JP2002527761A - An improved method for obtaining quantitative information on effects on cellular responses - Google Patents

Abstract

  (57) [Summary] Described are improved methods and means for quantifying the effects of cellular response effects. In particular, an improved method for detecting intracellular translocation or redistribution of a biologically active polypeptide is described. The invention also describes several methods of contacting a cell with a substance that affects a cellular response and obtaining quantitative information about the response at about the same time. This method uses simultaneous, high volume assay techniques available on the market, e.g., in connection with screening new drugs, testing substances for toxicity and identifying drug targets for known or new drugs. Can be used as a very effective method for testing or discovering the effect of a substance on water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] Summary of the Invention The present invention, effects on cellular responses, (cellular response, and are manifested in redistribution of at least one component in a cell), especially cell responses substance and cells and the contact or incubating that affect Improved methods and means for obtaining quantitative information on the consequences of doing so. In particular, the present invention relates to an improved method for obtaining quantitative information regarding effects on pathways involving redistribution of at least one component associated with an intracellular pathway. The method of the present invention uses the effect of a substance on a physiological process as a very effective tool for screening new drugs, testing toxicity of substances, testing or discovering related to identifying drug targets for known or new drugs, for example. Can be. In particular, the invention relates to an improved method for synchronizing test procedures, wherein multiple substances can be tested simultaneously using commercially available means. The present invention also relates to several methods of contacting cells with substances that affect cellular responses and to the actual cells before, during, and after contacting the cells with these substances to improve applicability. The use of methods to obtain quantitative information about modifications and, at the same time, about effects on intracellular pathways is described. Other useful uses of the methods and techniques of this invention will be apparent to those skilled in the art based on the following description. In a practical embodiment of the present invention, the present invention relates to a method for detecting the intracellular translocation or redistribution of a biologically active polypeptide, preferably an enzyme, that affects an intracellular process, and a DNA construct and a DNA construct. The cells used in the method.

BACKGROUND OF THE INVENTION The intracellular pathway is tightly regulated by a cascade of components that are modulated in a temporally and spatially characteristic manner. Some disease states involve individual signal components (i.e., protein kinases, protein phosphatases,
The transposition factor) contributes to the active polarization. Therefore, these components are attractive targets for therapeutic intervention. Protein kinases and phosphatases are well-described components of several intracellular signaling pathways. The catalytic activity of protein kinases and phosphatases appears to play a role in virtually all regulatable cellular processes. Although the involvement of protein kinases in cell signaling and regulation has been extensively studied, for example, precise knowledge of the precise timing and spatial characterization of signal events can be difficult to obtain due to the lack of convenient techniques There are many.

[0003] The measurement of intracellular enzyme activities, such as kinases and phosphatases, is a process that makes these tools useful for the study of new drug candidates by well-established techniques, both manually and in various automated forms. Can be done at a volume rate. In addition to measuring activity, measuring the distribution of these and other enzymes in cells has been found to be useful,
There are established techniques for this type of measurement. Protein kinases often exhibit specific subcellular distribution before, during and after activity. That is, monitoring of the translocation process and / or redistribution of individual protein kinases or subunits thereof may be indicative of their functional activity. The relationship between rearrangement and catalytic activity has been described by diacylglycerol (DAG) -dependent protein kinase C (PKC), cAMP-dependent protein kinase (PKA) [Debernardi et al., 1996] and mitogen-activated protein kinase Erk-1 [Sano Et al., 1995]. Such methods for detecting subcellular localization / activity of protein kinases and phosphatases include immunoprecipitation, western blotting and immunocytochemical detection.

[0004] One aspect of the function of intracellular enzymes that has not been fully characterized is the redistribution of the enzymes. Importance of subcellular redistribution of enzymes as a mechanism of enzyme specificity, and general considerations on the identification of novel drug targets and the measurement of subcellular redistribution as a tool for studying drug candidates affecting these targets Materiality was part of the priority application, as published in WO 9845704 during the priority year
A METHOD FOR EXTRACTING QUANTITATIVE INFORMATION RELATING TO AN INFLUENC
It is described in E ON A CELLULAR RESPONSE (both are incorporated herein by reference). Although the redistribution of subcellular components is known to be important, the immediate measurement of this phenomenon is not widely used. This is mainly because there is no appropriate technology. There is essentially only one direct technique: visualize subcellular components of interest with a fine image system, for example using a video or scientific CCD camera and appropriate software to collect and store the images. Microscopic imaging of cells that are labeled for recording. Novel methods of monitoring specific modulation of intracellular pathways in intact living cells would provide new opportunities for drug discovery, functional genetics, toxicology, patient monitoring, and the like.

Recently, green fluorescent proteins have been expressed in many different cell types, including mammalian cells
(GFP) was found to be highly fluorescent [Chalfie et al., 1994]. WO 95/07463
No. introduces into the cell a DNA molecule having a DNA sequence encoding the protein of interest linked to the DNA sequence encoding GFP, such that the protein produced by the DNA molecule has the protein of interest fused to GFP; The cells are then cultured under conditions that allow expression of the fusion protein, and cells that can express GFP based on detecting the localization of fluorescence in the cells, thereby positioning the protein of interest in the cells, and A method for detecting a protein of interest in a cell is described. However, no examples of such fusion proteins are provided, and translocation of biologically active polypeptides that affect intracellular processes by, for example, activating proteins involved in signal pathways such as protein kinases or phosphatases. Or the use of fusion proteins using GFP to detect or quantify redistribution has not been proposed. WO 95/07463 further discloses that the regulatory element of a gene affected by a molecule of interest is operatively linked to GFP (the presence of the molecule affects the regulatory element,
Cells that are useful for the detection of molecules such as hormones or heavy metals in biological samples by subsequently affecting GFP expression). Thus, the gene encoding GFP is used as a reporter gene in cells constructed to monitor the presence of specific molecular identity.

[0006] Green fluorescent protein (GFP) has been used in translocation assays for the glucocorticoid receptor (GR) [Carey, KL et al., 1996]. The GR-S65TGFP fusion was used to study the mechanism involved in translocation of the glucocorticoid receptor (GR) in response to the agonist dexamethasone from the ligand-free cytosol through the nuclear pore to the nucleus that remains after ligand binding to the nucleus. ing. The use of GR-GFP fusions allows for immediate imaging and quantification of the nuclear / cytoplasmic ratio of the fluorescent signal. Array Scan ^™ designed for automated drug screening using similar genetic constructs in continuation
Dexamethasone, which induces GR translocation to the nucleus in HeLa cells, was quantified in a system called (WO 97/45730) (Guiliano, KA et al., 1997). In recent years, several other studies have shown that the labeling of specific proteins (or parts of proteins) involved in the intracellular signaling pathway with GFP provides a new means to measure and quantify the effects of substances on this pathway. Proven to provide. This concept is based on the androgen receptor (G
eorget V et al., 1997) and show that both the translocation of transcription factors such as NF-Atc (Beals CR et al., 1997) from cytoplasm to nucleus function similarly to those described above for GR. ing. Another related example is the β-arrestin-GFP construct, which has been shown to transduce activation of G-protein coupled receptors by translocation from the cytosol to the plasma membrane (Barak LS et al., 1997). Finally, a small portion of the pleckstrin homology domain-like protein of phospholipase Cδ1 (Stauffer TP et al., 1998) and the cysteine-rich domain of PKCγ (Oa
ncea E et al., 1998) has also been demonstrated to be useful for reporting on effects from substances by quantifying intracellular redistribution during activation of the specific signaling pathway to which they belong. I have.

[0007] Many screening programs currently used to find compounds that affect protein kinase activity include measuring the phosphorylation of kinases with respect to artificial or natural substances, receptor binding and / or reporter gene expression. Is based on However, interest in fluorescence measurements as a basis for future high-throughput drug screening has increased dramatically in recent years (Silverman L et al., 1998). Of particular interest to the present invention are FMs by Molecular Devices Inc.
Commercially available as LIPR ^TM, a scanning laser imager rapid screening of fluorescence changes in living cells (Schroeder K & Neagle B 1996)
.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a new and important dimension in the study of cell lines involved in redistribution, and by virtue of the present invention, affects, in general, chemicals or chemicals. Contact with the mixture, as well as redistribution responses or events caused by changes in the natural environment, are quantified on a large scale simultaneously. This quantification can establish a numerically significant association, or curve or graph, between the effect (or degree of effect) on the cell line and the redistribution response. This is very advantageous. For, as will be appreciated, quantification can be achieved in a rapid and reproducible manner, and perhaps more importantly, a system that can be quantified utilizing the methods of the invention is immense. It is a system that can provide a great deal of new information and insight. This screening assay screens for bioactive substances that have a completely new mode of action (e.g., inhibition or promotion of redistribution / translocation of a bioactive polypeptide) as a process of modulating the action, rather than inhibiting / activating, the enzyme activity. , A system that can be identified in a way that guarantees a very high selectivity for a particular isoform of the bioactive polypeptide and a further development of the compound selectivity for the same bioactive polypeptide or other components of the same signaling pathway over other isoforms Are clearly more useful than other screening assays, such as receptor binding assays, enzyme assays and reporter gene assays.

In its broadest aspect, the present invention relates to an improved method for obtaining quantitative information on the effect on a cellular response, which has a higher throughput compared to previous methods, which method comprises: It consists of recording the changes caused by effects on living cells that are mechanically intact in the spatially dispersed light emitted by the luminophore. The luminophore can be present in the cell and modulated by components that can be redistributed in relation to the degree of influence and / or redistributed in relation to the degree of influence. The coupling modulates the luminescent properties of the luminophore, and changes in spatially scattered light from the luminophore are detected and recorded as changes in fluorescence intensity using an instrument designed to measure changes in fluorescence intensity, and the spatially scattered light The recorded changes therein are processed to obtain quantitative information in which the spatial variance or changes in the spatial variance are correlated with the degree of influence. In one aspect of the invention, viable cells that are mechanically intact are rendered permeable for some time after they have been affected, but before or during the actual experimental recording. In another aspect, the present invention relates to an improved method for obtaining quantitative information regarding the effect on a cellular response. The method consists in recording the changes caused by effects on permeabilized metaplastic cells in the spatially dispersed light emitted by the luminophore. The luminophore can be present in the cell and modulated by components that can be redistributed in relation to the degree of influence and / or redistributed in relation to the degree of influence. The binding modulates the emission characteristics of the luminophore, and the spatially scattered light from the luminophore is detected and recorded as a change in fluorescence intensity using an instrument designed to measure the change in fluorescence intensity, and in the spatially scattered light. The recorded changes are processed to obtain quantitative information in which the spatial variance or changes in the spatial variance are correlated with the degree of influence. In a preferred embodiment of the invention, the luminophore present in the cell may be redistributed by modulating the intracellular pathway so as to be associated with the redistribution of at least one component of the intracellular pathway. In another preferred embodiment of the invention, the luminophore is a fluorophore.

Generally, the cells and / or cells live mechanically intact during the experiment. In another embodiment of the invention, the cells are fixed at a time after the effect (time at which the response is scheduled to be significant) and recorded at any later time. In another embodiment, the cells and / or cells live mechanically intact during the experiment, but are physically or chemically disrupted or permeable as a first step in the experimental analysis. In another aspect of the invention, the cells have a permanent plasma membrane and are stably rendered permeable prior to the start of the experiment such that the plasma membrane is permeable during the experiment. This allows the components of the intracellular pathway to be brought into contact with substances that normally do not penetrate the cell plasma membrane, such as peptides, proteins and hydrophilic organic compounds. Living cells that are mechanically intact or permeabilized can be selected from the group consisting of fungal cells, such as yeast cells; invertebrate cells, including insect cells, and vertebrate cells, such as mammalian cells. it can. These cells may be at a temperature of 30 ° C. or higher, preferably a temperature of 32-39 ° C., more preferably a temperature of 35-38 ° C., most preferably about
Incubate at a temperature of 37 ° C for a period of time in which the effect is observed. In one aspect of the invention, the viable cells that are mechanically intact or permeabilized are part of a matrix of identical or non-identical cells. In one embodiment of the invention, the cells comprise a group or group of cells contained within a spatial restriction or classes of spatial restrictions. In one embodiment, the cells consist of multiple groups of cells that are quantitatively the same, but have been subjected to different effects. In another embodiment, the cells are comprised of multiple groups of cells that are quantitatively different but are subject to the same effect.

In one embodiment of the present invention, spatial limitations are:
The range is limited on the substrate on which the cell exists. Spatial restrictions may be arranged in one or more arrays on a common carrier. The spatial restriction may be the wells in a microtiter plate, for example 96, 384, 864 and 15
36 wells are located on a common carrier. In another embodiment, the spatial restriction is a well in a plate of a different type than the microtiter type. In one embodiment of the invention, the area is established by the presence of cells on the substrate in a pattern that defines the area. In another aspect of the invention, the extent is alternatively established by a spatial pattern or array of effects or classes of effects used or contacted by the cells. This embodiment is described in WO 9845704 during the priority year.
A METHOD whose contents were part of a priority application as published in
FOR EXTRACTING QUANTITATIVE INFORMATION RELATING TO AN INFLUENCE ON AC
ELLULAR RESPONSE (both incorporated herein by reference). Briefly, in this aspect of the invention, mechanically intact or permeabilized living cells are cultured on optically clear and flat surfaces that are generally optimized for cell culture. Part of a continuous or discontinuous sheet of cells to be removed. The optically clear, flat surface may be a porous membrane that allows for the processing of cells to grow cells through the pores of the membrane and allows direct capillary flow of liquid through the pores.

[0012] The cells used in the present invention should contain a nucleic acid construct capable of encoding the fusion polypeptide as defined herein and expressing the sequence encoded by the construct. The cell is a eukaryotic cell selected from the group consisting of a fungal cell such as a yeast cell; an invertebrate cell including an insect cell; a vertebrate cell such as a mammalian cell. Preferred cells are mammalian cells. In another aspect of the invention, the cell may be derived from an organism having a nucleic acid sequence encoding a fusion polypeptide as defined herein in at least one cellular component and capable of expressing the nucleic acid sequence. . The organism is selected from the group consisting of unicellular organisms and multicellular organisms such as mammals.

A luminophore is a component that allows the visualization and / or recording of redistribution by emitting light into a spatial dispersion related to the degree of influence. The term redistribution is intended to cover all aspects of changes in spatial location, such as dislocations of luminophores or other components. In one embodiment of the invention, the luminophores can be redistributed in a physiologically relevant manner to the extent of the effect. In another embodiment, the luminophore can be associated with a component that can be redistributed in a manner that is physiologically relevant to the extent of the effect. In another embodiment, the correlation between luminophore redistribution and the degree of impact can be demonstrated experimentally. In a preferred aspect of the invention, the luminophore can be redistributed in substantially the same manner as at least one component of the intracellular pathway. In another embodiment of the invention, the luminophore can be quenched by spatial coupling with components redistributed by modulation of the pathway. Quenching is measured as a change in emission intensity. In another embodiment of the invention, the luminophore is stationary, but has some spatial dispersion, in a manner that is physiologically relevant to the extent of the effect, the luminescent properties of one or more luminophores near or near the luminophore. It may interact with at least one component that can be redistributed to be modulated as a component acting away from the luminophore.

