of secretory traps to convey a set of time-resolvable F_qnt signals; (g) detecting the set of time- resolvable Fq_nt signals; (h) (optionally) repeating steps (c) through (g); (i) (optionally) after step (c), selectively conveying, light to the set of secretory traps to excite the FL1 s (or optionally the FL3s) in the set of molecular traps to convey a set of one or more F_ref baseline signals, detecting the set of F_ref baseline signals, and calculating, from the set of F_ref signals, a F_ref baseline; (j) (optionally) after step (c), selectively conveying, light to the cell to excite the FL2s (or optionally the FL3s) of the set of molecular traps to convey a set of one or more F_qnt baseline signals, detecting the set of F_qnt baseline signals, and calculating, from the set of F_qnt signals, a F_qnt baseline; and (k) (optionally) stimulating the cell to secrete one or more trap proteins or one or more reporter proteins.

[0049] In some embodiments, any of the methods of aspects 39 to 43 may provide for measuring cellular secretion of a protein-of-interest of any of aspects 1 to 43.

[0050] In a preferred embodiment, a difference in fluorescence (F) is calculated by measuring the difference in any of the F_ref signals of aspects of 1 to 43 or any of the F_qnt signals of any of aspects 1 to 43 between a first time period and second time period.

[0051] In a preferred embodiment, a F_min value is calculated by measuring the F in any of the F_ref signals of aspects of 1 to 43 or any of the F_qnt signals of any of aspects 1 to 43 after photo-releasing reporter proteins to dissociate them from a secretory trap. As used herein, the terms “F_min value” and “F_ref baseline” indicate the same value and used interchangeably. [0052] In a preferred embodiment, a F_max value is calculated by measuring, measuring the F of any of the F_ref signals of aspects of 1 to 43 or any of the F_qnt signals of any of aspects 1 to 43 at an end time point.

[0053] In a preferred embodiment, a secretion value is calculated by the formula: secretion value = (F - F_min value)/( F_max value - F).

[0054] In a preferred embodiment, any of the F_ref signals of aspects of 1 to 43 or any of the F_qnt signals of any of aspects 1 to 43 may be normalized to the fluorescence of any one of the secretory traps of aspects 1 to 43. In some embodiments, a 3-fold accumulation of fluorescent signal is sufficient to provide an effective secretory trap.

[0055] In a preferred embodiment, the illuminations system of any of aspects 28 to 37 and any of the methods of aspects 38 to 43, is a confocal microscope, such as Olympus FV1200 dual scanner confocal microscope. One having skill in the art will know that one scanner of a Olympus FV1200 dual scanner confocal microscope may be used to convey light to switch any of the optogenetic tools of any of aspects 1 to 43 to the lit confirmation.

EXAMPLES

[0056] The following examples are for illustration only. In light of this disclosure, those of skill in the art will recognize that variations of these examples and other embodiments of the disclosed subject matter are enabled without undue experimentation.

Example 1 - Monitoring Glucagon Secretion from Single Cells

[0057] Glucagon is a critical hormone secreted from a-cells of the endocrine pancreas in response to hypoglycemia. Despite being subject to study for several decades, the mechanisms controlling glucagon secretion from pancreatic a-cells are not completely understood. Glucagon is derived from the prohormone proglucagon which is cleaved in a tissue-specific manner to produce several hormone products. [0058] FIGS. 1A and 1 B each provide schematics of, respectively, proglucagon and a fluorescent-protein (FP)-tagged proglucagon construct. As shown in FIG. 1A, the prohormone proglucagon is cleaved by Pcsk2 and Pcskl in a tissue-specific manner to produce several hormone products. As shown in FIG. 1 B, (i) a FP-tagged glucagon vector construct encoding a MScarlet molecule (SEQ ID NO. 2), and (ii) a FP-tagged glucagon vector construct encoding a GFP molecule (SEQ ID NO. 3) were provided as reporter protein. As shown in FIG. 1 B, processing of proglucagon featuring an mScarlet-proglucagon-LOV2 construct in addition to the flanking cleavage sites and sequences generated a glucagon-like peptide (GLP1).

[0059] FIG. 2 provides an illustration showing a generalized and non-limiting representation of a reporter protein self-cleaving prior to secretion and subsequent sequestration by an optogenetic tool that switches to a lit conformation to dissociate the dissociate the reporter protein and thereby release it from the secretory trap. As shown in FIG. 2, a reporter protein (a) self-cleaves prior to secretion (b) through a plasma membrane of a cell where it (c) subsequently reversibly heterodimerizes with an optogenetic tool formed by a set of trap proteins in a dark conformation where, upon light being conveyed to an optogenetic tool, (d) the optogenetic tool then switches to a lit conformation to dissociate the complex and thereby release the reporter protein.

[0060] FIGS. 3 provides an illustration showing: (a) a trap protein reversibly heterodimerized to a reporter protein when the optogenetic tool is in a dark conformation to form a trap protein/reporter protein complex, and (b) a reporter protein dissociating from the complex when the optogenetic tool is in a lit conformation. Photo-release of the reporter protein provide for removal of residual Zdk1 to remove accumulated reporter proteins and thereby reduce the saturation of fluorescent signal that had in a secretory trap.

[0061] FIGS 4A and 4B are illustrations showing generalized and non-limiting examples of a set of reporter proteins being selectively released from a secretory trap; Fig. 4A shows (i) a set of reporter proteins accumulating in the secretory trap, creating a saturation of signal, which are then (ii) released by selectively conveying 490 nm light to the secretory trap, and Fig. 4B shows (iii) an substantially empty secretory trap and (iv) the set of reporter protein reaccumulating in the trap.

