WO2013151511A1 - Modified Dual-Colour Protein - Google Patents

Abstract

A modified GLUT4 comprising GLUT4, and two fluorophores, one fluorophore being a pH sensitive fluorophore. The modified GLUT4 is useful in ratio-metric and high throughput assays to analyse GLUT4 exocytosis in cells.

Description

MODIFIED DUAL-COLOUR PROTEIN

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of Singapore patent application No. 201202512-8, filed 5 April 2012, the contents of it being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[002] The present invention relates to the fields of molecular biology and biochemistry. In particular, the present invention refers modified dual-colour protein comprising GLUT4 that are useful in high-throughput assays.

BACKGROUND OF THE INVENTION

[003] Insulin regulation of GLUT4 glucose transporter trafficking plays a central role in peripheral actions of insulin. The stepwise molecular dissection of this process is essential in pinpointing key regulatory nodes that contribute either to insulin regulation or to insulin resistance.

[004] Insulin mediated glucose uptake into these tissues is achieved via the translocation of GLUT4 from intracellular storage compartments to the cell surface. The intricate regulation of glucose uptake is compromised in metabolic disease and thus a detailed understanding of the processes controlling GLUT4 surface levels is critical to improving patient outcomes.

[005] The study of GLUT4 trafficking steps has been greatly enhanced by advances in microscopy and development of fluorescent proteins. Labelled GLUT4 appears to behave in -a manner comparable to endogenous GLUT4 and as such as proven to be an invaluable tool for investigating GLUT4 trafficking.

[006] Currently available assays based on assessing a response in cell populations mask important biological information by averaging the response of multiple individual cells. Inter-cellular heterogeneity is present in many, if not all biological systems. Such heterogeneity may contain important information into the molecular regulation of processes such as GLUT4 trafficking; insight that can only be achieved through the measurement of large numbers of single cells.

[007] The challenge of performing a large screen of single cells is limited by current methodologies. Such methods include assessment of ³H 2-deoxuglucose uptake, cell fractionation, plasma membrane sheet assays, counting radioactivity that binds to adherent cells, Fluorescence Activated Cell Sorting (FACS) and immunofluorescence microscopy. For example, high frequency TIRFM experiments are technically difficult and impose a large analysis burden on the researcher.

[008] Thus, there is a need to provide for new method for assessing a response in multiple individual cells, particularly insulin stimulated GLUT4 exocytosis that overcomes, or at least ameliorates, one or more of the disadvantages described above.

[009] In a first aspect, there is provided a modified protein comprising a) GLUT4 or a protein having a 95% sequence identity to GLUT4 , b) at least one cytosolic fluorophore comprised in the GLUT4 sequence defined under a) and c) at least one lumenal pH-sensitive fluorophore comprised in the GLUT4 sequence defined under a)

[010] In a second aspect, there is provided an isolated nucleic acid molecule encoding the modified protein according to any of the preceding claims.

[Oil] In a third aspect, there is provided a recombinant vector comprising a nucleic acid molecule as defined herein.

[012] In a fourth aspect, there is provided a host cell, wherein the host cell is capable of expressing the modified protein as defined herein when comprising the vector as described herein.

[013] In a fifth aspect, there is provided a method for screening a compound that affects exocytosis of a protein of interest in a plurality of individual mammalian cells, comprising the use of a modified protein of interest, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH-sensitive fluorophore and at least one cytosolic fluorophore.

[014] In a sixth aspect, there is provided a method of assessing whether a condition or a stimulus affect exocytosis of a protein of interest ^"in a plurality of individual mammalian cells, comprising:

a. culturing mammalian cells expressing a modified protein of interest under a condition or stimulus to be assessed for its effect on exocytosis of said protein, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH-sensitive fluorophore and at least one cytosolic fluorophore, wherein the cells are referred to as test cells;

b. detecting a signal from the cytosolic fluorophore, which is indicative of total modified protein of interest in the cells and a signal from the luminal pH-sensitive fluorophore, which is indicative of fusion events from an intracellular compartment to an extracellular environment of the test cells;

c. determining a ratio of the signal from the cytosolic fluorophore and the signal from the pH-sensitive fluorophore of the modified protein of interest in the test cells of (b) , thereby producing a test value, which is indicative of the plasma membrane localisation of the modified protein of interest normalised to said protein total expression level;

d. comparing the test value with a control value, wherein the control value corresponds to a ratio of modified protein of interest at the surface of the cell in control cells to total modified protein of interest in control cells, and the control value is determined from control cells which are the same cell as cultured in (a) , and which are cultured under the same conditions as in (a) , expect that the control cells are not cultured under the condition or stimulus to be assessed, wherein if the test value is greater than the control value, then the condition or stimulus affects exocytosis of the modified protein of interest in the cells.