[0014] The luminophore may be a fluorophore. In a preferred embodiment of the invention, the luminophore is a polypeptide encoded by and expressed from a nucleotide sequence residing in a cell. The luminophore, as defined herein, may be a hybrid polypeptide consisting of at least a portion of each of two polypeptides, one consisting of a luminescent polypeptide and the other consisting of a biologically active polypeptide. The luminescent polypeptide may be GFP as defined herein, or F64L-GFP, F64L-GFP.
F64L mutant and EGFP as defined herein, such as 64L-Y66H-GFP, F64L-S65T-GFP
May be selected from the group consisting of green fluorescent proteins having GFP can be added to the bioactive polypeptide or to a portion of its subunits, optionally via a peptide linker, to provide N-
It may be labeled at the terminus or C-terminus. The fluorescent probe may be a component of an intracellular signaling pathway. The probe is encoded by the nucleic acid construct. In one aspect of the invention, the pathway studied is an intracellular signaling pathway.

In a preferred embodiment of the invention, the effect is contacting the chemical or chemicals with a group or group of living cells that are mechanically intact or permeabilized, and / or mechanically intact. Alternatively, it may be an incubation of a group or group of permeabilized living cells with a chemical in solution. As published during the priority year as WO 9845704,
A METHOD FOR EXTRACTING QUANTITATIVE I whose contents were part of the priority application
In one aspect of the invention fully disclosed in NFORMATION RELATING TO AN INFLUENCE ON A CELLULAR RESPONSE (both incorporated herein by reference), the chemical is associated with an underlying matrix. In this regard, the chemical may be produced, secreted from the genetically engineered cell sheet, or bound to the plasma membrane surface of the sheet. In this aspect of the invention, the chemicals may be binary separated on a non-denaturing gel using electrophoresis, the gel being directly and mechanically intact, so that the chemicals can contact cells through diffusion or transmission. Very close or direct contact with living cells that have been or have been rendered permeable. The effect will modulate intracellular processes. In one aspect, modulation may be activation of an intracellular process. In another respect,
Modulation may be the inactivation of an intracellular process. In yet another aspect, the effect may inhibit or promote redistribution without directly affecting the metabolic activity of components of the intracellular process.

In one aspect, the invention is used to establish a dose-response relationship for one or many chemicals. In one embodiment, the invention is used as the basis of a screening program, where the effects of an unknown effect, such as a compound library, can be compared under standard conditions to the effects of a known standard compound. In addition to strength, modulated by the effects of cellular phenomenon-based effects,
There are several fluorescence and emission parameters that can be used in the invention for this purpose. Examples include resonance energy transfer, fluorescence lifetime, polarization and wavelength shift. Each of these methods requires a particular type of filter in the outgoing light path to select the desired light component and reject other components. The recording of the optical properties may be in the form of a value ordering array such as a CCD array or a vacuum tube device such as a vidicon.
Furthermore, the translocation mobility or free movement of the luminophore that binds to the protein of interest is
It may be an important property affected by the effect on the underlying cellular phenomena and can therefore be used in the invention.

In one embodiment of the invention, the spatially dispersed light emitted by the luminophore is detected by a change in resonance energy transfer between the luminophore and another luminescent substance capable of transmitting energy to the luminophore. . Each is selected or designed to be part of a particular component of the intracellular pathway and to bind or associate with the component. In this embodiment, either the luminophore or a luminescent substance capable of transmitting energy to the luminophore is:
Receive redistribution depending on impact. Resonance energy transfer is measured as a change in emission intensity from the luminophore, and is preferably detected by a single-channel photodetector that is non-spatially resolved and responds only to the average intensity of the luminophore. In one embodiment of the invention, the spatially dispersed light emitted by the luminophore includes the case of uniform spatial dispersion of the light. In one aspect of the invention, the luminophore is a fluorophore that is redistributed over a non-homogeneous excitation light field and changes the light intensity emitted from the luminophore as a result of a change in the amount of excitation light intensity at different field points. Let it.

In one embodiment of the invention, the spatially dispersed light may be recorded at some point during the time after the effect. In another embodiment, the time may be recorded at two points, one before the effect and one after. The result or change is measured from the change in fluorescence compared to the fluorescence measured prior to the effect or modulation. In another embodiment of the invention, the recording may be performed at a sequence of time points affected some time after the first time point in the sequence. Recording is, for example, 0.1 seconds to 1
Time, preferably 1 to 60 seconds, more preferably 1 to 30 seconds, especially 1 to 10 seconds, at predetermined time intervals, 1 second to 12 hours, e.g. 10 seconds to 12 hours, e.g. 10 seconds to 1 hour, e.g. 60 seconds ~
This takes 30 or 20 minutes. The result or change is measured from the change in fluorescence over time. The result or change may be measured as a change in the spatial dispersion of the fluorescence over time.

In one embodiment, the recording consists of a time series of total luminescence for one or several spatially restricted cells. In one embodiment, the signals from all spatial constraints are:
Once in time, by making a record made at each spatial limit by means of a device that successively locates each limit within the field of view of the detector, by repeating the positioning and measuring process until all spatial limits have been measured, Measured. The detector may be a photomultiplier tube. In a preferred embodiment of the invention, one or more spatial restrictions are measured simultaneously. This is done by a one-dimensional or two-dimensional array detector, whereby multiple spatial constraints are imaged on the array detector, and individual subsets of detection units (pixels) in the array detector are one and one. The signal from any one of the spatial constraints is measured from only one of the spatial constraints and the signal from the pixel receiving the image from one of the spatial constraints. The array detector may be a linear diode array, a video camera (according to current or future standards and definitions for image capture and transmission) or a charge transfer device such as a charge coupled device (CCD). In one embodiment, recording the signal requires multiple spatially restricted illuminations so that the luminophores are excited and they emit light. In one embodiment, all spatial constraints are illuminated simultaneously during the measurement. In another embodiment, the space restriction is illuminated once only during the time being measured. In a preferred embodiment, the illumination occurs with a laser scanned in a raster fashion over some or all of the measured spatial limitations.
Scanning may be performed at a substantially faster rate than the measurement process, so that the illumination is temporally and spatially uniform and continuous over the measurement area for the measurement process.

The recording of the spatially dispersed luminescence emitted by the luminophore is performed by a device for measuring the fluorescence distribution in the cells and thereby recording any changes in the fluorescence distribution in the cells. The device includes, at a minimum, the following components: (a) a light source, (b) a method of selecting a light wavelength from a source that excites the emission of the luminophore, (c) a rapid cutoff of the excitation light for the rest of the system. Or a device that can pass, (d) sending the excitation light to the test piece, collecting the emitted fluorescent light spatially resolved, a series of optical members that form an image from the emitted fluorescent light (or,
(E) another type of intensity map associated with the detection and measurement method), (e) a bench or stand holding the cell containers measured in a predetermined arrangement with respect to a series of optical elements, (f) a spatial map in the form of an image. (G) a computer or electronic system for capturing and storing the recorded image and calculating the degree of redistribution from the recorded image, and related software. In a preferred embodiment of the invention, the system is automated. In one specific example, d and e
The components described above include a fluorescence microscope. In one specific example, the component of f is a CCD camera. In one embodiment, component f is an array of photomultiplier tubes / devices. In one embodiment, the image is formed by an optical scanning system,
Be recorded.

In one embodiment, an optical scanning system is used to illuminate the bottom of a microtiter plate, and changes in emission or fluorescence are time-resolved simultaneously from all spatial constraints. Can be recorded. In a preferred embodiment, the actual emission or fluorescence measurements are made on a FLIPR ^™ instrument commercially available from Molecular Devices, Inc. In one embodiment of the invention, the actual fluorescence measurements are made on a standard fluorimeter (fluorescence plate reader) for microtiter plates. In one embodiment, a liquid addition system is used to add a known or unknown compound to any or all cells in a cell holder at a predetermined time. Preferably, the liquid addition system is under the control of a computer or electronic system. Such automated systems can be used in screening programs because of the ability of an operator person to produce results from a larger amount of test compound than would be produced using the device manually.

The method of physically contacting the detection layer of a cell with a compound involves transmitting / diffusing the compound bound to an inorganic or organic support (eg, glass, plastic or the plasma membrane of a whole living cell) with the cell. Alternatively, it may be another conceptual method of delivering the compound to the cell through the pore membrane by direct contact with the cell. These methods were described in A Method FOR EXTRACTING QUANTITATIVE INFORMATI
Although fully disclosed in ON RELATING TO AN INFLUENCE ON A CELLULAR RESPONSE (both incorporated herein by reference), they are also outlined in the following paragraphs.

In one aspect of the invention, the cell detection layer is a continuous or discontinuous sheet of cells that is not divided into test units or wells at all. The compound is printed as a solution in a solvent on a non-absorbent sheet of porous material and dried. This printed compound sheet then defines the test pattern of the experiment to be brought very close to or in direct contact with the detection layer of the underlying cells. The compound newly dissolved by the liquid layer on the cell comes into contact with the cell through the membrane pore by transmission. The porous membrane on which the compound was printed was optically transparent, and its contents were part of a priority application, as published in WO 9845704 during the priority year
A METHOD FOR EXTRACTING QUANTITATIVE INFORMATION RELATING TO AN INFLUENC
It is preferably configured as described in E ON A CELLULAR RESPONSE (both incorporated herein by reference). In another embodiment of this aspect of the invention, the detection layer of cells is a continuous or discontinuous sheet of cells that are not separated into test units or wells, preferably porous and optically of the type described above. Grow on a transparent film. The pore membrane may allow cells to carry cellular processes through the pores. The compound is printed as a solution in a solvent on an optically clear substrate such as glass, plastic or quartz and dried. During the experiment, a cell sheet on a membrane covered with a thin liquid film is laminated on top of the printed compound pattern.
The compound is then lysed and the cells are contacted via diffusion and transmission. The compound is
It may be made using combinatorial chemistry techniques and may be a peptide. The compound may be covalently linked to an optically transparent substrate or pore membrane.
Compounds are secreted or bound by the plasma membrane of genetically modified cells that grow as continuous or discontinuous sheets on a flat optically transparent surface or an optically transparent porous membrane May be a modified protein, polypeptide or peptide.

[0024] The recording of the change or result with respect to the light emitted by the luminophore is performed by recording the spatially dispersed light as one or more digital images, and the recorded change is reduced to one or more typical examples for the degree of redistribution. The process of reducing includes a digital image processing method or a combination of digital image processing methods. Quantitative information indicating the extent of the cellular response to an effect or consequence of an effect in an intracellular pathway may be derived from a record, or a record according to a predetermined calibration based on the response or result, to provide a known degree of appropriate specific effect. Recorded similarly. This calibration method is developed according to the principles described below (Developing an Image-based Assay Technique). A detailed description of specific assay techniques is provided in the Examples.

Although the stepwise approach that requires reducing the image or images to the typical value of the response caused by the effect is unique to each assay, the individual steps are generally well-known methods for image processing. Some examples of individual steps include point manipulation such as subtraction, ratioing and thresholding, digital filtering methods such as smoothing, sharpening and edge detection, Fourier filtering, image correlation And space frequency methods such as image auto-calibration, object discovery and classification (blob analysis), and color space manipulation for visualization.
In addition to algorithmic methods, recursive methods such as neutral networks can also be used. In a preferred embodiment of the invention, a dose-response relationship is established based on quantification of the response caused by a particular effect typical of the underlying intracellular signaling pathway using the methods described above and in the Examples. Is done. The dose-response relationship for a particular effect was then determined by performing the same assay in a microtiter plate such as FLIPR ^™ or a device that allows simultaneous monitoring of all wells in a conventional fluorescent plate reader for microtiter plates. Compare to the resulting dose-response relationship. A simultaneous measurement format is valid if the correlation between the dose-response relationships obtained from the two different measurement systems is good (see Examples 8, 9, 10, and 11).
This means that the simultaneous measurement format can be used as a primary basis for screening assays with the potential utility of screening a significantly larger number of substances per time unit of impact on response. For example, if at least 96 experiment chambers are operated simultaneously in a single experiment performed with a microscope, the throughput of the person conducting the experiment increases by a factor of 96.

The image plate reader adjusts the signal from each well to one value per time point. Thus, the data resulting from a single "actuation" of the device is a time series of one numerical value for each well, with the injection of the test compound occurring at a known point in the series. The main advantage of this type of device is that the number of samples (throughput) that can be processed in a given time is greatly increased. This is extremely effective when used in assays in a screening program for new drug lead compounds. The first step in data analysis is to normalize the results from each well so that they can be compared to each other or to previously analyzed known compounds. This is always
Instrument bias from all data points based on wells
) Is started by calibrating the signal by subtracting). In this regard, each of the two techniques can be continued depending on the design of the assay: Method 1: Prior to the addition of the test compound, the average of the signal is subtracted from all data points on a well-by-well basis. Method 2: Correct the data for any known background by subtracting the background value from all data points on a well-by-well basis. The resulting background correction data is normalized by dividing each data set by the average of the pre-injection data values of the test compound on a well-by-well basis.

The time series data to be corrected or standardized is then further reduced by a technique for converting the time series into one value. There are at least three such methods: For transient responses, measure the maximum deviation from baseline. this is,"
Also known as "peak height" technology. Alternatively, the signal is adjusted over a time between predetermined ranges. If the data is processed according to method 2 above, the deduction is subtracted and the unresponsive integral is zero within the erroneous measurement range. This is the "peak area
(peak area) technology. If the response is a cumulative response, eg, an exponential change to a new level, the result is considered as either the difference or the ratio between the signal after a predetermined time and the signal before the addition of the test compound. All of the above methods reduce the data for a given well to one or more single values. For screening purposes, these values are examined for values greater than some statistically determined cut-off value. For characterization, the values represent a quantitative response and are further processed in sets by techniques such as fitting a dose-response curve.

In another embodiment of the invention, the measurement of redistribution is by taking advantage of the fact that the probe is subjected to some change in autonomy or restriction on movement within the intracellular environment to produce redistribution. Achieved indirectly. The degree of transposition will correlate with the amount of luminophores that move freely in the cytoplasm. At a point in time after which the test compound has any effect, the limited amount or fraction of luminophores is reduced by disintegrating or permeating the plasma membrane of the cell and diffusing freely moving luminophores. Can be measured. If the detector volume is limited to the area immediately around the cell and the total amount of free-moving luminophore that can diffuse is quite large, the free-moving luminophore will essentially disappear from the detector's field of view, The signal is not recorded. In one aspect of the invention, the above measurement of redistribution is performed on cells having a permanently permeabilized plasma membrane impregnated with a solution that mimics the cytoplasmic environment. Thus, the effects of compounds that cannot normally enter the cytoplasm of a cell can be tested. The nucleic acid construct used in the present invention comprises a bioactive polypeptide or a part thereof, which is a component of an intracellular signal pathway, and a bioactive polypeptide or a part thereof, and an N- or C-terminal optionally via a peptide linker. GFP, preferably F6 of GFP
Encodes a nucleic acid sequence fusion polypeptide consisting of the 4L mutant. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a protein kinase or phosphatase. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a transcription factor or a portion thereof that changes cell localization upon activation.