[0062] FIG. 5 provides a graph representing a time-course experiment showing first, a saturation of fluorescent signal that is reduced after release of reporter protein from a secretory trap and the re-accumulation of fluorescent signal after application of a stimulus. A vector construct encoding a trap protein (SEQ ID NO: 1) was provided and expressed in aTC1c9 cells to form a secretory trap at the surface of the cells. Ex-intergranule proteolysis and subsequent secretion produced a glucagon derivative was instantaneously trapped at the plasma membrane by of GPI-anchored Zdk-mPlum protein encoded by. Excitation with 490 nm light released the fluorescently tagged glucagon for further analysis by changing the conformation of the LOV2 domain.

[0063] FIG. 6 shows exocytosing FP-gcg-Lov2 fusion protein bound to Zdk1 at the plasma membrane of aTC1 c9 cells that to form a secretory trap.

[0064] FIG. 7 shows glucagon-bearing reporter proteins (encoded by the vector constructs of SEQ ID NO: 2 and SEQ ID NO: 3) secreting at the plasma membrane of aTC1c9 cells. Glucagon secretioin was measured by quantifying F_min and F_ref signals.

Example 2 - GPI-GFP-Zdk1 Forming Secretory Traps on The Surface of

HeLA Cells

[0065] FIGS. 8A, 8B, and 8C, show GPI-GFP-Zdk1 expressed and secreted in HeLa cells to demonstrate that the functionality of a secretrap system on the surface of intact cells. As shown in FIG. 8A, HeLa cells expressed prototype GPI-GFP-Zdk1 as an outer membrane-bound anchor. Notably, FIG.8A shows additional fluorescence the trafficking compartment, mainly the Golgi. As shown in FIG. 8B), addition of 200 nM recombinant GFP-LOV2 in HEPES buffer (pH 7.4) significantly increased the fluorescent signal at the plasma membrane, but not in Golgi membranes in the cell’s interior, indicating strong binding of the LOV2 domain to the Zdk1 peptide at the outer membrane. As shown in FIG. 8C., intensity subtraction of the cell images in FIGS. 8A and 8B confirmed a strong increase in plasma membrane fluorescence and little change in the internal membranes. As shown in FIG. 8D, a drop of fluorescence was detected after illumination of the Zdk1-LOV2 complex at the plasma membrane.

[0066] FIGS. 9A and 9B are each graphs of normalized fluorescence over time showing a drop of fluorescence after illumination of Zdk1-LOV2 complexed to the GPI-GPF-Zdk1 trap protein forming the secretory trap.

Example 3 - Insulin and/or Glucagon Secretrap System

[0067] Any of the systems of aspects 28 to 37 or any of the methods of aspects 38 to 43 may be used to provide an insulin and/or glucagon secretrap system. Here, the trapping principle of the secretrap systems will be used to solve two major problems when measuring hormone peptide secretion: 1) to overcome the rapid dissolution of the secreted peptide after cellular release, which prevents measurements at a single cell level; 2) light-induced release will empty the trap from randomly released peptide fusions at the beginning of the experiment.

[0068] A glucagon, glucagon-like, or insuslin peptide-of-interest will be modified by the addition of a fluorescent protein as well as a LOV2 domain that will recognize a short Zdk1 peptide with an affinity of 26 nM. Zdk1 will be tethered to a GPI anchor at the outer membrane of a cell of interest and tagged with mPlum for controlling expression levels. The high affinity of the LOV2-Zdk1 peptide interaction will ensure that the fluorescently tagged peptide will be readily trapped at the plasma membrane. The fluorescence accumulation will be normalized to the mPlum fluorescence of the “trap”. The mPlum fluorescence will also serve as a mask for determining membrane pixel. All other fluorescence will be discarded. The quantification will be performed using a FIJI macro FluoQ, may be used to distinguish between fluorescence in the cell body and the plasma membrane (Stein et al. , (2013), Acs Chemical Biology). Alternatively, TIRF microscopy may be used. Illumination with 490 nm light will change the affinity of the LOV2 domain forZdkl to over 4 mM, thereby releasing any peptide bound to the anchor on the plasma membrane.

[0069] Molecular Biology. Initially, a GPI-CFP-Zdk1 construct (the “Trap”) will be built as well as a fluorophore-insulin (A-and B-peptide)-LOV2 construct and a fluorophore-proglucagon-LOV2 construct. The “trap” will be transfected into the a- cell line aTC1c9 or MIN6 b-cells, available in our lab. We will check for proper plasma membrane location of the fluorophore-insulin (A-and B-peptide)-LOV2 construct and fluorophore-proglucagon-LOV2 construct by confocal microscopy. An appropriate construct bearing the GPI signaling peptide is available in provided herein as SEQ ID NO. 1. The LOV2 domain is available commercially (E.g., Addgene.org or Addgene Headquarters, 490 Arsenal Way, Suite 100, Watertown, MA 02471 , USA).

[0070] The fluorophore-proglucagon-LOV2 construct and the fluorophore-insulin (A-and B-peptide)-LOV2 construct will be expressed in aTC1c9 cells and MIN6 cells, respectively. It is known in the art that fluorophore-proglucagon fusions are exclusively located in granules and insulin fusions are readily secreted with endogenous insulin (Schifferer et al., Cell Chem Biol (2017)). Alternatively, shift to alternative constructs such as a proglucagon-fluorophore-LOV2 may be used. If location problems occur, one having skill in the art will understand that the routine addition one or more linker sequences (E.g., a set of one or more flexible glycine linkers) for increased flexibility between domains. If none of these measures produces a properly located and well secreting construct, reducing the size of any Zdk1 domains of any of aspects 1 to 43 may be used, replacing the LOV2 domain.