[015] In a seventh aspect, there is provided a method of assessing the steps affected in the trafficking pathway of a modified protein of interest by a condition or a stimulus in a plurality of individual mammalian cells, comprising:

a. culturing mammalian cells expressing a modified protein of interest under a condition or stimulus that activate translocation of said protein from an intracellular location to the plasma membrane of the cells, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH- sensitive fluorophore and at least one cytosolic fluorophore thereof, wherein the cells are referred to as control cells;

b. detecting a first signal from the cytosolic fluorophore, which is indicative of the total amount of modified protein of interest in the cells and a second signal from the lumenal pH-sensitive fluorophore, which is indicative of the presence of the modified protein of interest in the plasma membrane of the control cells, thereby producing a first control value from the cytosolic fluorophore and a second control value from the pH- sensitive fluorophore;

c. providing the control cells with the condition or stimulus that affect the trafficking of the modified protein of interest, thereby producing test cells;

d. detecting a third signal from the cytosolic fluorophore, which is indicative of the total amount of modified protein of interest in the cells and a fourth signal from the pH-sensitive fluorophore, which is indicative of the presence of the modified protein of interest in the plasma membrane of the test cells, thereby producing a first test value from the cytosolic fluorophore and a second test value from the pH-sensitive f1uorophore ;

e. determining a first proportion of the first test value and the first control value and a second proportion of the second test value and the second control value;

f. comparing the first proportion with the second proportion, wherein if the first proportion is less than 1, the intracellular movement of the modified protein of interest is inhibited, and if the second proportion is less than 1, the fusion of the modified protein of interest is inhibited and wherein if the first proportion is greater than the second proportion, then the condition or stimulus mainly effects the fusion of the modified protein of interest to the plasma membrane in the cells.

[016] In an eight aspect, there is provided a method of assessing a fusion event of a modified protein of interest affected by a condition or a stimulus in at least one mammalian cell, comprising:

a. culturing mammalian cells expressing a modified protein of interest, wherein the modified protein of interest comprises the protein of interest and at _; least one lumenal pH-sensitive fluorophore and at least one cytosolic fluorophore thereof;

b. detecting a signal from the lumenal pH- sensitive fluorophore, which is indicative of the presence of the modified protein of interest in the plasma membrane of the at least one mammalian cell, thereby localizing the site of fusion from the presence of the pH- sensitive fluorophore;

c. capturing multiple images of the at least one cell over a period of time prior to providing the cell with the condition or stimulus that affects fusion, the multiple images comprising the signal from thereby producing a basal imaging period;

d. providing the at least one cell with the condition or stimulus that affect the fusion event;

e. capturing multiple images of the at least one cell over a period of time after providing the cell with the condition or stimulus as in (d) ;

f. analysing the images obtained in (c) and (e) , thereby quantifying the total number of fusion events and generating a fusion event rate ( F_ER ) ;

g. comparing the F_ER during the basal imaging period and after providing the at least one cell with the condition or stimulus, wherein if the F_ER is increased after providing the at least one cell with the condition or stimulus, then the condition or stimulus activate the fusion of the modified protein of interest to the plasma membrane in the cells .

[017] In a ninth aspect, there is provided a method of assessing a pre-fusion event of a modified protein of interest affected by a condition or a stimulus in at least one mammalian cell, comprising:

a. culturing mammalian cells expressing a modified protein of interest under a condition or stimulus to be assessed for its effect on exocytosis of said protein, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH-sensitive fluorophore and at least one cytosolic fluorophore;

b. detecting a signal from the cytosolic fluorophore, which is indicative of total modified protein of interest in the cells and a signal from the luminal pH-sensitive fluorophore, which is indicative of fusion events from an intracellular compartment to an extracellular environment of the cells;

c. capturing multiple images of the at least one cell over a period of time prior and after providing the cell with the condition or stimulus that affects fusion, wherein the images comprising a first signal from the cytosolic fluorophore, and a second signal from the lumenal pH-sensitive fluorophore;

d. determining the time of presence of the first signal or absence thereof of the first signal at the site of fusion, thereby providing a dwell-time; wherein the dwell- time is representative of whether the condition or stimulus affect the pre-fusion events of the modified protein of interest in the at least one cell.