In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a protein or a portion thereof that binds to the cytoskeletal network and changes cell localization upon activation. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a protein kinase or a portion thereof that changes cell localization upon activation. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a serine / threonine protein kinase or a portion thereof that can alter cellular localization upon activation. In one embodiment, the bioactive polypeptide encoded by the nucleic acid construct is a tyrosine protein kinase or a portion thereof that can alter cellular localization upon activation. In one specific example,
The biologically active polypeptide encoded by the nucleic acid construct is a phospholipid-dependent serine / threonine protein kinase or a portion thereof that can change cellular localization upon activation. In a specific embodiment, a method for obtaining quantitative information on the effect of a construct listed in Table 1 on a cellular response in living cells that are mechanically intact or permeabilized, comprising: Redistribution in relation to the extent of impact, and / or
Or a change in spatially dispersed fluorescence emitted from a fluorophore that can be modulated by a component that can be redistributed in relation to the degree of influence, preferably a fluorescence measured by an instrument designed to measure a change in fluorescence intensity. Used in a method consisting of recording as a change in intensity. Names described herein, constructs and DNs encoding full-length amino acid sequences
Fusion constructs of the invention by reference to the relevant SEQ ID NO of the A sequence

[Table 1]

As exemplified in Examples 8, 9 and 11, the redistribution of PKA and PKC is, for example, FLIP
It can be easily detected as a change in fluorescence intensity as measured by an ^RTM device. In one embodiment, for patterns similar to those observed microscopically for PKA or PKC (see Examples 1, 2, 8, and 11), ie, for luminophores that produce aggregated to dispersed form or redistribution Any new luminophores that are measured to redistribute from their dispersed form to their influence on the aggregate form are expected to be detectable as changes in light intensity as measured, for example, on a FLIPR ^™ instrument. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a cAMP-dependent protein kinase or a portion thereof that can alter cellular localization upon activation. In a preferred embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a PKAc-F64L-S65T-GFP fusion. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a cGMP-dependent protein kinase or a portion thereof that can alter cellular localization upon activation.

In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a calmodulin-dependent serine / threonine protein kinase or a portion thereof that can alter cellular localization upon activation. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a mitogen-activated serine / threonine protein kinase or a portion thereof that can alter cellular localization upon activation. In a preferred embodiment, the biologically active polypeptide encoded by the nucleic acid construct is an ERK1-F64L-S65T-GFP fusion or an EGFP-ERK1 fusion. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a cyclin-dependent serine / threonine protein kinase or a portion thereof that can alter cell localization upon activation. In one embodiment, the biologically active polypeptide encoded by the nucleic acid construct is a protein phosphatase or a portion thereof that can alter cellular localization upon activation. In a preferred embodiment of the invention, the nucleic acid construct may be a DNA construct.

In one embodiment, the biologically active polypeptide is encoded by a nucleic acid construct. In one embodiment, the gene encoding GFP in the nucleic acid construct is from Aequorea victoria. In a preferred embodiment, the gene encoding GFP in the nucleic acid construct is EGFP or F64L-GFP, F64L-Y66H-GFP.
And GFP variants selected from F64L-S65T-GFP. In a preferred embodiment of the invention, the DNA construct identifiable by any of the DNA sequences set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and 17 encodes the same fusion polypeptide Variants of these sequences, or fusion polypeptides that are biologically equivalent thereto, such as isoforms or splice variants or homologues from another species. The present invention describes a method that can be used to establish a screening program for the identification of bioactive substances that directly or indirectly affect intracellular signaling pathways and, due to this property, may be useful as pharmaceuticals. Based on measuring spatially resolved luminescence redistribution in living cells from luminophores whose distribution has been altered by activation or inactivation of intracellular signaling pathways, individual Measurements indicate its potential biological activity.

In one embodiment of the invention, the screening program is used to identify a biotoxic agent as defined herein that exerts a toxic effect by interfering with an intracellular signaling pathway. Based on measuring spatially resolved luminescence redistribution in living cells from luminophores whose distribution has been altered by activation or inactivation of intracellular signaling pathways, the individual Measurements indicate its potential biotoxic activity. In one embodiment of the screening program, a compound that modulates a component of the intracellular pathway as defined herein can be found, and the therapeutic amount of the compound can be estimated by methods according to the methods of the present invention. In a preferred embodiment, the invention provides for administering to a patient suffering from a condition or disease relating to the intracellular function of a biologically active polypeptide an effective amount of a compound found by any of the methods according to the invention. The discovery of new methods of treating the condition or disease. In another preferred embodiment of the invention, a method is established for identifying a new drug target or several new drug targets from a group of bioactive polypeptides that are components of an intracellular signaling pathway.

[0034] In another embodiment of the invention, an individual treatment regimen is established for elective treatment of a selected patient suffering from a disease. Here, transfecting cells or cells having at least one DNA sequence encoding a fluorescent probe according to the invention,
The transfected cells or cells are transferred back to the patient, or the cells or cells are cultured under conditions that allow expression of the probe and exposed to an array of available drugs, and then Comparing changes in the fluorescent or redistribution pattern of the fluorescent probe to detect cellular response to a particular agent (resulting in a cellular action profile) and then based on the desired activity and acceptable levels of side effects, 1
Related progenitor cells or cells obtained from one or several tissues of a patient using a method comprising selecting the above drugs or drugs and administering to the selected patient an effective amount of these drugs. The class is tested for available drugs used to treat the disease. The present invention describes a method that can be used to establish a screening program for the conversion pathway of a back-tracking signal as defined herein. In one embodiment, a screening program is directed to the redistribution of spatially resolved luminescence from several luminophores that have undergone a distribution change due to activation or inactivation of an intracellular signaling pathway under study. By testing the effects of compounds or compounds sequentially or simultaneously, one or several compounds can be used to more accurately establish the level at which a particular signal transduction pathway is affected.

Generally, a probe, ie, a “GeneX” -GFP fusion or a GFP- “GeneX” fusion,
Using GeneX specific primers, PCR
It is built by integrating FP and in-frame. Fusions include between GeneX and GFP (
For example, a part of the multi-cloning site region in the plasmid) contains a sequence derived from the short vector, which is expressed as "GeneX
And GFP with a peptide linker. Some of the steps involved in probe development include: Identification of the gene sequence. This is most easily accomplished by searching a genetic information repository, such as the GenBank Sequence Database, which is widely used and commonly used by molecular biologists. The detailed example below shows the GenBank accession number of the gene in question.

Design of gene specific primers. By examining the gene sequence, gene-specific primers used for the PCR reaction can be designed. Typically, the gene
When fused after GFP, i.e., for a GFP- "GeneX" fusion, the top strand primer contains the ATG start codon of the gene followed by about 20 nucleotides and the bottom strand primer precedes the stop codon. It contains about 20 nucleotides. If the gene is fused before GFP, ie, in the case of the "GeneX" -GFP fusion, the stop codon must be avoided. Optionally, the full-length sequence of GeneX is not used for fusion, and only a portion that is localized and redistributed like GeneX in response to a signal may be used. In addition to the gene-specific sequence, the primer contains at least one recognition sequence for a restriction enzyme, which allows for subsequent cloning of the PCR product. The sites are chosen to be unique in the PCR product and compatible with the sites of the cloning vector. Further, to establish the correct reading frame and / or translation initiation consensus sequence for the fusion gene, it is necessary to include the correct number of nucleotides between the restriction enzyme site and the gene-specific sequence. Finally, primers always contain a few nucleotides before the restriction enzyme site to make digestion with that enzyme efficient.

Identification of the source of the gene to be amplified. In order for a PCR reaction to produce a product with gene-specific primers, the gene sequence must first be present in the reaction, for example, in the form of a cDNA. GenBank or scientific literature information will usually indicate in which tissues the gene is expressed. Also, cDNA libraries from a wide variety of tissues or cell types from various species are available, for example, from Clontech (Palo Alto), St.
It is commercially available from ratagene (La Jolla) and Invitrogen (San Diego). Many genes are also cloned in the American Type Tissue Collection (Virginia)
Available from Optimization of PCR reaction. Several factors are known to affect the efficiency and specificity of the PCR reaction, including primer annealing temperature, ionic concentration in the reaction, notably Mg2 ⁺ and K ⁺ concentrations, and the pH of the reaction. Have been. If the results of the PCR reaction are not satisfactory, it may be because the above parameters are not optimal. Various annealing temperatures were tested on a temperature gradient built-in PCR instrument available from, for example, Stratagene (La Jolla) and / or various buffer compositions, such as Stratagene (
La Jolla) should try the OptiPrime buffer system.

A clone of the PCR product. When designing primers, vectors for cloning the amplified gene product and fusing it with GFP have already been considered. When selecting a vector, at least consider in which cell type the probe will be expressed,
The promoter-controlled expression of the probe should be compatible with the cell. Most expression vectors also include one or more selectable markers, for example, conferring drug resistance, a useful feature when it is desired to produce stable transfectants. The selectable marker should also be compatible with the cells used. The actual cloning of the PCR product should not be difficult as it generally involves cloning a fragment digested with two different restriction enzymes into a vector digested with the same two enzymes in one step. If you have problems with this cloning,
It may be because the restriction enzyme did not work well with the PCR fragment. In this case, it is possible to add a long extension to the end of the primer to overcome indigestion near the end of the fragment, or to introduce an intermediate cloning step that is not based on restriction enzyme digestion. Some companies, such as Invitrogen (San
Diego) and Clontech (Palo alto) provide systems for this method. Once the gene has been cloned and fused with the GFP gene in the process, the resulting product, usually a plasmid, should be carefully checked to confirm that it is as expected. The most accurate test would be to obtain the nucleotide sequence of the fusion gene.

Once a DNA construct for a probe has been generated, its functionality and utility can be evaluated by transfecting it into cells capable of expressing the probe. The fluorescence of the cells is examined immediately, generally the next day. at this point,
Note two features of cellular fluorescence: intensity and subcellular localization. The intensity should usually be at least equal to the intensity of the unfused GFP in the cell. If not, the sequence or quality of the probe DNA may be incorrect and should be checked carefully. Subcellular localization is an indicator of whether the probe works well. If it is localized as expected in the gene in question, e.g. if it is excluded from the nucleus,
It can be immediately subjected to a functional test. If the probe is not localized immediately after the transfection procedure, it may be due to overexpression at this point. This is because cells usually take up a large number of plasmid copies and localization occurs with a decrease in plasmid copy number and expression level, e.g., within a few weeks. . If localization does not occur with extended time,
May have disrupted the localization function, for example, by masking a protein sequence essential for its interaction with normal cell fixation-protein.
In this case, the opposite fusion works, for example, GFP-GeneX works when GeneX-GFP does not work, because the two different parts of GeneX are affected by the proximity to GFP. Will. If the opposite fusion does not work, there may be a problem with the proximity of the GFP at either end and extend the distance by incorporating a longer linker between GeneX and GFP in the DNA construct Can be.

If there is no prior knowledge of localization and no localization is observed, it may be because the probe should not be localized at this point, because of the nature of the protein fused to GFP There is. This should then be subjected to a functional test. In a functional test, cells expressing the probe are treated with at least one known compound and, typically, redistribution within the cell activates the signal pathways that the probe is expected to report, and agitation ( perturb). If the redistribution is as expected, for example, if, due to prior knowledge, the redistribution should be transposed from position X to position Y, it is subjected to a first criticality test. In this case, the response can be further characterized and quantified. If redistribution does not work as expected, it may be because the cell lacks at least one component of the signaling pathway, e.g., a cell surface receptor, or, for example, if the probe is a human gene product sequence If the model was designed based on the information and the cells were derived from hamsters, this may be due to interspecies incompatibility. In any case, other cell types should be identified for test processes that do not cause these problems. Without prior knowledge of the pattern of redistribution, conduct a more thorough analysis of the redistribution to identify what is essential and indicative, and when it becomes apparent,
The response can be further characterized and quantified. If the characteristics of the redistribution are not identified, the problems described above may arise and the probe should be retested under more optimal cellular conditions. If the probe does not work at optimal cell conditions, then return to the first step.

The process of initiating an image-based redistribution assay may involve unplanned experimental observations that can visualize the redistribution phenomena, or specific probes that specifically track redistribution phenomena that are already known to occur. Start with one of the designs. In both cases, the first and best research technique for a skilled scientist or technician is to observe the phenomenon. Despite the rapid advances in computer technology, the human eye-brain combination is still the most powerful pattern recognition system known and requires no prior knowledge of the system to provide interesting and useful information in raw data. For potential patterns. This is remarkable when the data is presented in a “data type” image form that is natural for human visual processing. Because human visual processing works most effectively in a relatively narrow range of frequencies (i.e., it cannot see either very fast or very slow changes in the field of view), it records data and extends or extends time. You may need to play it with any of the compressions. Some luminous phenomena are not directly visible to the human eye. For example, polarization and fluorescence duration are included. However, with appropriate filters or detectors, these signals can be recorded as an image or a series of images and displayed to humans just as described. Thus, the pattern is detected and the same method is applied.

Once redistribution has been determined to be a reproducible phenomenon, one or more data sets arise to develop a method of obtaining quantitative information from the data. At the same time, the biological and optical conditions that give the best quality raw data for the assay are determined. This allows for an iterative process: it may be necessary to develop a quantitative approach to assess the effect on the assay in which the assay conditions have been manipulated. The data sets are tested by persons or persons with knowledge and skill in biological phenomena in the application of image processing techniques. The purpose of this implementation is to determine, or at least to propose, a way to reduce the image or series of images making up the "response" record to a value corresponding to the degree of response. By repeatedly using either image processing software or an image processing toolbox and a programming language, the method takes the image or images as input and reduces the degree of response (
It is encoded (in any unit) as a technique or algorithm that occurs as its output. Some criteria for assessing the effectiveness of a particular method are as follows: The extent of the response changes in a biologically significant manner, ie, it depends on the stimulating agent concentration or condition. Show known or inferred dependencies on

Is the degree of response reproducible, that is, does the same concentration or level of stimulant or condition produce the same response where changes are acceptable? Is the dynamic range of the response sufficient for the purpose of the assay? If not, can a method change or one of its parameters improve the dynamic range? Does the method show any obvious "medical conditions", i.e. does the method produce an outrageous value for the response if the image processing is generally defective? Can these conditions be excluded, controlled or attacked? Can the method cope with normal changes in the number and / or size of cells in the image? In some cases the method will be self-evident and in other cases a number of possible approaches are suggested. Even though some methods are clearly superior to others, parameter optimization will be required. Various methods are applied to the data set until the above proposed criteria are determined or one method is used repeatedly until the most sufficient signal, noise, width, etc. combination is achieved. , Parameters or parameters are adjusted. This is equivalent to calibrating any type of single channel sensor. The number of ways to get a single value from an image is very large, so that the first step in reducing this number to a small and finite number of possible approaches is to take a smart way. No. This does not mean that the method achieved is necessarily the best method, but the overall research for the best method is a complete number (sheer num
Easily get out of the problem because the potential of ber) is involved.