In this case, the “trap” will be equipped with the fluorescent protein and the LOV2 domain. One having skill in the art will understand that the risk of failure due to a lack of processing is small because the GPI-anchored “trap” does not require processing and will express well on the surface of b-cells.

[0071] Probe Validation. Secretion of the hormone fusion proteins (HFPs) will be determined by live cell imaging after stimulation of aTC1c9 or MIN6 cells with phorbol ester (Ono et al., Diabetes Res Clin Pract (1986)), KCI (Gromada et al., Diabetes (2004)), glycine (Li et al., J Biol Chem (2013), or by reducing or elevating glucose levels, respectively. Secretion levels of the HFPs and endogenous glucagon or insulin and the glucagon or insulin-LOV2 fusion quantified by an ELISA will be correlated. Secretion levels will be compared by Westerns using anti- fluorescent protein and anti-glucagon or anti-insulin antibodies. This will tell of how well the SecreTrap system reflects on the release of endogenous glucagon or insulin, respectively. The trapped hormone will be further released by activating the optogenetic LOV2 domain with 490 nm light (for 1 , 5, or 10 sec), after which the supernatant will be collected to perform Western blots to validate the “trap” system and to quantify the amount secreted. The difference in fluorescence (F) after releasing the trapped protein optogenetically will provide the F_min value, addition of an access of LOV2-fluorescent protein at the end of the experiment (not shown) will provide F_max for quantification. Hormone secretion will be determined by [hormone] = F - Fmin / Fmax - F. The fluorescence values will be normalized to the fluorescence of the “trap” construct. The number of “trap” molecules on the cell surface” will be estimated by titrating in recombinant mScarlet-LOV2. A 3-fold accumulation at the plasma membrane will be considered a success but one having skill in the art will expect much higher trapping efficiencies, at least until all Zdk1 binding sites are saturated. [0072] Currently, it is difficult to predict how much change in plasma membrane fluorescence vs intracellular fluorescence will be detectable in confocal imaging experiments as usually only a fraction of the peptide hormone is secreted after a given stimulus. One having skill in the art would know that the trapping mechanism will help to significantly increase the contrast between plasma membrane and cytosol, especially as the signal can be normalized to the mPlum fluorescence on the plasma membrane. In addition, employing total internal reflection microscopy (TIRF) which will permit monitoring fluorescence build up at the plasma membrane. To boost the output signal, the optogenetic release of all accumulated fluorescent fusions by a flash of 490 nm light at the end of the experiment will provide a large quantifiable signal (see FIG. 5 for a t. This should be clearly detectable by TIRF as well as by regular confocal microscopy.

[0073] Any of the systems of aspects 28 to 37 and any of the methods of aspects 38 to 43 may be used to report on glucagon release in correlative fashion between endogenous hormone and the artificially added fusion. Therefore, the release of the fusion constructs versus glucagon release from cell batches will correlated by Western blot. The outcome will elucidate how much more or less efficient the artificially added proteins are secreted.

[0074] Probe Application and Data Analysis. aTC1c9 or MIN6 cells will initially be treated with buffers containing various glucose concentrations and monitor glucagon secretion by confocal microscopy. All imaging experiments will be performed at least in biological triplicates with recordings from >10 cells per dish. Biochemical experiments (Western) will be performed in triplicate.

[0075] aTC1c9 cells kept at high glucose levels will be stimulated by changing the media to a low glucose concentration (3 mM) or by adding phorbol ester (PMA, 500 nM), 1 .2 mM glycine, or 30 mM KCI, known stimuli of glucagon secretion. The time courses of glucagon release will then be monitored. It has been shown that cultured and primary b-cells stop calcium signaling and insulin secretion when washed (Hauke et al., Diabetes , (2018)), but this has not been shown for a-cells. Therefore, after washing aTC1c9 cells extensively with low or high glucose buffers, respectively, glucagon secretion will be monitored and then supernatant from another batch of a- cells and monitor glucagon secretion will be added in response. If a- and b-cells regulate their respective secretion by similar mechanisms, the removal of extracellular signaling molecules will stop glucagon secretion and the adding back such factors will start the secretion again (Hauke et al., Diabetes , (2018)).

[0076] It is well established that a- and b-cells are not electrically connected, i.e. via gap junctions (Hughes, et al., Diabetes Obes Metab, (2018)). Therefore, following the hypothesis that insulin and other signaling molecules secreted from b- cells influence glucagon secretion, aTC1c9 cells will again be washes and supernatants of cultured MIN6 or INS1 b-cells will be added. Similarly, single components known to be released from b-cells including fatty acids, ATP and neuropeptide Y will be tested (Hauke et al., Diabetes , (2018), and Brothers et al., EMBO Mol Med , (2010)). As these agonists act via G-protein coupled receptors, suitable receptor antagonists will be used to determine which signaling pathway was triggered.

[0077] A FIJI macro FluoQ, may be used to distinguish between fluorescence in the cell body and the plasma membrane (Stein et al., (2013), Acs Chemical Biology). Alternatively, TIRF microscopy may be used. In brief, direct excitation of mPlum on the plasma membrane will be used to identify pixels classified as “membrane” in which we will measure an increase of fluorescence in the mScarlet channel. The time course of the experiments should permit monitoring of pulsatile secretion. For this, the first derivative of the fluorescence increase over time will be plotted.

[0078] These experiments are will demonstrate that use of any of aspects 1 to 43 may elucidate how glucagon and insulin secretion is regulated and what role the interplay with b-cells plays.