[018] In a tenth aspect, there is provided a method of assessing whether a condition or a stimulus affect exocytosis of GLUT 4 in a plurality of individual mammalian cells, comprising:

a. Culturing mammalian cells expressing the modified protein according to claim 1 under a condition or stimulus to be assessed for its effect on exocytosis of GLUT , wherein the cells are referred to as test cells;

b. detecting a signal from the cytosolic fluorophore, which is indicative of total modified GLUT4 in the cells and a signal from and a signal from the lumenal pH- sensitive fluorophore, which is indicative of fusion events from an intracellular compartment to an extracellular environment of the test cells;

c. determining a ratio of the signal from the cytosolic fluorophore and the signal from the lumenal pH-sensitive fluorophore of the modified protein in the test cells of (b) , thereby producing a test value;

d. comparing the test value with a control value, wherein the control value corresponds to a ratio of the modified protein at the surface of the cell in control cells to the total modified protein in control cells, and the control value is determined from control cells which are the same cells as cultured in (a) , and which are cultured under the same conditions as in (a) , expect that the control cells are not cultured under the condition or stimulus to be assessed; wherein if the test value is greater than the control value, then the condition or stimulus causes exocytosis of the modified GLUT4 in the cells.

[019] In an eleventh aspect, there is provided a method of assessing the steps affected in the trafficking pathway of the modified protein of claim 1 by a condition or a stimulus in a plurality of individual mammalian cells, comprising: a. culturing mammalian cells expressing the modified protein under a condition or stimulus that activate translocation of GLUT4 from an intracellular location to the plasma membrane of the cells, wherein the cells are referred to as control cells;

b. detecting a first signal from the cytosolic fluorophore, which is indicative of the total amount of modified ^"protein in the cells and a second signal from the lumenal pH- sensitive fluorophore in the lumenal, which is indicative of the presence of the modified protein in the plasma membrane of the control cells, thereby producing a first control value from the cytosolic fluorophore and a second control value from the lumenal pH- sensitive fluorophore ;

c. providing the control cells with the condition or stimulus that affect the trafficking of the modified protein, thereby producing test cells;

d. detecting a third signal from the cytosolic fluorophore, which is indicative of the total amount of modified protein in the cells and a fourth signal from the lumenal pH- sensitive fluorophore in the lumenal, which is indicative of the presence of the modified protein in the plasma membrane of the test cells, thereby producing a first test value from the cytosolic fluorophore and a second test value from the lumenal pH- sensitive fluorophore;

e. Determining a first proportion of the first test value and the first control value and a second proportion of the second test value and the second control value;

f. Comparing the first proportion with the second proportion; wherein if the first proportion is less than 1, the intracellular movement of the modified protein is inhibited, and if the second proportion is less than 1, the fusion of the modified protein of interest is inhibited and wherein if the first proportion is greater than the second proportion, then the condition or stimulus mainly effects the fusion of the modified protein of interest to the plasma membrane in the cells.

BRIEF DESCRIPTION OF DRAWINGS

[020] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[021] Figure 1 relates to the characterization of a novel GLUT construct, rGLUTpHluor.

[022] Figure 1 A and B are a schematic diagram and cartoon of rGLUTpHluor embedded in a lipid bilayer. The construct contains the pH sensitive eGFP derivative super ecliptic pHluorin in the first exofacial loop of GLUT4 and is therefore located in the vesicle lumen or exposed to the extracellular environment. The tdTomato is located at the cytoplasmic carboxyl- terminus .

[023] Figure 2 A is a pair of immunoblot exposure of lysates of 3T3-L1 adipocytes transiently expressing the construct that were subjected to SDS-PAGE and subsequently probed with anti-GLUT4 and anti-GFP antibodies. The two- colour construct and endogenous GLUT4 are indicated.