An image-based assay is a method in which utility is a parameter, eg, specificity of a desired component of a sample.
Sex, kinetic range, variability, sensitivity, concentration range over which the assay operates, and other such
There is no difference from other assay techniques in being characterized by parameters. Up
It is not necessary to characterize each and all of these prior to use of the sail, but some
Only one way to compare a sail to another is shown. Second, the final step improves the usefulness of the screening program as a basis.
In order to increase the throughput of the assay.
To this end, the underlying intracellular signals are generated using the methods described above and in the Examples.
Based on quantification of the response caused by a particular effect, typical of the pathway
Establish a volume-response relationship. The dose-response relationship for a particular effect is then^{T M} Allows simultaneous monitoring of all wells in a microtiter plate, such as
Instruments or fluorescent plates for normal imaging or microtiter plates
Compare the dose-response relationship obtained by performing the same assay on a leader. Two
If there is a good correlation between the dose-response relationships obtained from different measurement systems,
It can be said that the fixed form is effective (see Examples 8, 9 and 11). This is a simultaneous measurement type
Condition screens a significantly larger number of substances for their effect on the response per unit of time.
Used as a primary basis for screening programs that may have
Means that it can be used.

In this specification and in the claims, the term “effect” includes any effect that the cellular response consists of redistribution. Thus, for example, heating, cooling, pH, high pressure, low pressure, humidification or drying are the effects on the cellular response that can quantify the redistribution, but, as noted above, perhaps the most important effects are the cells involved in the redistribution contribution. It is the effect of contacting or incubating cells with a substance known or suspected to have an effect on the response. In another embodiment of the invention, the effect may be a substance from a compound drug library. As used herein, the term "green fluorescent protein" is intended to indicate a protein that, when expressed by a cell, fluoresces when exposed to light at the exact excitation wavelength.Chalfie, M. et al., ( 1994) Science 263, 802-805.
In the following, GFP in which one or more amino acids are substituted, inserted or deleted,
Almost often referred to as "modified GFP". As used herein, `` GFP '' includes wild-type GFP from Jellyfish Aequorea Victoria and GFP modifications such as the blue fluorescent mutant of GFP disclosed by Heim et al. (Heim, R. et al. (1994) Proc. Natl. Acad. Sci.
91:26, pp12501-12504｝, and other modifications that alter the spectral properties of GFP fluorescence, or PCT / DK96, published as WO 97/11094 on March 27, 1997 and incorporated herein by reference. / 0051, exhibit increased fluorescence when expressed in cells at temperatures above about 30 ° C., and exhibit a fluorescent protein from Aequorea green fluorescent protein (GFP) or any functional analog thereof (this (The amino acid at position 1 upstream from the chromophore has been mutated so as to increase the fluorescence intensity when the fluorescent protein of the invention is expressed in cells). Preferred GFP variants are F64L-GFP, F64L-Y66H-GFP and F64L-S65T-GFP. In all aspects of this invention, a particularly preferred variant of the GFP used is EGFP (DNA encoding EGFP, which is an F64L-S65T variant with codons optimized for expression in mammalian cells, is Clontech, The plasmid available from Palo Alto and containing the EGFP DNA sequence is GenBank accession numbers U55762, U55763).

The terms “intracellular signaling pathway” and “signal transduction pathway” are intended to indicate synergistic intracellular processes by which living cells translate external or internal signals into cellular responses. The signal transduction will involve an enzymatic reaction. The enzymes include, but are not limited to, protein kinases, GTPases, ATPases, protein phosphatases, phospholipases and cyclic nucleotide phosphodiesterases. Cellular responses include, but are not limited to, gene transcription, secretion, proliferation, mechanical activity, metabolic activity, cell death. The term "second messenger" is used to indicate low molecular weight components that are involved in early events in the intracellular signal transduction pathway. The term "luminophore" is used to indicate a chemical substance that has the property of emitting light, either intrinsically or upon stimulation by chemical or physical means.
This includes, but is not limited to, fluorescence, bioluminescence, phosphorescence and chemiluminescence. The term "viable mechanically intact cells" is considered to be alive according to standard criteria for that particular cell type, such as maintaining normal membrane potential, energy metabolism, proliferative capacity, and as in microinjection. Used to indicate cells that have not undergone any physically invasive treatments designed to introduce extraneous material into such cells.

As used herein, the term “permeabilized metaplastic cell” refers to a pore-forming agent, such as streptolysin O or Staphylococcus Aureus α-toxin, which is used to generate the cells. Used to indicate cells that have been introduced into the plasma membrane. This results in proteinaceous pores with a defined pore size in the plasma membrane of the exposed cells. The pores will also be made by electroporation, a procedure that exposes cells to a high voltage discharge, a procedure that creates small holes in the plasma membrane by coagulating the required membrane proteins. The same would be obtained by treatment with a mild detergent such as saponin. Common to all these treatments is the formation of pores only in the plasma membrane without affecting the integrity of cytoplasmic components and organelles. As used herein, the term "living" refers to a permeabilizing cell, such as an endoplasmic reticulum, that is capable of absorbing and releasing actively breathing mitochondria and calcium ions in a solution that mimics the intracellular environment. It means that it still has functional organelles and functional components. The advantage of this method is that substances that normally cannot cross the plasma membrane but can be introduced into cells can be introduced and their effects can be studied without cumbersome microinjection of the substance into single cells. . By using this method, responses to effects from many cells can be recorded at the same time.

As used herein, the term “permeabilization” is intended to indicate a selective disruption of the plasma membrane barrier such that soluble substances that are freely mobile in the cytosol are lost from the cells. Permeabilization can be achieved as described above for "permeabilized living cells" or by using other chemical detergents such as Triton X-100 or digitonin in carefully titrated amounts. The term "physiologically relevant", when applied to an experimentally measured redistribution of an intracellular component as measured by a change in luminescent properties or distribution, is the basis on which the redistribution produces a redistribution. It is used to indicate that a biological phenomenon can be explained. The terms "image processing" and "image analysis" refer to large digital data analysis techniques or combinations of such techniques that reduce an ordered numeric array (image) to quantitative information describing those ordered numeric arrays. Is used to describe Therefore,
When the above ordered numerical sequence represents measurements from a physical process, the derived quantitative information is a quantification of the physical process. The term "fluorescent probe" refers to a GFP fused to a biologically active polypeptide as defined herein at the N-terminus or C-terminus, optionally via a peptide linker consisting of one or more amino acids, or a functional moiety thereof. Used to indicate a fluorescent fusion polypeptide consisting of moieties. The size of the linker peptide itself is not critical as long as the desired function of the fluorescent probe is maintained. Fluorescent probes according to the invention are expressed in cells and essentially mimic the physiological behavior of the biologically active polypeptide molecule of the fusion polypeptide.

The term “mammalian cell” is intended to indicate any living cell of mammalian origin. Cells are mostly American Type Culture Collection (ATCC, Vir
ginia, USA) or a limited lifespan progenitor cell derived from a mammalian tissue, including a transgenic animal-derived tissue, or a newly established progenitor cell derived from a mammalian tissue, including a transgenic tissue. It may be an immortal cell line, or a hybrid cell or cell line (eg, a hybridoma cell line) derived by fusing different cell types of mammalian origin. The cell may optionally express one or more non-natural gene products (eg, receptors, enzymes, enzyme substrates) prior to or in addition to the fluorescent probe. Preferred cell lines are of fibroblast origin (eg BHK, CHO, BALB) or of endothelial origin eg HUVEC, BAE (bovine artery endothelium), CPAE (cow pulmonary artery endothelium), HLMVEC
(Human lung microvascular endothelial cells) あ る い は, or those of pancreatic origin (eg RIN, INS-1,
MIN6, bTC3, aTC6, bTC6, HIT) or of hematopoietic origin (eg, initially isolated human mononuclear cells, macrophages, neutrophils, basophils, eosinophils and lymphocyte populations, AML-193, HL-60, RBL-1) or those of adipocyte origin (eg 3T3-L1
), Of neuro / neuroendocrine origin {eg, AtT20, PC12, GH3}, muscle origin (eg, SKMC, A10, C2C12), kidney origin (eg, HEK293, LLC-PK1).

The term “hybrid polypeptide” refers to at least one of each of the two proteins
It is intended to indicate a polypeptide that is a fusion of portions (in this case, at least a portion of the green fluorescent protein and at least a portion of the catalytic and / or regulatory domains of the protein kinase). Further, a hybrid polypeptide is intended to indicate a fusion polypeptide consisting of at least a portion of a green fluorescent protein comprising GFP or a functional fluorophore, and at least a portion of a biologically active polypeptide. Thus, GFP may be labeled at the N- or C-terminus of the biologically active polypeptide, optionally with a linker moiety or peptide comprising one or more amino acid sequences. The hybrid or fusion polypeptide is
Under conditions that allow expression of the hybrid polypeptide, it will act as a fluorescent probe in whole living cells carrying the DNA sequence encoding the hybrid polypeptide. The term "kinase" is intended to indicate an enzyme that can phosphorylate cellular components. The term "protein kinase" is intended to indicate an enzyme capable of phosphorylating serine and / or threonine and / or tyrosine in peptides and / or proteins. The term "phosphatase" is intended to indicate an enzyme capable of dephosphorylating phosphoserine and / or phosphothreonine and / or phosphotyrosine in peptides and / or proteins. The term "cyclic nucleotide phosphodiesterase" is intended to indicate an enzyme capable of inactivating the second messengers cAMP and cGMP by hydrolysis of the 3'-ester bond.

As used herein, the term “biologically active polypeptides” refers to those enzymes that are active in intracellular processes or those that comprise a desired amino acid sequence that have a biological function or that exert a biological effect in a cell line. It is intended to indicate polypeptides, such as some, that upon activation affect intracellular processes. In the polypeptide, one or several amino acids may be deleted, inserted or substituted to alter its biological function, for example, by inactivating a catalytic site. Preferably, the biologically active polypeptide is derived from proteins that participate in intracellular signaling pathways, such as enzymes involved in intracellular phosphorylation and dephosphorylation processes, including kinases, protein kinases and phosphorylases as defined herein. Although selected from the group, proteins that make up the cytoskeleton also play an important role in intracellular signal transduction and are thus included within the meaning of "bioactive polypeptide". More preferably, the biologically active polypeptide is a protein that changes its intracellular localization according to an activated or deactivated state, such as an intermediate component, preferably in a signal transduction pathway. This preferred group of bioactive polypeptides includes cAMP-dependent protein kinase A.

The term “biologically active substance” is intended to indicate any sample that has a biological function or that exerts a biological effect in a cell line. The sample may be a sample of biological fluids, such as a sample of biological fluids such as blood, plasma, saliva, milk, urine or a sample of microorganisms or plant extracts, an environmental sample containing contaminants (including heavy metals or toxins). Or a sample containing a compound or a mixture of compounds prepared by organic synthesis or genetic techniques. The expression "any change in fluorescence" is any change in absorption properties, such as wavelength and intensity, or any change in the spectral properties of emitted light, such as a change in wavelength, fluorescence lifetime, intensity or polarity, or It means any change in the subcellular localization of the fluorophore. In this way, it may be localized to specific cellular components (eg, organelles, membranes, cytoskeleton, molecular structure) and may be uniformly distributed throughout the cell or part of the cell. The term "organism" as used herein refers to any unicellular or multicellular organism preferably from the animal kingdom, including protozoa, but is an organism that is a member of the plant kingdom such as algae, fungi, bryophytes. ), And vascular plants also
Included in this definition.

The term “nucleic acid” is intended to indicate any type of poly or oligo nucleic acid sequence, such as a DNA, cDNA or RNA sequence. The term `` biologically equivalent '' in reference to proteins refers to splices where the two proteins are capable of replacing the cellular functions of each other, e.g., where the two proteins are closely related isoforms encoded by different genes. A variant or an allelic variant from the same gene means that the first protein is equivalent to the second protein if they perform the same cell function in different cell types or different species. The term "biologically equivalent" for DNA
Means that the first DNA sequence encoding the polypeptide is equal to the second DNA sequence encoding the polypeptide if the functional proteins encoded by the two genes are biologically equivalent. The term "high throughput" is intended to mean an increase in the number of experiments per unit of time per person performing the actual experiment. As used herein, the term "high throughput screening assay" refers to a screening assay in which one of the skilled artisans has at least 100 individual experiments testing a compound for its effect on luminophore redistribution on a single working day. It is intended to mean how to do. In a preferred embodiment, a high-throughput screening assay comprises at least 500 individual experiments, eg, 750, 1000,
With up to 2000, 5000, or 10,000 individual experiments.

The expression “back-tracking of signal transduction pathways” refers to a process that more accurately reveals the levels at which signal transduction pathways are affected, either by the effects of chemical compounds or disease states in an organism. Intend. Consider the specific signal transduction pathway exhibited by the biologically active polypeptide ABCD, where signal transduction goes from A to D. In studying all components of this signal transduction pathway, compounds or disease states that affect the activity or redistribution of D alone may act on C or the C downstream, whereas in A and B Compounds or disease states that affect the activity or redistribution of C and D without affecting the underlying tissue of B are considered. The term "fixed cells" is used to mean cells that have been treated with a cell fixative such as glutaraldehyde or formaldehyde, which treatment chemically and chemically cross-links soluble and insoluble proteins within the structure of the cell. Helps to fix and fix. Once in this state, such proteins are not lost from the current structure of dead cells.

As used herein, a “screening assay” refers to a mechanically intact or permeabilized raw material associated with a material, cell, instrument, chemical, reagent, detection unit, effect on an intracellular pathway. By any measurement protocol, including calibration and quantification procedures used to measure the response from the cells. As used herein, the terms "dose-response relationship" and "screening program" refer to a clear correlation between the quantified cellular response in a screening assay for the application of an effect, such as a compound, and the concentration of the effect applied. . The response to the effect may be both up-regulation and down-regulation of the quantification parameters used in the screening assay. As used herein, the term "physiology" is intended to mean the normal functioning of biological and biochemical intracellular processes between cells and whole organisms or animals.

BRIEF DESCRIPTION OF THE DRAWINGS FIG . 1 CHO cells expressing the PKAc-F64L-S65T-GFP hybrid protein were transformed with HAM F
Treated with 50 μM forskolin at 37 ° C. in 12 media. Images of GFP fluorescence in these cells are shown at different time intervals after treatment: a) 40 seconds, b) 60
Seconds, c) 70 seconds, d) 80 seconds. In the (nucleated) cytoplasm, the fluorescence changes from speckled to a more uniform distribution. Figure 2. Time course analysis of PKAc-F64L-S65T-GFP redistribution induced by forskolin. PKAc-F6
CHO cells expressing the 4L-S65T-GFP fusion protein were analyzed by fluorescence microscopy over time. Fluorescence micrographs were taken at regular intervals from 2 minutes before and 8 minutes after addition of the agonist. Immediately after the upper left image was obtained (t = 0), cells were examined with 1 μM forskolin. The frames are the following times: i) 0, ii) 1, iii) 2, iv) 3, v) 4 and vi) 5
Collected in minutes. Measurement bar 10 μm.