DEFINITIONS

[0079] The term "affinity", as used herein, refers to the degree to which a binding domain, in particular a protein, such as a membrane protein, binds to a ligand so as to shift the equilibrium of ligand and protein binding domain toward the presence of a complex formed by their binding. Thus, for example, where an optogenetic tool and a binding partner are combined in relatively equal concentration, an optogenetic tool of high affinity will bind to the available binding partner so as to shift the equilibrium toward high concentration of the resulting complex. The dissociation constant (K_D) is commonly used to describe the affinity between the protein binding domain and the ligand. In some instances, the dissociation constant is reported as a KD value, (e.g., “10⁴ to 10⁶,” or “10⁷ to 10⁹” ) or as Molar concentration (sensitivity) (e.g., “Micromolar (mM)”, or “Nanomolar (nM)”).

[0080] The term "conformation" or "conformational state" of a protein refers generally to the range of structures that a protein may adopt at any instant in time. One of skill in the art will recognize that determinants of conformation or conformational state include a protein's primary structure as reflected in a protein's amino acid sequence (including modified amino acids) and the environment surrounding the protein. The conformation or conformational state of a protein also relates to structural features such as protein secondary structures (e.g., a-helix, b- sheet, among others), tertiary structure (e.g., the three dimensional folding of a polypeptide chain), and quaternary structure (e.g., interactions of a polypeptide chain with other protein subunits). Post-translational and other modifications to a polypeptide chain such as ligand binding, phosphorylation, sulfation, glycosylation, or attachments of hydrophobic groups, among others, can influence the conformation of a protein. Furthermore, environmental factors, such as pH, salt concentration, ionic strength, and osmolality of the surrounding solution, and interaction with other proteins and co-factors, among others, can affect protein conformation. The conformational state of a protein may be determined by either functional assay for activity or binding to another molecule or by means of physical methods such as X- ray crystallography, NMR, or spin labeling, among other methods. For a general discussion of protein conformation and conformational states, one is referred to Cantor and Schimmel, Biophysical Chemistry, Part I: The Conformation of Biological. Macromolecules, .W.H. Freeman and Company, 1980, and Creighton, Proteins: Structures and Molecular Properties, W.H. Freeman and Company, 1993. A "specific conformational state" is any subset of the range of conformations or conformational states that a protein may adopt.

[0081] In some embodiments provided herein, each claim feature in a set is configured identically (e.g., “A set of proteins, each protein in the set configured to [X]”). In some embodiments provided herein, each object described in the detailed description in a set is configured identically (e.g., “A set of proteins, each protein in the set configured to [X]”). In some embodiments provided herein, substantially all of the claim features in a set are configured identically. In some embodiments provided herein, substantially all of the object described in the detailed description in a set are configured identically (e.g., “A set of proteins, each protein in the set configured to [X]”).

[0082] The terms “substantially” and "substantially all" as used herein are intended to mean greater than 90% of the members or units indicated (for instance 90% to 100%). Typically, “substantially all” is at least 95% of the members or units indicated, more typically at least 99% of the members or units indicated. For instance, in embodiments in which substantially all of the cells in the set of cells is configured to secrete at least one protein selected from the set of trap proteins and the set of secretion proteins, at least 90% of the cells in the set are configured to secrete the at least one protein. In other embodiments, substantially all of the cells in the set would indicate at least 95% of the cells, while in other embodiments, substantially all of the cells in the set would indicate at least 99% of the cells, while in other embodiments.

[0083] The term “dimerization,” as used herein, refers to an addition reaction in which two molecules of the same compound react with each other to give an adduct.”

[0084] The terms “embodiment” and “aspect” used herein each refer to an example, an instance, or an illustration of the present disclosure and may be used interchangeably. In some instances, as described, one may further define, limit, or serve as a subset, subgeneric description, or specific example of the other. In other instances, one embodiment or aspect may provide a comparison to another or a distinction from the other.

[0085] A "functional conformation" or a "functional conformational state", as used herein, refers to the fact that proteins possess different conformational states having a dynamic range of activity, in particular ranging from no activity to maximal activity.

It should be clear that "a functional conformational state" is meant to cover any conformational state of a protein, in particular a membrane protein, having any activity, including no activity; and is not meant to cover the denatured states of proteins.

[0086] The term “heterodimerization,” refers to an addition reaction in which two non-identical compounds react with each other to give an adduct.

[0087] The terms “polypeptide," "protein," and "peptide," are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0088] As used herein, the terms "nucleic acid molecule", "polynucleotide", "polynucleic acid", "nucleic acid" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.

[0089] The term "membrane protein," as used herein, refers to a protein that is attached to or associated with a membrane of a cell or an organelle. Specific non limiting examples are provided further in the specification.

[0090] The term "protein binding domain" or simply "binding domain" refers generally to any non-naturally occurring molecule or part thereof that is able to bind to a protein or peptide using specific intermolecular interactions. A variety of molecules can function as protein binding domains, including, but not limited to, proteinaceous molecules (protein, peptide, protein-like or protein containing), nucleic acid molecules (nucleic acid, nucleic acid-like, nucleic acid containing), and carbohydrate molecules (carbohydrate, carbohydrate-like, carbohydrate containing). A more detailed description can be found further in the specification.

[0091] The term “protein domain,” as used herein, refers generally to a conserved part of a given protein sequence and tertiary structure that can evolve, function, and exist independently of the rest of the protein chain. For example, as disclosed herein, an optogenetic tool comprises a LOV2 domain that may be fused to either a trap protein or a reporter protein and still independently function.