FIG. 3. Time course analysis of PKAc-F64L-S65T-GFP redistribution for various agonists. PKAc-F6
1 μM forskolin (A), 50 μL for localization of 4L-S65T-GFP fusion protein
The effect of M forskolin (B), 1 mM dbcAMP (C) and 100 μM IBMX (D) (addition indicated by white arrow) analyzed by fluorescence microscopy of CHO / PKAc-F64L-S65T-GFP cells over time did. The effect on the localization of the PKAc-F64L-S65T-GFP fusion protein by adding 10 μM forskolin (open arrow), and immediately thereafter, repeating washing with a buffer (black arrow) was analyzed in the same cells (E ). 10 μM in parallel experiments
Was added (white arrow), and the effect of repeated washing with a buffer containing 100 μM IBMX (black arrow) was analyzed (F). Removal of forskolin returns the PKAc-F64L-S65T-GFP fusion protein to its cytoplasmic aggregate, which is prevented by the continued presence of IBMX (F)
. The effect of 100 nM glucagon on the localization of the PKAc-F64L-S65T-GFP fusion protein (Figure 3G, white arrow) is also shown for BHK / GR, PKAc-F64L-S65T-GFP cells. PK
10 μM norepinephrine for localization of Ac-F64L-S65T-GFP fusion protein (
The effect of (black arrow) was pre-treated with 10 μm forskolin (white arrow) and analyzed in transiently transformed CHO and PKAc-F64L-S65T-GFP cells to increase [cAMP]. . Note that in FIG. 3H, the x-axis calculates the number of images every 12 seconds. The raw data for each experiment consisted of 60 fluorescence micrographs taken at regular intervals, including several images obtained before the addition of buffer or agonist. Charts (AG) each show the quantification of the response seen in all 60 images, performed as described in Method 2. The change in the total area of the highly fluorescent aggregate relative to the initial area of the fluorescent aggregate was plotted in each experiment as the ordinate versus time in all graphs of FIG. Measurement bar 10 μm.

FIG. 4. Dose-response curves for forskolin-induced redistribution of PKAc-F64L-S65T-GFP fusions (two experiments). Fig. 5 Time from the start of response until the redistribution of PKAc-F64L-S65T-GFP reaches half the maximum (t1 _{/ 2max} ) and the maximum ( _tmax ). Data was extracted from curves as shown in "Figure 2". All t _{1/2 max} and t _max values are mean ± SD and are based on the sum of 26-30 cells from 2-3 independent experiments at each forskolin concentration. Since the observed redistribution is maintained for a long time, the _tmax value is the earliest point at which complete redistribution is reached. Note that this value is independent of the degree of redistribution. FIG. 6. Parallel dose-response analysis of forskolin-induced cAMP elevation and PKAc-F64L-S65T-GFP redistribution. The effect of buffer or five-step increasing concentrations of forskolin on the localization of the PKAc-F64L-S65T-GFP fusion protein in CHO / PKAc-F64L-S65T-GFP cells grown in 96-well plates was determined as described above. Was analyzed as follows. Computer calculation of the SD ratio of fluorescence micrographs taken in the same field of cells before and 30 minutes after the addition of forskolin provides a reproducible measure of PKAc-F64L-S65T-GFP redistribution. Was done. The graph shows a trace of 48 individual measurements and their mean ± sem at each forskolin concentration. For comparison, the effect of buffer or 8-step increasing concentrations of forskolin on [cAMP] i was analyzed by a scintillation proximity assay of cells grown under the same conditions. The graph shows a trace of the mean ± sem shown for any unit of the four experiments.

FIG. 7 BHK cells stably transfected with a fusion of human muscarinic (hM1) receptor and PKCα-F64L-S65T-GFP. Carbachol (100 μM added in 1.0 second) is PKCα-F6
Transient redistribution of 4L-S65T-GFP from the cytoplasm to the plasma membrane was induced. Images were taken at the following times: 1) 1 second before carbachol addition, b) 8.8 seconds after addition, and c) 52.8 seconds after addition. Figure 8 B stably transfected with hM1 receptor and PKCα-F64L-S65T-GFP fusion
HK cells were treated with carbachol (1 μM, 10 μM, 100 μM). In one cell, intracellular [Ca ²⁺ ] was observed simultaneously with the redistribution of PKCα-F64L-S65T-GFP. The dashed line indicates when carbachol was added. The upper panel shows the time course of the intracellular Ca ²⁺ concentration of each cell in each treatment. The middle panel shows the time course of the average cytoplasmic GFP fluorescence of each cell in each treatment. The lower panel shows the change in fluorescence of one cell, as seen in normal images, especially at the periphery within the area that includes the cell periphery. The best area to observe changes in fluorescence intensity is the cell membrane.

FIG. 9 hERK-1-F64L-S65T-GFP fusion expressed in HEK293 cells treated with 100 μM MEK1 inhibitor PD98059 in HAM F-12 (without serum) for 30 minutes at 37 ° C. During this process, the nuclei lose fluorescence. Cells similar to (a) were treated with 10% fetal bovine serum at 37 ° C. for 15 minutes. The time profile of GFP fluorescence redistribution in HEK293 cells was determined using Hepes buffer (HA
MF-12 results from treatment with various EGF concentrations in Hepes buffer just before the experiment). Fluorescence redistribution is expressed as a change in the nuclear to cytoplasmic area ratio of a single cell. Each time profile is the average of the changes seen in six single cells. The bar graph measuring the change in fluorescence (nucleus: cytoplasm) at the end point, which is 600 seconds after the start of EGF treatment, according to various concentrations of EGF. FIG. 10. SMAD2-EGFP fusion expressed in HEK293 cells serum starved overnight in HAM F-12. HAM F-12 was subsequently replaced with pH 7.2 Hepes buffer just prior to the experiment. Measurement bar 10 μm. HEK293 cells expressing the SMAD2-EGFP fusion were treated with various concentrations of TGF-β as described.
And observed the redistribution of fluorescence over time. The time profile plot represents the increase in fluorescence in the nucleus and is normalized to the initial value of each cell measured. Each trace is a single cell nucleus time profile. The bar graph shows the change in the end point of fluorescence in the nucleus for different concentrations of TGF-β (
850 seconds after processing). Each bar is the value of one nucleus in each treatment.

FIG. 11 V in CHO cells stably transfected with the human insulin receptor
ASP-F64L-S65T-GFP fusion. Cells were starved for 2 hours in HAM F-12 without serum and then treated with 10% fetal calf serum. Images show fluorescence redistribution obtained 15 minutes after processing. GFP fluorescence shows local attachment along the length of the actin stress fiber (fo
cal adhesion). FIG. 12. Dose-response relationship of translocation of PKCα-GFP in BHKhM1 cells stimulated with the muscarinic agonist carbamylcholine using FLIPR ^™ to perform actual experiments. Figure 13 CHO / P stimulated with forskolin using FLIPR ^™ to perform the actual experiment
Dose-response relationship of translocation of PKCα-GFP in KAc-F64L-S65T-GFP cells. FIG. 14 CHO cells stably expressing human insulin receptor and mouse cPKA-labeled S65T-GFP were examined in more detail on a FLIPR ^™ instrument. Six different wells were imaged over time for each concentration to obtain a dose-response of forskolin, a substance that increases the production of adenylate cyclase of cAMP in cells. Fluorescence changes were calculated as AUC (area under the curve) at 9 minutes of stimulation. Conclusion: Despite the fact that all wells containing approximately 50-100,000 cells are illuminated and simultaneously image single cells or subcellular events at a spatial resolution that cannot be resolved, the redistribution of mouse cPKA-BioST is FLIPR ^Can be detected by ^TM . This method can be used for simultaneous measurement of intracellular cAMP levels and should be used as a screening assay to measure the effect of ligand on G-protein coupled receptors that bind Gi and Gq-type G-proteins. Can be.

FIG. 15. Permeabilized CHO / when pre-exposed to different doses of forskolin
Dose-response relationship of the quenched fluorescence from PKAc-F64L-S65T-GFP. FIG. 16 CHO cells stably expressing human insulin receptor and human PKCβ1 labeled with EGFP were examined microscopically. A series of cells were imaged over time at each concentration, resulting in a dose-response. Fluorescence changes were calculated as AUC over 4 minutes of stimulation. The following data was extracted from the images: All images: A mere analysis of the change in intensity of the whole image, including cells and background. Single cells: 5 independent cells were analyzed after background compensation. Analysis was performed on whole cells. Cytoplasm: The same 5 cells as above were analyzed after background compensation. The analysis was performed on a small area of cytoplasm near the nucleus. Conclusion: Redistribution of human PKCβ1-EGFP is detectable only when analyzing a subregion of each cell. This event is clearly visible when viewing a sequence of images like a movie. However, no redistribution is detected when the fluorescence change of the whole image or the whole cell is analyzed.

FIG. 17 CHO cells stably expressing human insulin receptor and human PKCβ1 labeled with EGFP were tested with FLIPR ^™ . Six different wells were imaged over time for each concentration to obtain a dose-response. Fluorescence changes were calculated as AUC with a 5-minute stimulation. Conclusion: Due to the fact that all cells, including about 50-100,000 cells, are illuminated and imaged by the detector at a much lower resolution than required to resolve single cells or subcellular structures. Nevertheless, redistribution of human PKCβ1-EGFP is detectable on the FLIPR ^™ instrument. This phenomenon cannot be clearly predicted from the microscope data of FIG. FIG. 18 CHO cells stably expressing the insulin receptor and the human NFkB-GFP protein hybrid were stimulated with various concentrations of IL-1 for 1 hour, then in a low osmosis buffer (TRI
S-base 10 mM, MgCl _{2 2} mM, PMSF (phenylmethylsulfonyl fluoride) 0.5 m
M, pH 7.4) and mounted on a microscope. A series of images is obtained during the addition of 0.05% Triton X-100, followed by gentle mixing and short incubation. The treatment disrupts the cell membrane and leaves a fraction of NFkB-GFP that later translocates to the nucleus, but some of the cytoplasmic probe leaves the cells more quickly and is quickly diluted indefinitely in the surrounding medium (focus Mismatch-this part of the total fluorescence from the probe is thereby lost)
. At predetermined times before and after this processing, all intensity values for all images were extracted. Dividing the later value by the previous value to normalize each experiment shows that more NFkB has been translocated to the nucleus, thereby indicating a higher proportion of cells contributing to total fluorescence after transmission. Results: This protocol is a good example that may indicate translocation of a fluorescent probe from cytoplasm to nucleus or translocation from nucleus to cytoplasm.

[0064] EXAMPLES Example 1: Construction of cAMP assays based on PKA activation, by activation of the test method and the execution example G _s or G _l binds to a class of G- protein G- protein coupled receptors, the cAMP Useful for monitoring the activity of signal pathways that cause concentration changes. Murine cAMP-dependent protein kinase catalytic subunit (PKAc)
F64L-S65T derivative at the C-terminus. The obtained fusion (PKAc-F64L-S65T-GF
P) was used to monitor translocation in vivo and thereby monitor PKA activation. To construct a PKAc-F64L-S65T-GFP fusion, convenient restriction endonuclease sites were added to mouse PKAc (Gen Bank accession number: M12303) and F64L-S65T-GFP (W
Polymerase chain reaction (PCR) to the cDNA encoding the sequence disclosed in O97 / 11094)
Introduced by The PCR reaction was performed using the following primers based on a standard protocol. The amplified product of PKAc was then digested with HindIII + Ascl, and the F64L-S65T-GFP product was digested with Ascl +
Digested with Xhol. The two digested PCR products are then ligated with a plasmid (pZeoSV ^™ mammalian expression vector, Invitrogen, San Diego, CA, USA) digested with HindIII + Xhol. The resulting fusion constructs (SEQ ID NOs: 1 and 2) were under the control of the SV40 promoter.

Transfection and cell culture conditions: Chinese hamster ovary cells (CHO) were transfected with a plasmid containing the PKAc-F64L-S65T-GFP fusion by using calcium phosphate precipitation in HEPES buffered saline (Sambrook Et al., 1989). Growth medium (Technologies Inc., Ga
1000 mg / l glucose, 10% fetal bovine serum (F) purchased from ithersburg, MD, USA
Stable transfectants were selected using 1000 μg / ml zeocin (Invitrogen) in 100 μg / ml penicillin-streptomycin mixture, DMEM containing 2 mM L-glutamine). Untransfected CHO cells were used as controls. To evaluate the effect of glucagon on fusion protein translocation, a PKAc-F64L-S65T-GFP fusion was stably expressed in human hamster kidney cells (BHK / GR cells) overexpressing the human glucagon receptor. . Untransfected BHK / GR cells were used as controls. GR expression was maintained at 500 μg / ml G418 (Neo marker) and PKAc-F64L-S65T-GFP was maintained at 500 μg / ml zeocin (Shble marker). CHO cells were also co-transfected with the PKAc-F64L-S65T-GFP fusion and a vector containing the human α2a adrenal receptor (hARa2a).

For fluorescence microscopy, cells were allowed to adhere to Lab-Tek chamber coverslips (Nalge Nunc Int., Naperville, Ill., USA) for at least 24 hours and were allowed to reach approximately 80%
Cultured to confluence. Prior to the experiment, cells were glutamax (Life Technologies
), HAM containing 100 μg / ml penicillin-streptomycin mixture and 0.3% FBS
The cells were cultured overnight in F-12 medium without selection pressure. This medium has low autofluorescence that allows for cell fluorescence microscopy directly from the incubator. Simultaneous monitoring of PKA activity: Capture live cell images with Fluar 40X, NA: 1.3 oil immersion lens, Photometrics C
Collection was performed using a Zeiss Axiovert 135M fluorescence microscope connected to an H250 charge coupled device (CCD) camera. Cells were irradiated with a 100 W HBC arc lamp. In the light path there is a 470 ± 20 nm excitation filter, a 510 nm dichroic mirror and a 515 ± 15 nm emission filter to minimize image background. Cells were maintained at 37 ° C. with a custom stage heater.

The images were processed and analyzed in the following way: Method 1: Stepwise procedure for quantifying PKA dislocations Dark images (taken under the same conditions as the real images, except that the camera shutter is not opened) The image was corrected for dark current by removing pixel by pixel. The non-uniformity of the illuminance of the image was corrected by performing a pixel-by-pixel ratio with the flat-field corrected image (the image captured under the same conditions as the actual image of the uniform fluorescent specimen). The frequency of occurrence of each intensity value in the bar graph of the image, that is, the image was calculated. A smooth quadratic function (smoothed, second derivative) of the bar graph was calculated, and a second order 0 (second zero) was determined. This 0 corresponds to the inflection point of the bar graph at the higher side of the main peak representing the bulk of the image pixel values. The value determined in step 4 is deleted from the image. All negative values were removed. The square deviation (standard deviation squared) of the remaining pixel values was determined. This value represents the "response" of the image. Scintillation proximity assay (SPA) for individual quantification of cAMP.

Method 2: Another Method for Quantifying PKA Redistribution Using a threshold obtained automatically based on maximizing an information measure between subject and background, the set of fluorescence is Each image was split. Use a priori entropy in the image bar graph as an information criterion. The area occupied by the set in each image was calculated by counting the pixels in the divided regions. The values obtained in step 2 for each image in the continuous or treatment pair are normalized to the values found in the first (unstimulated) image collected. A value of zero (0) indicates that the fluorescence has not been redistributed from the starting state. A value of 1 (1) according to this method is equivalent to a perfect redistribution. Cells were cultured in HAM F-12 medium as described above except that they were performed in 96-well plates. The medium was replaced with Ca ²⁺ -HEPES buffer containing 100 μM IBMX and cells were stimulated with different concentrations of forskolin for 10 minutes. The reaction was stopped by adding NaOH to 0.14 M, and the amount of cAMP produced was measured using cAMP-SPA kit RPA538 (Amersham) as described by the manufacturer. Manipulation of cAMP at the intracellular level to test PKAc-F64L-S65T-GFP fusions. The following compounds were used to alter cAMP levels: forskolin, an activator of adenylate cyclase; dbcAMP, a membrane-permeable cAMP analog that is not degraded by phosphodiesterase; IBMX, an inhibitor of phosphodiesterase.

In CHO cells stably expressing PKAc-F64L-S65T-GFP, 1 μM forskolin (
Spotted distribution of fusion protein after stimulation with 10 μM forskolin (n = 4), 50 μM forskolin (n = 4) (FIG. 1), or 1 mM dbcAMP (n = 6) Dramatically translocated from to a uniform distribution throughout the cytoplasm. FIG. 2 shows the time course of the response after treatment with 1 μM forskolin. In Figure 3,
Comparison of mean time profiles of fusion protein redistribution, and the absence of three forskolin concentrations (Figures 3A, E, B), similar but slow responding 1 mM dbcAMP (Figure 3C), and no adenylate cyclase stimulation Also slows response 100 μM IBMX (n = 4, Figure 3
The measurement results of the degree of each response to the addition of D) are shown. Addition of buffer (n = 2) had no effect (data not shown). As a control for the behavior of the fusion protein, only F64L-S65T-GFP was expressed in CHO cells and given 50 μM forskolin (n = 5). GFP in these cells
Was not affected by such treatment (data not shown). Translocation of PKAc-F64L-S65T-GFP induced by forskolin showed a dose-response relationship (FIGS. 4 and 6). See quantification procedure above.

Reversibility of PKAc-F64L-S65T-GFP translocation The release of PKAc probe from cytoplasmically fixed hot spots was reversible. Ten
After treatment with μM forskolin, the cells were washed repeatedly with buffer (5-8 times) and the spotted pattern was completely restored in 2-5 minutes (n = 2, FIG. 3E). In fact,
The fusion protein returned to a pattern of intracellular fluorescent assembly that was almost indistinguishable from that seen before forskolin stimulation. To test whether fusion protein reversion to cytoplasmic aggregates reflects reduced [cAMP] _i , cells were incubated with 10 μM forskolin and 100 μM IBMX (n
= 2) and then repeated with buffer containing 100 μM IBMX (5-8
Times) (FIG. 3F). In these experiments, the fusion protein did not return to its pre-stimulatory position after forskolin removal.

Testing of PKA-F64L-S65T-GFP Probe with Physiologically Relevant Agents To test the response of the probe to receptor activation of adenylate cyclase, it was stably transfected with the glucagon receptor. BHK cells and PKA-F64L-S6
The 5T-GFP probe was exposed to glucagon stimulation. Glucagon receptors increases cAMP levels and coupled to G _s protein which activates adenylate cyclase. In these cells, addition of 100 nM glucagon (n = 2) resulted in P
Release of the KA-F64L-S65T-GFP probe occurs, resulting in translocation of the fusion protein within 2-3 minutes and more even distribution within the cytoplasm (FIG. 3G). Similar but less pronounced effects were seen at lower glucagon concentrations (n = 2, data not shown)
. Addition of buffer (n = 2) had no effect over a long period of time (data not shown). Transiently transfected CHO cells expressing hARα2a and the PKA-F64L-S65T-GFP probe were treated with 10 μM forskolin for 7.5 minutes, and then subsequently exposed to 10 μM norepinephrine in the presence of forskolin for exogenous Stimulates sex adrenal receptors. It binds to G _I protein, adenylate cyclase is inhibited.
This treatment re-emits fluorescence in cytoplasmic aggregates indicating a decrease in [cAMP] _i (Fig.
3H).

Fusion Protein Translocation Correlated to [cAMP] _i As described above, the time to complete the response was dependent on the forskolin dose (FIG. 5). The extent of the response was also dose dependent. To test PKA-F64L-S65T-GFP fusion protein translocation in a slightly higher throughput system, use PKA-F64L-S6
CHO cells stably transfected with the 5T-GFP fusion were stimulated with buffer and 5 escalating doses of forskolin (n = 8). Utilizing the image analysis algorithm described above (method 1), a dose-response relationship was observed in the forskolin range of 0.01-50 μM (FIG. 6).
Half-maximal stimulation was seen at about 2 μM forskolin. In parallel, cells were stimulated with buffer and forskolin in increasing concentrations in eight steps in the range of 0.01-50 μM (n = 4). The amount of cAMP produced was measured in a SPA assay. A sharp increase was seen between 1-5 μM forskolin, which coincided with the steepest part of the fusion protein translocation curve (FIG. 6).

Example 2 Construction of Probe PKC-GFP Fusion for Detection of PKC Activity cDNA of mouse PKCα (GenBank accession number: M25811) and F64L-S65T-GFP (WO97 / 1109)
Probes were constructed by ligating two products, each of which was amplified by polymerase chain reaction (PCR) and treated with a restriction enzyme. Taq® polymerase and the following oligonucleotide primers were used for PCR. As the HindIII-Xhol cassette described in Example 1, the hybrid DNA strand was pZeoSV (
®) mammalian expression vector. BHK cells that express the human M1 receptor under the control of the inducible metallothionein promoter and are maintained by the dihydrofolate reductase marker were purified from HEPES-buffered saline (HBS [pH
7.10]) and transfected with the PKCα-F64L-S65T-GFP probe using the calcium phosphate precipitation method. Stable transfectants were grown at 1000 μg / ml in growth medium (DMEM containing 1000 mg / l glucose, 10% fetal bovine serum (FBS), 100 μg / ml penicillin-streptomycin mixture, 2 mM L-glutamine). Was selected using Zeocin®. The hM1 receptor and PKCα-F64L-S65T-GFP fusion protein were combined with 500 nM methotrexate and 500 μg / ml Zeocin®, respectively.
Maintained. Twenty-four hours before all experiments, cells were transferred to HAM F-12 medium containing glutamax, 100 μg / ml penicillin-streptomycin mixture and 0.3% FBS. Since this medium relieves the selection pressure, it induces less signal transduction pathways and has low autofluorescence at the appropriate wavelength to allow cytofluorescence microscopy directly from the incubator.

Method 1: Simultaneous Monitoring of PCAα Activity Digital images of live cells were taken with a 40 ×, NA: 1.3 oil immersion lens and Photometrics CH
Collection was performed using a Zeiss Axiovert 135M fluorescence microscope connected to a 250 charge coupled device (CCD) camera. Cells were irradiated with a 100 W HBC arc lamp. In the light path there is a 470 ± 20 nm excitation filter, a 510 nm dichroic mirror and a 515 ± 15 nm emission filter to minimize image background. Cells were monitored at 37 ° C. with a custom stage heater. Images were analyzed using the Macintosh IPLab software package. Stimulation of M1-BHK cells stably expressing the PKCα-F64L-S65T-GFP fusion with carbachol resulted in a dose-dependent transient translocation from the cytoplasm to the plasma membrane (FIGS. 7a, b, c)
. Simultaneous measurement of the cytosolic free calcium concentration shows that carbachol-induced calcium mobilization precedes translocation (FIG. 8). A step-by-step method for quantifying PKCα dislocations: The dark currant of an image is removed pixel by pixel from a dark image (an image taken under the same conditions as the actual image except that the camera shutter is not opened) Corrected.

The illuminance non-uniformity of the image was corrected by performing a pixel-by-pixel ratio with a flat-field corrected image (an image captured under the same conditions as the actual image of the uniform fluorescent specimen). A copy of the image with defined edges was made. The edge of the image is a standard edge that involves an image with a nucleus that removes all large non-changing elements (ie, background) and emphasizes any small changes (ie, sharp edges). Was found by the part detection method. This image was then converted to a binary image by setting a threshold. Subjects that were too small to represent cell edges in the binary image were removed. The binary image was extended to cover all gaps at the edges of the image. All edge objects in the image that touched the edges of the image were removed. This binary image represents the edge mask. Another copy of the image was made by the procedure of Step 3. This copy is further processed to detect objects surrounding the "hole" and to make all pixels in the hole a binary value at the edge, i.e. one. This image represents the whole cell mask. Mask the first image with the edge mask from step 3 and measure the sum of all pixel values. The first image was masked with the whole cell mask from step 4 and the sum of all pixel values was measured. Dividing the value of step 5 by the value of step 6 gives the final result, a fraction of the fluorescence intensity in the cells that were localized at the edges.

Example 3 Probes for Detecting Redistribution of Mitogen-Activated Protein Kinase Erk1 Useful for monitoring signal pathways involving MAPK, for example, compounds that modulate the activity of pathways in living cells. Identify. Erk1, a serine / threonine protein kinase, is a component of a signaling pathway that is activated, for example, by many growth factors. Probe for simultaneous detection of ERK-1 activity in living cells: N- or C-terminal fusion of a kinase (ERK-1, mitogen-activated protein kinase, MAPK) regulated by an extracellular signal to a GFP derivative I do. Fusions expressed in different mammalian cells are used to monitor nuclear translocation in vivo and thus ERK1 activation in response to stimuli that activate the MAPK pathway. The human Erk1 gene (GenBank accession number: X60188) was subjected to PCR based on a standard protocol.
Was amplified using primers. Erk 1-Top And Erk1-bottom / + stop The PCR product was digested with restriction enzymes EcoR1 and BamH1, and p digested with EcoR1 and BamH1.
Ligation to EGFP-C1 (Clontech, Palo Alto; GenBank accession number U55763). This produces an EGFP-Erk1 fusion (SEQ ID Nos. 5 and 6) under the control of the CMV promoter.

The plasmid containing the EGFP-Erk1 fusion was prepared using the FUGENE transfection reagent (
HEK293 cells were transfected using Boehringer Mannheim). Prior to the experiment, cells were grown to 80-90% confluence in DMEM containing 10% FCS in 8 well chambers. MEK, a kinase that activates Erk1 in HAM F-12 medium without any cells (without FCS), washed in HAM F-12 medium without any cells (without FCS)
Incubated with 100 μM of 1 inhibitor PD98059 for 30-60 minutes. This step effectively exports the nucleus of EGFP-Erk1. HAM F-12 just before starting the experiment
Is replaced with Hepes buffer, followed by washing with Hepes buffer. As a result, PD98
[059] The inhibitor is removed. If further blocking of MEK1 is required (eg, in a control experiment), the inhibitor is included in Hepes buffer. The experimental equipment for the microscope was as described in Example 1. Sixty images were collected at 10 second intervals each. From image number 10 onwards, test compounds were added. Addition of EGE (1-100 nM) resulted in redistribution of EGFP-Erk1 from cytoplasm to nucleus within minutes (FIGS. 9a, b). Responses were quantified as described below and a dose-dependent relationship between EGF concentration and nuclear translocation of EGFP-Erk1 was found (FIGS. 9c, d). In this example, the redistribution of GFP fluorescence is shown as a change in the area ratio of the cell's nucleus to cytoplasmic component. Each time profile is the average of the nucleus to cytoplasm ratio from 6 cells in each treatment.

Example 4 Probes for Detecting Smad2 Redistribution Useful for monitoring signaling pathways activated by some members of the transforming growth factor β family, eg, the activity of the pathway in living cells Compounds that modulate are identified. Smad2, a signal transducer, is a component of the signaling pathway triggered by several members of the TGFβ family of cytokines. a) The human Smad2 gene (GenBank accession number: AF027964) was amplified using primers by PCR based on a standard protocol. Smad2-top And Smad2-bottom / + stop The PCR product was digested with restriction enzymes EcoR1 and Acc65I and ligated to pEGFP-C1 (Clontech, Palo Alto; GenBank accession number U55763) digested with EcoR1 and Acc65I. This produces an EGFP-Smad2 fusion (SEQ ID Nos: 7 and 8) under the control of the CMV promoter.

B) The human Smad2 gene (GenBank accession number: AF027964) was amplified using primers by PCR based on a standard protocol. Smad2-top And Smad2-bottom / -stop The PCR product was digested with restriction enzymes EcoR1 and Acc65l and ligated to pEGFP-N1 (Clontech, Palo Alto; GenBank accession number: U55762) digested with EcoR1 and Acc65l. This produces a Smad2-EGFP fusion (SEQ ID Nos: 9 and 10) under the control of the CMV promoter.

Transfection of HEK293 cells with a plasmid containing the EGFP-Smad2 fusion resulted in cytoplasmic redistribution. Prior to the experiment, cells were placed in an 8-well Nunc chamber for 10
The cells were grown to 80% confluency in DMEM containing% FCS and starved overnight in HAM F-12 medium without FCS. In the experiment, HAM F-12 medium was replaced with Hepes buffer at pH 7.2. The experimental equipment for the microscope was as described in Example 1. Ninety images were collected at 10 second intervals each. From image number 5 onwards, test compounds were added. After serum deprivation of the cells, each nucleus had lower GFP fluorescence compared to the surrounding cytoplasm (FIG. 10a). Addition of TGFβ resulted in redistribution of EFGP-Smad2 from cytoplasm to nucleus in minutes (FIG. 10b). The redistribution of fluorescence in treated cells was determined by nuclear GF in each unstimulated cell.
It was quantified as a mere increase in the fraction of nuclear fluorescence normalized to the initial value of P fluorescence. Dose-dependent changes to TGFβ were displayed (FIG. 10c).

Example 5 Probes for Detecting VASP Redistribution Identify compounds that are useful for monitoring signal pathways that involve rearrangement of cytoskeletal structures, for example, that modulate the activity of the pathway in living cells. VASP, a phosphoprotein, is a component of the cytoskeletal structure and redistributes in response to signals affecting local adhesion. The human VASP gene (GenBank accession number: Z46399) was amplified using primers by PCR based on a standard protocol. VASP-Top And VASP-bottom / + stop

The PCR product was digested with the restriction enzymes Hind3 and BamH1 and p digested with Hind3 and BamH1.
It was linked to EGFP-C1 (Clontech, Palo Alto; GenBank accession number U55763). This produces an EGFP-VASP fusion (SEQ ID Nos: 11 and 12) under the control of the CMV promoter. The resulting plasmid was transfected into human insulin receptor expressing CHO cells using the calcium phosphate transfection method. Before the experiment,
Cells were grown in 8-well Nunc chambers and starved overnight in media without FCS. The experiment was performed using the microscope equipment described in Example 1. 10% FCS was added to the cells and images were collected. The EGFP-VASP fusion redistributed from a more or less uniform distribution near the periphery to a more localized structure and was identified as a local attachment point (FIG. 11).