[0092] The term "specificity", as used herein, refers to the ability of a binding domain, in particular protein that is attached to or associated with a membrane of a cell or an organelle to bind preferentially to one binding domain, versus a different binding domain, and does not necessarily imply high affinity.

ASPECTS OF THE DISCLOSURE [0093] A first aspect provided herein provides a set of one or more trap proteins, each trap protein in the set comprising: having a conformation- dependent affinity for an optogenetic tool; a first fluorescent-label (FL1) configured to, upon excitation, convey a set of one more fluorescent reference F_ref signals, and an outer-leaflet anchor to anchor the trap protein to an outer leaflet of a plasma membrane of a cell, whereby, upon secretion of the set of trap proteins by the cell, secreted trap proteins accumulate at the outer leaflet of the plasma membrane to form a set of one or more secretory traps.

[0094] A second aspect provides the set of trap proteins of aspect 1 , in which each in the set of trap proteins comprises a Zdark (Zdk1) domain.

[0095] A third aspect provides the set of trap proteins of aspect 1 , in which each FL1 in the set of trap proteins comprises a fluorescent protein or a fluorescently labeled protein.

[0096] A fourth aspect provides the set of trap proteins of aspect 1 , in which each FL1 in the set comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

[0097] A fifth aspect provides the set of trap proteins of aspect 1 , in which each outer-leaflet anchor in the set comprises at least one domain selected from a glycosylphosphatidylinositol (GPI) domain or a murine-macrophage-inflammatory-1 (Ml P1 ) domain.

[0098] A sixth aspect provides the set of trap proteins of any of aspects 1 to 5, in which each trap protein in the set comprises an optogenetic tool and does not comprise .

[0099] A seventh aspect provides the set of trap proteins of aspect 6, in which each optogenetic tool in the set comprises a light-oxygen-voltage (LOV) domain or a light-oxygen-voltage-2 (LOV2) domain.

[00100] An eighth aspect provides the set of trap proteins of aspect 7, in which each LOV domain or LOV2 domain in the set further comprises a third fluorescent label (FL3) to convey, upon excitation, a set of one or more FL3 signals.

[00101] An ninth aspect provides the set of trap proteins of aspect 8, in which each FL3 in the set comprises at least one label protein selected from a fluorescent protein and a fluorescently labeled protein. [00102] A tenth aspect provides the set of trap proteins of aspect 9, in which the fluorescent protein or the fluorescently labeled protein are fused to the LOV domain or the LOV2 domain.

[00103] A eleventh aspect provides the set of trap proteins of any of aspects, 8, 9, and 10, in which each FL3 in the set comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

[00104] A twelfth aspect provides the set of trap proteins of any of aspects 1 to 10, in which, each trap protein in the set is a recombinant protein expressed by the cell. [00105] A thirteenth aspect herein provides a set of one or more reporter proteins for measuring cellular secretion of a protein-of-interest from a plasma membrane of a cell, each reporter protein in the set comprising: a protein-of-interest (POI) component comprising the protein-of-interest, a second fluorescent-label (FL2) configured to, upon excitation, convey a set of one or more fluorescent quantification (Fqn_t) signals, and an optogenetic tool configured to, in a dark conformation, reversibly heterodimerize with a binding partner, and, in a lit conformation, photo release the binding partner.

[00106] A fourteenth aspect provides the set of reporter proteins of aspect 13, in which each protein-of-interest in the set comprises at least one amino acid sequence selected from an insulin sequence, a glucagon sequence, a leptin sequence, a neuropeptide sequence, and a hormone sequence.

[00107] A fifteenth aspect provides the set of reporter proteins of aspect 13, in which the POI portion is genetically encoded and fused to the FL2 and the optogenetic tool.

[00108] A sixteenth aspect provides the set of reporter proteins of aspect 13, in which each FL2 in the set comprises a fluorescent protein or a fluorescently labeled protein.

[00109] A seventeenth aspect provides the set of reporter proteins of aspect 13, in which each FL2 in the set comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label. [00110] An eighteenth aspect provides the set of reporter proteins of aspect 13, in which each POI portion in the set is genetically encodable and fusible to the FL2 and the optogenetic tool.

[00111] A nineteenth aspect provides the set of reporter proteins of aspect 13, in which each optogenetic tool in the set comprises a light-oxygen-voltage (LOV) domain or a light-oxygen-voltage-2 (LOV2) domain.

[00112] A twentieth aspect provides the set of reporter proteins of aspect 19, in which each LOV domain or LOV2 domain in the set further comprises a third fluorescent label (FL3) to convey, upon excitation, a set of one or more FL3 signals. [00113] A twenty-first aspect provides the set of reporter proteins of aspect 20, in which each FL3 in the set comprises a fluorescent protein or a fluorescently labeled protein.

[00114] A twenty-second aspect provides the set of reporter proteins of aspect 21 , in which each FL3 in the set comprises the fluorescent protein or the fluorescently labeled protein and is fused to the LOV domain or the LOV2 domains.

[00115] A twenty-third aspect provides the set of reporter proteins of any of aspects 20, 21 , and 22, in which each FL3 in the set is configured to, upon excitation, combinedly convey the set of FL3 signals with the set of fluorescent quantification (F_qnt) signals.

[00116] A twenty-fourth aspect provides the set of reporter proteins of any of aspects 20, 21 , 22, and 23, in which each FL3 in the set comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

[00117] A twenty-fifth aspect provides the set of reporter proteins of any of aspects

12 to 23, in which each reporter protein in the set comprises and does not comprise an optogenetic tool.

[00118] A twenty-sixth aspect provides the set of reporter proteins of aspect 25, in which each reporter protein in the set comprises a Zdk1 domain.