Example 7 Probes for Detecting NF-Kappa B Redistribution Compounds useful for monitoring the signaling pathways leading to the activation of NF-kappa B, for example, compounds that modulate the activity of living cell pathways Identify. NF-kappa B, an activator of transcription, is a component of a signaling pathway that responds to a variety of triggers, including cytokines, lymphokines and some immunosuppressants. a) The human NF kappa B p65 subunit gene (GenBank accession number: M62399) was amplified using primers by PCR based on a standard protocol. NF kappa B-top And NF kappa B-bottom / + stop PCR product digested with restriction enzymes Xho1 and BamH1, pEG digested with Xho1 and BamH1
It was linked to FP-C1 (Clontech, Palo Alto; GenBank accession number U55763). This is C
Produce an EGFP-NF kappa B fusion (SEQ ID Nos: 13 and 14) under the control of the MV promoter.

B) The human NF kappa B p65 subunit gene (GenBank accession number: M62399)
Amplification was performed using primers in PCR based on standard protocols. NF kappa B-top And NF kappa B-bottom / -stop The PCR product was digested with restriction enzymes Xho1 and BamH1, and digested with Xho1 and BamH1.
It was linked to EGFP-N1 (Clontech, Palo Alto; GenBank accession number U55762). This produces an NF kappa B-EGFP fusion (SEQ ID Nos: 15 and 16) under the control of the CMV promoter.

When the resulting plasmid is transfected into a suitable cell line, such as Jurkat, the EGFP-NF kappa B probe and / or the NF kappa B-EGFP probe activates the signal pathway, eg, by IL-1 Alters cell distribution from the cytoplasm to the nucleus in response to CHO cells stably expressing the insulin receptor and the human NFkB-GFP protein hybrid are stimulated with different concentrations of IL-1 for 1 hour and then in a low osmosis buffer (TRIS
Base 10 mM, MgCl _{2 2} mM, PMSF (phenylmethylsulfonyl fluoride) 0.5 mM, pH
Washed in 7.4) and placed on a microscope. Image continuations were obtained while adding 0.05% Triton X-100, followed by gentle mixing and brief incubation. This treatment ruptures the cell membrane and leaves a fraction of NFkB-GFP that later translocates to the nucleus, but a certain amount of cytoplasmic probe leaves the cell faster and is immediately diluted indefinitely in the surrounding medium (out of focus) -This part of the total fluorescence from the probe is therefore lost). At a predetermined time before and after this processing, a total intensity value of all images was extracted. Divide the post-treatment value by the pre-treatment value to standardize each experiment. More
Higher ratios were seen in cells where NFkB translocated to the nucleus and contributed to total fluorescence after permeabilization. The actual data from this experiment performed twice is shown in FIG. Conclusion: This protocol may reveal the translocation of the fluorescent probe from cytosol to nucleus or from nucleus to cytosol by measurements taken immediately before and immediately after plasma membrane penetration recorded as a series of images. There is a good example.

Example 8 : Simultaneous redistribution of protein kinase Cα Protein kinase in response to carbamylcholine stimulation of muscarinic M1 receptor
Measurement of simultaneous redistribution of Cα isoform-GFP fusion (PKCα-GFP, SEQ ID No: 3 and 4); 96 simultaneous redistribution measurements in microtiter plates. BHK cells are recombinant human muscarinic under selection of 500 μg / ml methotrexate
The type 1 receptor was stably expressing the PKCα-GFP construct (KaA048) under the further selection of 500 nM zeocin. Cells were grown in 96-well plates (Packard ViewPlate, black and clear bottom), washed and pre-cultured in Hank's buffered saline (HBSS) without phenol red, containing 20 mM HEPES and 5.5 mM glucose. Plates were read on a Molecular Devices FLIPR ^™ (fluorescence imaging plate reader). A 488 nm emission line generated at 0.4-0.8 W from an argon ion laser is
Used to excite fluorescence from GFP. The emission wavelength was collected with a 510-565 nm bandpass filter.

Cells were tested with three doses of carbamylcholine, a known M1 receptor agonist from previous studies, and redistribution of the PKCα-GFP construct was detected microscopically [Almholt et al., 1997]. The measurement was performed every 10 seconds for 5 minutes. After data processing including normalization of reference fluorescence in different wells, background subtraction and averaging of the 6 wells used at each concentration, the data shown in FIG. 14 was obtained. It is clearly shown that carbamylcholine caused a time- and dose-dependent transient decrease in fluorescence that was very similar to that seen in microscopic fluorescence measurements (see Almholt et al., 1997) (FIG. 1).
2). This experiment was repeated twice with the same batch of cells, and the results were similar.

Example 9 : Simultaneous redistribution of a cyclic AMP-dependent protein kinase catalytic subunit-GFP fusion Cyclic AMP-dependent protein kinase catalytic subunit-GFP fusion in response to forskolin stimulation of adenylate cyclase (C-GFP ^LT SEQ ID No: 1 and 2)
Of simultaneous redistribution: 96 parallel redistribution measurements in a microtiter plate. CHO cells were stably transfected with hybrid DNA of the PKA catalytic subunit-F64L + S65T GFP (C-GFP ^LT ) fusion protein under continuous selection, usually with 1000 μg / ml zeocin (Invitrogen). Grow cells without selection in 96-well plates (Packard ViewPlate, black, clear bottom) for 2 days, wash, and preserve in Hank's buffered saline (HBSS) without phenol red, 20 mM HEPES and 5.5 mM glucose Cultured. Plates were read on a Molecular Devices FLIPR ^™ (fluorescence imaging plate reader). A 488 nm emission line generated at 0.4-0.8 W from an argon ion laser is
Used to excite fluorescence from GFP. The emission wavelength was collected with a 510-565 nm bandpass filter.

Cells were tested with three doses of forskolin (FIG. 13) and an adenylate cyclase agonist known from previous studies, and the redistribution of the C-GFP ^LT construct was detected microscopically. The measurement was performed every 10 seconds for 6 minutes after the addition of forskolin.
After data processing including normalization of reference fluorescence in different wells, background subtraction and averaging of the 6 wells used at each concentration, the data shown below were obtained. Figure 1
In FIG. 5, it is clearly shown that forskolin caused a time- and dose-dependent decrease in fluorescence that was very similar to that seen in microscopic fluorescence measurements (see Almholt et al., 1997). This experiment was repeated twice with the same batch of cells, and the results were similar. As seen in FIG. 14, a broader dose-response study quickly showed that the method was sensitive and reproducible enough to be used as the basis for a high-throughput screening assay. I have.

Example 10 Cyclic AMP-dependent protein kinase catalytic subunit-GFP fusion Cyclic AMP-dependent protein kinase catalytic subunit-GFP fusion after forskolin stimulation of adenylate cyclase (C-GFP ^LT SEQ ID No: 1 And 2) measurement of redistribution response: measurement of change in total fluorescence due to permeabilization of cells treated with agonist. CHO cells were stably transfected with hybrid DNA of PKA catalytic subunit-F64L + S65T GFP (C-GFP ^LT ) fusion protein under continuous selection, usually with 1000 μg / ml zeocin (Invitrogen). In the experiments reported here, cells were grown without selection to 90% confluence in 8-well tissue-cultured Lab-Tek® chamber coverslip units (chambers were from Nunc, Inc. Illinois,
USA). Immediately before the experiment, the growth medium is washed from the cells and 20 wells per well
Replaced with 0 μl HEPES buffer. In the results reported here, the chamber was a cooled CCD camera (KAF1400) equipped with an inverted microscope (Diaphot 300, Nikon, Japan) equipped with an x40 oil immersion Fluar lens NA1.4.
Chip, Photometrics Ltd., USA). Cells were illuminated with 450-490 nm light from a 50 W HBO lamp and the emitted light was collected between 510-560 nm.

Cells were tested with four doses of forskolin, an adenylate cyclase agonist known from previous studies, and the redistribution of the C-GFP ^LT construct was detected microscopically. Images were collected for 10 minutes at 10 second intervals for each treatment. Six minutes after the addition of Forskolin or control buffer, Triton X-100 was added to a final concentration of 0.1%. This detergent releases C-GFP ^LT, which is free to move from the cells. The change in fluorescence resulting from this loss was measured after 1 minute equilibration. After data processing including background removal and normalization to pre-denaturation values, the data shown in FIG. 16 was obtained. The permeation treatment reduced the fluorescence, the magnitude of which was dependent on forskolin treatment. This experiment was repeated twice with the same batch of cells with similar results.

Example 11 Probe for Detecting PKCβ1 Redistribution Identify compounds that are useful for monitoring signal pathways, including protein kinase C, and that modulate the activity of the pathway in living cells. PKCβ1, a serine / threonine protein kinase, is closely related, but not identical, to PKCα and PKCβ2. It is a component of the signaling pathway activated by elevated intracellular calcium contaminants with increasing diacylglycerol species. a) Human PKCβ1 gene (GenBank accession number: X06318) based on standard protocol
Amplified with primers by PCR. PKCβ1-top And PKCβ1-bottom PCR product digested with restriction enzymes Xho1 and BamH1, pEG digested with Xho1 and BamH1
It was linked to FP-C1 (Clontech, Palo Alto; GenBank accession number: U55763). This produces an EGFP-PKCβ1 fusion (SEQ ID Nos: 17 and 18) under the control of the CMV promoter.

B) CHO cells stably expressing the human insulin receptor and human PKCβ1 labeled with EGFP were examined microscopically. A series of cells was imaged over time at each concentration to obtain a dose-response. The change in fluorescence was calculated as AUC for a 4 minute stimulus. According to FIG. 16, it can be seen that using microscopic measurements, the redistribution of human PKCβ1-EGFP can be detected only when analyzing the subregion of each cell. This event is clearly visible when viewing a sequence of images like a movie. However, no redistribution is detected when the fluorescence change of the whole image or the whole cell is analyzed. CH stably expresses human insulin receptor and human PKCβ1 labeled with EGFP
O cells were examined with FLIPR ^™ . For each concentration, six independent wells were imaged over time to obtain a dose-response. Fluorescence changes were calculated as AUC for a 5 minute stimulus. As shown in FIG. 17, all wells containing about 50-100,000 cells were illuminated and imaged at a much lower resolution than required to resolve single cells or subcellular components. Despite the fact, redistribution of human PKCβ1-EGFP is detectable on the FLIPR ^™ instrument. This phenomenon cannot be clearly predicted from the microscope data of FIG. Based on these findings, it is clear that screening assays can be established with the FLIPR ^™ instrument. Further optimization could also establish high throughput screening assays. References

[0094]

[Sequence list]

[Brief description of the drawings]

FIG. 1. CHO cells expressing PKAc-F64L-S65T-GFP hybrid protein were transformed with HAM F12
The medium was treated with 50 μM forskolin at 37 ° C. Images of GFP fluorescence in these cells were taken at different time intervals after treatment: a) 40 seconds, b) 60 seconds, c) 70 seconds, d) 80 seconds. In the (nucleated) cytoplasm, the fluorescence changes from speckled to a more uniform distribution.

FIG. 2. Time course analysis of PKAc-F64L-S65T-GFP redistribution induced by forskolin. PKAc-F64L-
CHO cells expressing the S65T-GFP fusion protein were analyzed by fluorescence microscopy over time. Fluorescence micrographs were taken at regular intervals from 2 minutes before and 8 minutes after addition of the agonist. Immediately after the upper left image was obtained (t = 0), cells were examined with 1 μM forskolin. Flames were collected at the following times: i) 0, ii) 1, iii) 2, iv) 3, v) 4 and vi) 5 minutes. Measurement bar 10 μm.

FIG. 3. Time course analysis of PKAc-F64L-S65T-GFP redistribution for various agonists. PKAc-F64L-
Effect of 1 μM forskolin (A), 50 μM forskolin (B), 1 mM dbcAMP (C) and 100 μM IBMX (D) on the localization of S65T-GFP fusion protein (
The addition is indicated by the white arrow) was analyzed by time-lapse fluorescence microscopy of CHO / PKAc-F64L-S65T-GFP cells. The effect on the localization of the PKAc-F64L-S65T-GFP fusion protein by adding 10 μM forskolin (open arrow), and immediately thereafter, repeating washing with a buffer (black arrow) was analyzed in the same cells (E ). In a parallel experiment, the effect of repeated washing with a buffer containing 100 μM IBMX (black arrow) after addition of 10 μM forskolin and 100 μM IBMX (open arrow) was analyzed (F). Removal of forskolin returns the PKAc-F64L-S65T-GFP fusion protein to its cytoplasmic aggregate, which is prevented by the continued presence of IBMX (F). PKA
Effect of 100 nM glucagon on the localization of c-F64L-S65T-GFP fusion protein (Fig.
3G, white arrow) are also shown for BHK / GR, PKAc-F64L-S65T-GFP cells. PKAc-F64
The effect of 10 μM norepinephrine (black arrow) on the localization of L-S65T-GFP fusion protein was determined by transiently transforming CHO, PKAc-F64L-S65T, pretreated with 10 μm forskolin (white arrow). [CAMP] was increased by the same analysis in -GFP cells. Figure 3H
Note that in, the x-axis calculates the number of images every 12 seconds. The raw data for each experiment consisted of 60 fluorescence micrographs taken at regular intervals, including several images obtained before the addition of buffer or agonist. Charts (AG) each show the quantification of the response seen in all 60 images, performed as described in Method 2. The change in the total area of the highly fluorescent aggregate relative to the initial area of the fluorescent aggregate was plotted in each experiment as the ordinate versus time in all graphs of FIG. Measurement bar 10 μm.

FIG. 4. Dose-response curves for forskolin-induced redistribution of PKAc-F64L-S65T-GFP fusions (two experiments).

FIG. 5: Time from the start of response until the redistribution of PKAc-F64L-S65T-GFP reaches half the maximum (t _{1 / 2max} ) and the maximum (t _max ). Data was extracted from curves as shown in "Figure 2". All t _{1/2 max} and t _max values are mean ± SD and are based on the sum of 26-30 cells from 2-3 independent experiments at each forskolin concentration. Since the observed redistribution is maintained for a long time, the _tmax value is the earliest point at which complete redistribution is reached. Note that this value is independent of the degree of redistribution.

FIG. 6. Parallel dose of forskolin-induced cAMP elevation and redistribution of PKAc-F64L-S65T-GFP
-Response analysis. P in CHO / PKAc-F64L-S65T-GFP cells grown in 96-well plates
The effect of buffer or 5-step increasing concentrations of forskolin on the localization of the KAc-F64L-S65T-GFP fusion protein was analyzed as described above. Computer calculation of the SD ratio of fluorescence micrographs taken in the same field of cells before and 30 minutes after the addition of forskolin provides a reproducible measure of PKAc-F64L-S65T-GFP redistribution. Was done. The graph shows a trace of 48 individual measurements and their mean ± sem at each forskolin concentration. For comparison, the effect of buffer or 8-step increasing concentrations of forskolin on [cAMP] i was analyzed by a scintillation proximity assay of cells grown under the same conditions. The graph shows a trace of the mean ± sem shown for any unit of the four experiments.

FIG. 7: BHK cells stably transfected with a human muscarinic (hM1) receptor and a PKCα-F64L-S65T-GFP fusion. Carbachol (100 μM added in 1.0 second) is PKCα-F64L-S
Transient redistribution of 65T-GFP from the cytoplasm to the plasma membrane was induced. Image is next time: 1)
One second before carbachol addition, 8.8 seconds after b) addition, and 52.8 seconds after c) addition.

FIG. 8. BHK stably transfected with hM1 receptor and PKCα-F64L-S65T-GFP fusion
Cells were treated with carbachol (1 μM, 10 μM, 100 μM). In one cell,
Intracellular [Ca ²⁺ ] was observed simultaneously with the redistribution of PKCα-F64L-S65T-GFP. The dashed line indicates when carbachol was added. The upper panel shows the time course of the intracellular Ca ²⁺ concentration of each cell in each treatment. The middle panel shows the time course of the average cytoplasmic GFP fluorescence of each cell in each treatment. The lower panel shows the change in fluorescence of one cell, as seen in normal images, especially at the periphery within the area that includes the cell periphery. The best area to observe changes in fluorescence intensity is the cell membrane.

FIG. 9. hERK-1-F64L-S65T-GFP fusion expressed in HEK293 cells treated with 100 μM MEK1 inhibitor PD98059 in HAM F-12 (no serum) for 30 minutes at 37 ° C. During this process, the nuclei lose fluorescence. Cells similar to (a) were treated with 10% fetal bovine serum at 37 ° C. for 15 minutes. The time profile of GFP fluorescence redistribution in HEK293 cells was determined using Hepes buffer (HAM
F-12 results from treatment with various EGF concentrations in Hepes buffer just before the experiment). Fluorescence redistribution is expressed as a change in the nuclear to cytoplasmic area ratio of a single cell. Each time profile is the average of the changes seen in six single cells. The bar graph measuring the change in fluorescence (nucleus: cytoplasm) at the end point, which is 600 seconds after the start of EGF treatment, according to various concentrations of EGF.

FIG. 10. SMAD2-EGFP fusions expressed in HEK293 cells serum starved overnight in HAM F-12. HAM F-12 was subsequently replaced with pH 7.2 Hepes buffer just prior to the experiment. Measuring bar
10 μm. HEK293 cells expressing the SMAD2-EGFP fusion were treated with various concentrations of TGF-β as described.
And observed the redistribution of fluorescence over time. The time profile plot represents the increase in fluorescence in the nucleus and is normalized to the initial value of each cell measured. Each trace is a single cell nucleus time profile. The bar graph shows the change in the end point of fluorescence in the nucleus for different concentrations of TGF-β (
850 seconds after processing). Each bar is the value of one nucleus in each treatment.

FIG. 11. VASP- in CHO cells stably transfected with the human insulin receptor.
F64L-S65T-GFP fusion. Cells were starved for 2 hours in HAM F-12 without serum and then treated with 10% fetal calf serum. Images show fluorescence redistribution obtained 15 minutes after processing. GFP fluorescence shows localized attachment (focal
(adhesion).

FIG. 12: Dose-response relationship of PKCα-GFP translocation in BHKhM1 cells stimulated with the muscarinic agonist carbamylcholine using FLIPR ^™ to perform actual experiments.

FIG. 13: CHO / PKAc-stimulated with forskolin using FLIPR ^™ to perform actual experiments
Dose-response relationship of translocation of PKCα-GFP in F64L-S65T-GFP cells.

FIG. 14. CHO cells stably expressing human insulin receptor and mouse cPKA-labeled S65T-GFP were examined in more detail on a FLIPR ^™ instrument. Six different wells were imaged over time for each concentration to obtain a dose-response of forskolin, a substance that increases the production of adenylate cyclase of cAMP in cells. The change in fluorescence is
Minutes were calculated as AUC (area under the curve). Conclusion: Despite the fact that all wells containing approximately 50-100,000 cells are illuminated and simultaneously image single cells or subcellular events at a spatial resolution that cannot be resolved, the redistribution of mouse cPKA-BioST is FLIPR ^Can be detected by ^TM . This method can be used for simultaneous measurement of intracellular cAMP levels and should be used as a screening assay to measure the effect of ligand on G-protein coupled receptors that bind Gi and Gq-type G-proteins. Can be.

FIG. 15. Permeabilized CHO / PKAc when pre-exposed to different doses of forskolin
-Dose-response relationship of the quenched fluorescence from F64L-S65T-GFP.

FIG. 16: CHO cells stably expressing human insulin receptor and human PKCβ1 labeled with EGFP were examined microscopically. A series of cells were imaged over time at each concentration, resulting in a dose-response. Fluorescence changes were calculated as AUC over 4 minutes of stimulation.
The following data was extracted from the images: All images: A mere analysis of the change in intensity of the whole image, including cells and background. Single cells: 5 independent cells were analyzed after background compensation. Analysis was performed on whole cells. Cytoplasm: The same 5 cells as above were analyzed after background compensation. The analysis was performed on a small area of cytoplasm near the nucleus. Conclusion: Redistribution of human PKCβ1-EGFP is detectable only when analyzing a subregion of each cell. This event is clearly visible when viewing a sequence of images like a movie. However, no redistribution is detected when the fluorescence change of the whole image or the whole cell is analyzed.

FIG. 17: CHO cells stably expressing human insulin receptor and human PKCβ1 labeled with EGFP were tested with FLIPR ^™ . Six different wells were imaged over time for each concentration to obtain a dose-response. Fluorescence changes were calculated as AUC with a 5-minute stimulation. Conclusion: Due to the fact that all cells, including about 50-100,000 cells, are illuminated and imaged by the detector at a much lower resolution than required to resolve single cells or subcellular structures. Nevertheless, redistribution of human PKCβ1-EGFP is detectable on the FLIPR ^™ instrument. This phenomenon cannot be clearly predicted from the microscope data of FIG.

FIG. 18: CHO cells stably expressing the insulin receptor and the human NFkB-GFP protein hybrid are stimulated with various concentrations of IL-1 for 1 hour, followed by a low permeability buffer (TRIS-base 10 mM, MgCl _{2 2} mM, PMSF (phenylmethylsulfonyl fluoride) 0.5 mM, p
H7.4) and mounted on a microscope. A series of images is obtained during the addition of 0.05% Triton X-100, followed by gentle mixing and short incubation. The treatment disrupts the cell membrane and leaves a fraction of NFkB-GFP that later translocates to the nucleus, but some of the cytoplasmic probe leaves the cells more quickly and is rapidly diluted indefinitely in the surrounding medium (focus Mismatch-this part of the total fluorescence from the probe is lost thereby). At predetermined times before and after this processing, all intensity values for all images were extracted. Dividing the later value by the previous value to normalize each experiment shows that more NFkB has been translocated to the nucleus, thereby indicating a higher proportion of cells contributing to total fluorescence after transmission. Results: This protocol is a good example that may indicate translocation of a fluorescent probe from cytoplasm to nucleus or translocation from nucleus to cytoplasm.

[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] November 1, 2000 (2000.11.1)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 21/78 G01N 33/483 C 4B063 33/483 C12M 1/00 A 4B065 // C12M 1/00 G01N 33 / 15 Z C12N 5/10 33/50 Z G01N 33/15 C12N 15/00 ZNAA 33/50 5/00 B (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, U, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (71) Applicant Morkhoj Bygade 28, DK-2860 Soborg DENMARK (72) Inventor Scudder, Kurt, Marshall Denmark, Decay-2830 Wilm, Ravendel Her Ven 70 (72) Inventor Björn, Sarah, Petersen, Decay-2800 Ringby, Denmark, Klampenborgvey 102 (72) Inventor Sastrap, Ole Denmark, Decay-3460 Birkerad, Birkvey 37 (72) Inventor Hager , Grease Denmark, Decay -2791 Dragal, Halle Vangette 109 F-term (reference) 2G043 AA03 BA16 EA01 EA06 FA02 FA03 JA02 KA02 KA09 LA02 LA03 2G045 BB20 BB29 BB51 CB01 FA16 FB02 FB12 GC15 2G054 AA08 EB08 CA04 CA07 CA09 DA02 DA12 FA10 GA11 GA18 HA13 4B029 AA07 AA21 AA23 BB07 BB11 CC03 FA15 4B063 QA01 QA05 QQ07 QQ08 QQ13 QQ20 QR56 QR57 QR66 QR76 QR77 QR80 QR82 QS03 QS24 QS36 QS39 QX02 ABXA AXA AXA AXA AXA AXA AXA AXA AXA AXA AXA AXA AXA AXA AXA AXA XA AXA AX

Claims (39)

[Claims] 1. A method for obtaining quantitative information on the effect of a mechanically intact or permeabilized living cell on a cellular response, wherein the method is present in the cell and relates to the degree of the effect. Changes in the scattered light of the space emanating from the luminophore that can be redistributed and / or modulated by components that can be redistributed in relation to the degree of influence are designed to measure changes in fluorescence intensity Recording as a change in light intensity measured by a modified device. 2. A method according to claim 1, wherein the quantitative information indicating the degree of the cellular response to the effect or the consequence of the subcellular component is based on a response or result similarly recorded for a known degree of the particular effect involved. 2. The method according to claim 1, wherein the method is derived from the changes recorded according to the calibration of. 3. The method according to claim 1, wherein the effect is mechanically intact or permeabilized living cell contact with the chemical and / or incubation of the mechanically intact or permeabilized live cell with the chemical. A method according to claim 1 or claim 2, comprising: 4. The cell according to claim 1, wherein the cell comprises a group of cells included within the space limitation.
3. The method according to any one of 3. 5. The method according to claim 1, wherein the cells comprise a plurality of groups of cells contained within a plurality of space restrictions. 6. The method according to claim 1, wherein the cells consist of a plurality of groups of cells that are quantitatively the same but have been subjected to different effects. 7. The method according to claim 1, wherein the cells consist of a plurality of cell groups that are quantitatively different but have the same effect. 8. A plurality of spatial constraints are simultaneously measured by a one-dimensional or two-dimensional array detector, whereby the plurality of spatial constraints are imaged on the array detector and a detection unit (pixel) in the array detector ) Measures the signal from any one of the one or more spatial constraints and the signal from any one of the spatial constraints combined with the signal from the pixel receiving the image from one of the spatial constraints A method according to any of the preceding claims. 9. The method according to claim 8, wherein the detector is a linear diode array. 10. The method according to claim 8, wherein the detector is a video camera. 11. The method according to claim 8, wherein the detector is a charge transfer device. 12. The method according to claim 8, wherein the charge transfer device is a charge coupled device. 13. The method according to claim 1, wherein all of the plurality of spatial restrictions are illuminated simultaneously during the measuring operation. 14. The method according to claim 1, wherein the individual space restrictions are illuminated only once during the time to be measured. 15. The illumination occurs with a laser that is scanned in a raster fashion over some or all of the measured spatial constraints, and the illumination is temporally and spatially uniform and continuous over the measurement area for the measurement process. The method according to any of the preceding claims, wherein the scanning is performed at a substantially faster rate than the measurement process. 16. The method according to claim 1, wherein the spatial restrictions are spatial restrictions arranged in one or more arrays on a common carrier. 17. The method according to claim 16, wherein the space restriction is a well in a microtiter plate. 18. The method according to any one of claims 1 to 17, wherein the space restriction is a range limited on the substrate on which the cell is present. 19. The method according to claim 18, wherein the area is the area established by the presence of cells on the substrate in a pattern defining the area. 20. The method according to claim 18, wherein the area is an area established by a spatial pattern of effects applied to or in contact with the cell. 21. The recording is performed at a series of time points and is effected some time after the first time point of the series of recordings, wherein the recording is for example from 0.1 seconds to 1 hour, preferably 1 second.
1 to 12 hours, for example, 10 seconds to 12 hours, for example, 10 seconds to 1 hour, for example, 60 seconds to 30 minutes, at a predetermined time interval of ~ 60 seconds, more preferably 1 to 30 seconds, especially 1 to 10 seconds. 21. A method according to any of the preceding claims, which takes place over a period of 20 minutes. 22. The method according to claim 21, wherein the recording is made at two points in time, before the effect is effected and at a point in time after that. 23. The method according to any one of claims 1 to 22, wherein the cells are fixed at a time after the effect (time when the response is scheduled to be significant) and recorded at any later time. 24. The method according to claim 1, wherein the redistribution causes quenching of the fluorescence, and the quenching is measured as a decrease in fluorescence intensity. 25. The method according to claim 1, wherein the redistribution causes an energy transfer, and the energy transfer is measured as a change in emission intensity. 26. The illumination required to excite fluorescence is non-homogeneous, such that redistribution results in more or less excited molecules, and the result is measured as a change in fluorescence intensity. The method according to any one of 1 to 24. 27. The method according to any of the preceding claims, wherein the recorded light intensity is a function of the fluorescence duration, polarization, wavelength shift or other property that is modulated as a result of the underlying cellular response. . 28. The method according to claim 1, wherein the light to be measured passes through a filter which selects the desired light component to be measured and rejects other components. 29. The method according to any of claims 1-28, wherein the fluorescence originates from a fluorophore encoded and expressed by a nucleotide sequence resident in the cell. 30. The method wherein the fluorescence is generated from a fluorophore introduced into the cell by any of a variety of techniques, such as transfection, incubation, scraping, electroporation, or by a variety of techniques for loading substances into the cell. Item 30. The method according to any one of Items 1 to 28. 31. The method according to any of the preceding claims, wherein the fluorescence originates from a luminescent polypeptide such as GFP. 32. The method according to any one of claims 1 to 31, wherein the cell is selected from the group consisting of a fungal cell such as a yeast cell; an invertebrate cell including an insect cell; and a vertebrate cell such as a mammalian cell. Method. 33. The living cells that are mechanically intact or permeabilized are at a temperature of 30 ° C. or higher, preferably a temperature of 32 to 39 ° C., more preferably a temperature of 35 to 38 ° C., most preferably 33. The method according to claim 32, wherein the mammalian cells are cultured at a temperature of about 37 [deg.] C for a time during which the effect is observed. 34. The nucleic acid construct encodes a DNA construct having a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15 and 17, or the same fusion polypeptide. A method according to any of the preceding claims, which is a variant thereof or a fusion polypeptide biologically equivalent thereto. 35. The method according to claim 1, which is used as a screening program.
4. The method according to any one of 4. 36. A screen, wherein the method is a screening program for the identification of a bioactive substance that directly or indirectly affects an intracellular signaling pathway and may be useful as a drug, and indicates its potential biological activity. The individual measurement results of each substance to be subjected have undergone a change in distribution due to activation of intracellular signaling pathways,
36. The method according to claim 35, which is based on measuring spatially resolved luminescence redistribution in living cells. 37. A screen, wherein the method is a screening program for the identification of a biotoxic agent as defined herein, which exerts a toxic effect by interfering with an intracellular signaling pathway, and exhibits its potential biotoxic activity. 36. The method according to claim 35, wherein the individual measurement of each of the substances performed is based on a measurement of the redistribution of a fluorescent probe in living cells, which has undergone a change in distribution due to activation of an intracellular signaling pathway. 38. The method according to any one of claims 1 to 37, wherein a fluorescent probe is used for back-tracking the signal conversion pathway as defined herein. 39. The method according to claim 39, which is present in the cell and can be redistributed in relation to the degree of influence.
And / or the change in spatially scattered light emitted from the luminophore that can be modulated by a component that can be redistributed in relation to the degree of influence, the light intensity measured by a device designed to measure changes in fluorescence intensity Series of data on the effect on cell response in mechanically intact or permeabilized living cells, recorded as changes in