[00119] A twenty-seventh aspect provides the set of reporter proteins of aspects

13 to 26, in which each reporter protein in the set is a recombinant protein expressed by the cell.

[00120] A twenty-eighth aspect herein provides a secretion trap (secretrap) system for measuring cellular secretion of a protein-of-interest, the system comprising: a set of trap proteins of any of aspects 1 to 11 ; a set of reporter proteins of any of aspects 11 to 27; a set of one or more cells, each cell in the set of cells configured to secrete at least one protein selected from the set of trap proteins and the set of reporter proteins, whereby, upon secretion: the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membranes to form a set of one or more secretory traps, and the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps, and in the lit conformation photo-release the of sequestered reporter proteins (or optionally sequestered trap proteins) to facilitate release of the sequestered reporter proteins from the set of secretory traps; an illumination system configured to: (a) selectively convey light to the set of secretory traps to selectively switch optogenetic tools between the dark and lit conformations, and (b) selectively excite FL1s in the set of trap proteins and FL2s in the set of reporter proteins; an optical detection system for detecting the sets of F_ref signals and the sets of F_qnt signals conveyed, respectively, from the set of secretory-trap proteins and the set of the reporter proteins; and a processor coupled to the optical detection system and configured to analyze the sets of F_ref signals and the sets of F_qnt signals to determine the quantity of the POI component secreted by the cell.

[00121] A twenty-ninth aspect provides the system of aspect 28, in which the optical detection system is configured to use total internal reflection (TIRF) microscopy.

[00122] A thirtieth aspect provides the system of aspect 28, in which the optical detection system and the processor are configured perform time-resolved fluorescence spectroscopy.

[00123] A thirty-first aspect provides the system of aspect 27, in which the processor is further configured to determine one or more locations of one or more secretory traps.

[00124] A thirty-second aspect provides the system of aspect 27, in which the processor is further configured to detect the set of F_ref signals or the set of F_qnt signals throughout a time period to generate a set of time-resolvable F_ref signals or a set of time-resolvable F_qnt signals.

[00125] A thirty-third aspect provides the system of aspect 27, in which the processor is further configured to calculate, from the set of time-resolvable F_ref signals the set of time-resolvable F_qnt signals, one or more rates of secretion.

[00126] A thirty-fourth aspect provides the system of aspect 27, in which the processor is further configured to calculate from the set of time-resolvable F_ref signals a F_ref minimum and a F_ref maximum.

[00127] A thirty-fifth aspect provides the system of aspect 27, in which the processor is further configured to calculate from the set of time-resolvable F_qnt signals and a F_qnt minimum and a F_qnt maximum.

[00128] A thirty-sixth aspect provides the system of aspect 27, in which each FL1 of any of aspects 1 to 11 and each FL2 of any of aspects claims 12 to 25, further comprise, respectively, first and second excitation ranges and first and second emission ranges in which the first excitation range essentially does not overlap the second excitation range or the first emission range essentially does not overlap the second emission range.

[00129] A thirty-seventh aspect provides the system of aspect 35, in which each FL3 of any of aspects of aspects further comprises a third excitation range and third emission range in which the third excitation range essentially does not overlap the first or second excitation range and the emission range essentially does not overlap the first or second emission range.

[00130] A thirty-eighth aspect herein providing a method for measuring cellular secretion of a protein of interest, the method comprising:

(a) expressing in a set of one or more cells, each cell in the set of cells having a plasma membrane with an outer leaflet, a set of one or more trap proteins of any of aspects 1 to 12 whereby, upon secretion by the set of cells, the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membrane to form a set of one or more secretory traps;

(b) expressing in the set of cells, a set of one or more reporter proteins of any of claims 13 to 27 whereby, upon secretion, the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps;

(c) selectively conveying, during a time period, light to the cell to excite the FL1s (or optionally the FL3s) of the set of secretory traps to convey a set of time-resolvable F_ref signals;

(d) detecting the set of time-resolvable F_ref signals;

(e) selectively conveying light to the set of secretory traps to selectively switch the optogenetic tools of the secreting trap proteins (or optionally the secreting reporter proteins) to the lit conformation to facilitate release of the sequestered reporter proteins from the set of secretory traps

(f) selectively conveying, during the time period, light to the set of secretory traps to excite the FL2s (or optionally the FL3) of the set of secretory traps to convey a set of time-resolvable F_qnt signals;

(g) detecting the set of time-resolvable F_qnt signals;

(h) (optionally) repeating steps (c) through (g);

(i) (optionally) after step (c), selectively conveying, light to the set of secretory traps to excite the FL1s (or optionally the FL3s) in the set of molecular traps to convey a set of one or more F_ref baseline signals, detecting the set of F_ref baseline signals, and calculating, from the set of F_ref signals, a F_ref baseline;

(j) (optionally) after step (c), selectively conveying, light to the cell to excite the FL2s (or optionally the FL3s) of the set of molecular traps to convey a set of one or more F_qnt baseline signals, detecting the set of F_qnt baseline signals, and calculating, from the set of F_qnt signals, a F_qnt baseline; and

(k) (optionally) stimulating the cell to secrete one or more trap proteins or one or more reporter proteins. [00131] A thirty-ninth aspect provides the method of aspect 38, further comprising: (I) calculating, from the set of time-resolvable F_ref signals, a F_ref minimum and a F_ref maximum.

[00132] A fortieth aspect provides the method of aspect 38, further comprising: (n) calculating, from the set of time-resolvable F_qnt signals, a F_qnt minimum and a F_qnt maximum.

[00133] A forty-first aspect provides the method of aspects 38, in which each FL1 , FL2, of any of aspects 38, 39, and 40, further comprise, respectively, first and second excitation ranges and first and second emission ranges in which the first excitation range essentially does not overlap the second excitation range or the first emission range essentially does not overlap the second emission range.

[00134] A forty-second aspect provides the method of aspect 41 , which each FL3 of aspect 41 further comprises a third excitation range and third emission range in which the third excitation range essentially does not overlap the first or second excitation range and the emission range essentially does not overlap the first or second emission range.

[00135] A forty-third aspect provides the method of any of aspects 38 to 42 further comprising the system of any of aspects 28 to 37.

REFERENCES CITED

[00136] All references cited in this disclosure are incorporated by reference in their entirety.

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[00139] Brumbaugh, J., Schleifenbaum, A., Gasch, A., Sattler, M., and Schultz, C. (2006) A dual parameter FRET probe for measuring PKC and PKA activity in living cells. J Am Chem Soc 128, 24-25.

[00140] Bulusu, V., Prior, N., Snaebjornsson, M. T., Kuehne, A., Sonnen, K. F., Kress, J., Stein, F., Schultz, C., Sauer, U., and Aulehla, A. (2017) Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development. Dev Cell 40, 331-341 e334. [00141] Gromada, J., Ma, X., Hoy, M., Bokvist, K., Salehi, A., Berggren, P. O., and Rorsman, P. (2004) ATP-sensitive K+ channel-dependent regulation of glucagon release and electrical activity by glucose in wild-type and SUR1-/- mouse alpha-cells. Diabetes 53 Suppl 3, S181-189.

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[00143] Hughes, J. W., Ustione, A., Lavagnino, Z., and Piston, D. W. (2018) Regulation of islet glucagon secretion: Beyond calcium. Diabetes Obes Metab 20 Suppl 2, 127-136.

[00144] Hiscox, S., Hallett, M. B., Morgan, B. P., and van den Berg, C. W. (2002) GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface. Biochem Biophys Res Commun 293, 714-721.

[00145] Gao, J.L., and Murphy, P.M. (1995) Cloning and Differential Tissue- specifc Expression of Three Mouse b Chemokine Receptor-like Genes, Including the Gene for Funcational Macrophage Inflammatory Protein-1 a Receptor. J. Biol Chem. 270, 17494-17501.

[00146] Li, C., Liu, C., Nissim, I., Chen, J., Chen, P., Doliba, N., Zhang, T., Nissim, I., Daikhin, Y., Stokes, D., Yudkoff, M., Bennett, M. J., Stanley, C. A., Matschinsky,

F. M., and Naji, A. (2013) Regulation of glucagon secretion in normal and diabetic human islets by gamma-hydroxybutyrate and glycine. J Biol Chem 288, 3938-3951. [00147] Ono, J., Kumae, S., Sato, Y., and Takaki, R. (1986) Effect of phorbol esters on glucagon secretion from a glucagon-secreting clonal cell line. Synergistic effects of A23187 and theophylline. Diabetes Res Clin Pract 2, 29-34.

[00148] Piljic, A., de Diego, I., Wilmanns, M., and Schultz, C. (2011) Rapid development of genetically encoded FRET reporters. ACS Chem Biol 6, 685-691. [00149] Schifferer, M., Yushchenko, D. A., Stein, F., Bolbat, A., and Schultz, C. (2017) A Ratiometric Sensor for Imaging Insulin Secretion in Single beta Cells. Cell Chem Biol 24, 525-531 e524.

[00150] Stein, F., Kress, M., Reither, S., Piljic, A., and Schultz, C. (2013) FluoQ: A Tool for Rapid Analysis of Multiparameter Fluorescence Imaging Data Applied to Oscillatory Events. Acs Chemical Biology S, 1862-1868.

[00151] Wang, H., Vilela, M., Winkler, A., Tarnawski, M., Schlichting, I., Yumerefendi, H., Kuhlman, B., Liu, R., Danuser, G., and Hahn, K. M. (2016) LOVTRAP: an optogenetic system for photoinduced protein dissociation. Nat Methods 13, 755-758.

[00152] It will be obvious to those having skill in the art that many changes may be made to the details of the above-described embodiments without departing from the underlying principles of the invention. The scope of the present invention should, therefore, be determined only by the following claims.

Claims

Claims

1. A set of one or more trap proteins, each trap protein in the set comprising: a binding-partner having a conformation-dependent affinity for an optogenetic tool; a first fluorescent-label (FL1) configured to, upon excitation, convey a set of one more fluorescent reference F_ref signals; and an outer-leaflet anchor to anchor the trap protein to an outer leaflet of a plasma membrane of a cell, whereby, upon secretion of the set of trap proteins by the cell, secreted trap proteins accumulate at the outer leaflet of the plasma membrane to form a set of one or more secretory traps.

2. The set of trap proteins of claim 1 , in which each binding-partner in the set comprises a Zdark (Zdk1) domain.

3. The set of trap proteins of claim 1 , in which each FL1 in the set comprises a fluorescent protein or a fluorescently labeled protein.

4. The set of trap proteins of claim 1 , in which each FL1 in the set comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

5. The set of trap proteins of claim 1 , in which each outer-leaflet anchor in the set comprises at least one domain selected from a glycosylphosphatidylinositol (GPI) domain or a murine-macrophage- inflammatory-1 (Ml P1 ) domain.

6. The set of trap proteins of claim 1 , in which each trap protein in the set comprises an optogenetic tool and does not comprise a binding partner.

7. The set of trap proteins of any of claims 1 to 6, in which each trap protein in the set is a recombinant protein expressed by the cell.

8. A set of one or more reporter proteins for measuring cellular secretion of a protein-of-interest from a plasma membrane of a cell, each reporter protein in the set comprising: a protein-of-interest (POI) component comprising the protein-of- interest; a second fluorescent-label (FL2) configured to, upon excitation, convey a set of one or more fluorescent quantification (F_qnt) signal; and an optogenetic tool configured to, in a dark conformation, reversibly heterodimerize with a binding partner, and, in a lit conformation, photo-release the binding partner.

9. The set of reporter proteins of claim 8, in which each protein-of-interest in the set comprises at least one amino acid sequence selected from an insulin sequence, a glucagon sequence, a glucagon-like sequence, a leptin sequence, a neuropeptide sequence, and a hormone sequence.

10. The set of reporter proteins of claim 8, in which each FL2 in the set comprises a fluorescent protein or a fluorescently labeled protein.

11.The set of reporter proteins of claim 8, in which each FL2 in the set comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

12. The set of reporter proteins of claim 8, in which each optogenetic tool in the set comprises a light-oxygen-voltage (LOV) domain or a light-oxygen-voltage- 2 (LOV2) domain.

13. The set of reporter proteins of claim 12, in which each LOV domain or LOV2 domain in the set further comprises a third fluorescent label (FL3) to convey, upon excitation, a set of one or more FL3 signals.

14. The set of reporter proteins of claim 12, in which each FL3 in the set comprises a fluorescent protein or a fluorescently labeled protein.

15. The set of reporter proteins of claim 14, in which each FL3 in the set comprises at least one label selected from a mScarlet label, a mPLum label, a yellow fluorescent protein (YFP) label, a mCherry label, a superfolder GFP (sfGFP) label, a meGFP label, and a Red Fluorescent Protein (RFP) label.

16. The set of reporter proteins of claim 8, in which each reporter protein in the set comprises and does not comprise an optogenetic tool.

17. The set of reporter proteins of claim 16, in which each binding-partner in the set comprises a Zdk1 domain.

18. The set of reporter proteins of claim 8, in which each reporter protein in the set is a recombinant protein expressed by the cell.

19. A secretion trap (secretrap) system for measuring cellular secretion of a protein-of-interest, the system comprising: a set of trap proteins of any of claims 1 to 7; a set of reporter proteins of any of claims 8 to 18; a set of one or more cells, each cell in the set of cells configured to secrete at least one protein selected from the set of trap proteins and the set of reporter proteins, whereby, upon secretion: the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membranes to form a set of one or more secretory traps, and the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps, and in the lit conformation photo-release the binding-partner of sequestered reporter proteins (or optionally sequestered trap proteins) to facilitate release of the sequestered reporter proteins from the set of secretory traps; an illumination system configured to: (a) selectively convey light to the set of secretory traps to selectively switch optogenetic tools between the dark and lit conformations, and (b) selectively excite: FL1s of the set of trap proteins, FL2s of the set of reporter proteins, (or optionally FL3s in the set of trap proteins or the set of reporter proteins); an optical detection system for detecting the sets of F_ref signals and the sets of Fq_nt signals conveyed, respectively, from the set of secretory- trap proteins and the set of the reporter proteins; and a processor coupled to the optical detection system and configured to analyze the sets of F_ref signals and the sets of F_qnt signals to determine the quantity of the POI component secreted by the cell.

20. A method for measuring cellular secretion of a protein of interest, the method comprising:

(a) expressing in a set of one or more cells, each cell in the set of cells having a plasma membrane with an outer leaflet, a set of one or more trap proteins of any of claims 1 to 7 whereby, upon secretion by the set of cells, the outer leaflet anchor of secreting trap proteins anchors the secreting trap proteins at the outer leaflet of secreting plasma membranes and the secreting trap proteins accumulate along the secreting plasma membrane to form a set of one or more secretory traps;

(b) expressing in the set of cells, a set of one or more reporter proteins of any of claims 8 to 18 whereby, upon secretion, the optogenetic tool of secreting trap proteins (or optionally the secreting reporter proteins), in the dark conformation, reversibly heterodimerizes with the binding partner of the secreting reporter proteins (or optionally the secreting trap proteins) to reversibly sequester the secreting reporter proteins at one of the secretory traps in the set of secretory traps;

(c) selectively conveying, during a time period, light to the cell to excite the FL1s (or optionally the FL3s) of the set of secretory traps to convey a set of time-resolvable F_ref signals;

(d) detecting the set of time-resolvable F_ref signals;

(e) selectively conveying light to the set of secretory traps to selectively switch the optogenetic tools of the secreting trap proteins (or optionally the secreting reporter proteins) to the lit conformation to facilitate release of the sequestered reporter proteins from the set of secretory traps;

(f) selectively conveying, during the time period, light to the set of secretory traps to excite the FL2s (or optionally the FL3) of the set of secretory traps to convey a set of time-resolvable F_qnt signals;

(g) detecting the set of time-resolvable F_qnt signals;

(h) (optionally) repeating steps (c) through (g);

(i) (optionally) after step (c), selectively conveying, light to the set of secretory traps to excite the FL1s (or optionally the FL3s) in the set of molecular traps to convey a set of one or more F_ref baseline signals, detecting the set of F_ref baseline signals, and calculating, from the set of F_ref signals, a F_ref baseline;

(j) (optionally) after step (c), selectively conveying, light to the cell to excite the FL2s (or optionally the FL3s) of the set of molecular traps to convey a set of one or more F_qnt baseline signals, detecting the set of F_qnt baseline signals, and calculating, from the set of F_qnt signals, a F_qnt baseline; and

(k) (optionally) stimulating the cell to secrete one or more trap proteins or one or more reporter proteins.