[024] Figure 2 B is an image by widefield epifluorescence microscopy of a representative 3T3-L1 adipocyte expressing the 2 colour construct imaged by widefield epifluorescence microscopy. The image shows the signal from pHluorin (green) , TdTomato (red) and the signal from pHluorin after pH neutralization with 50mM NH₄C1. Scale bar = 10 fim. [025] Figure 2 C is a confocal microscopy image of adipocytes expressing rGLUTpHluor. The adipocytes were incubated in the absence or in the presence of lOOnM insulin for 20 min then stained with anti-IRAP antibodies, and imaged by confocal microscopy. Representative slices at the base and through the middle of a cell are shown. Scale Bar = 5 μπι.

[026] Figure 2 D is a bar graph showing the co- localization of rGLUTpHluor and endogenous IRAP measured using a 3D structure based approach based on a deconvolved image of electroporated adipocytes obtained by confocal microscopy. Data is presented as the mean ±SEM of six cells (three independent experiments) .

[027] Figure 2 E is^' a series of immunoblot exposure of adipocytes lysates expressing rGLUTpHluor. Adipocytes expressing rGLUTpHluor were incubated in the absence or presence of 100 nM insulin for 20 min. Cell lysates were subjected to sub-cellular fractionation and analysed by immunoblotting with anti-GFP and anti-GLUT4 antibodies. The integrity of the fractions was verified by immunoblotting for markers of membrane (syntaxin 4) and cytosol (tubulin) . TCL-total cell lysate; Cyt- cytosolic/soluble fraction; IM-internal membrane fraction; PM-plasma membrane; M/N mitochondrial/nuclear fraction.

[028] Figure 2 F shows representative TIRFM images of adipocytes expressing either GLUT4 -eGFP or rGLUTpHluor in the absence or presence of insulin stimulation. Adipocytes expressing either GLUT4 -eGFP or rGLUTpHluor were mixed prior to replating and imaged by TIRFM. Representative cells imaged before (basal) and after 30 min of insulin stimulation (insulin) are shown. Scale Bar = 10 μπι.

[029] Figure 2 G is a scatter plot showing time- resolved fluorescence measurements of adipocytes expressing either GLUT4 -eGFP or rGLUTpHluor measured by TIRFM imaging over the time course of insulin stimulation from Figure 2 F. Data is presented as the mean + SEM of the fold response over basal (FOB) from 3 experiments (15 cells) .

[030] Figure 3 relates to the assessment of GLUT4 exocytosis by ratiometric epifluorescence microscopy of rGLUTpHluor .

[031] Figure 3 A-D is a series of images of rGLUTpHluor by widefield Epifluorescence can be used to assess GLUT4 exocytosis. When imaged by EpiM, excitation light penetrates the entire cell and all tdTomato fluorophores in the cell are excited. In contrast only pHluorin molecules exposed to the extracellular environment (pH 7.4) emit high levels of signal. pHluorin molecules within the lumen of intracellular compartments are effectively quenched by the lower pH (pH 5.0-6.5). Insulin stimulates the redistribution of GLUT4 to the PM. This results in an increase in the amount of excitable pHluorin and hence a greater signal is measured. No difference is observed in the signal derived from the tdTomato. The graph shows the theoretical signal derived from the pHluorin and tdTomato during the transition from the basal state (A) to insulin stimulated state as in (B) . The dotted line represents a theoretical transition between the two steady states. When fusion (C) and/or movement is blocked by experimental manipulation there is no change in pHluorin signal. D) Therefore any experimental manipulation that effects GLUT4 trafficking will be detected when imaging this construct by EpiM.

[032] Figure 3 E are a series of epifluorescence microscopy images of green and red channel overlays of a single 3T3-L1 adipocyte expressing rGLUTpHluor over the time course of stimulation with insulin (lOOnM) . Scale bar= 5 ra. [033] Figure 3 F is a scatter plot graph showing the time-resolved fluorescence trace of the green and red signal over the course of insulin stimulation from fig. 3 E .

[034] Figure 3 G is a scatter plot graph representing the pHluorin/tdTomato ratio and change in cell area over the time course of insulin stimulation from fig. 3 E.

[035] Figure 3 H is a scatter plot graph showing the time course of GLUT4 exocytosis measured in 3T3-L1 adipocytes expressing rGLUTpHluor and treated with either MK-2206 (10 μΜ) , Wortmannin (100 nM) or DMSO (0.1%)prior to stimulation with 100 nM insulin. Data represents the mean + SEM of 18 cells from three independent experiments.

[036] Figure 4 shows that tdTomato, but not pHluorin is highly correlated with cell area in a heterogeneous cell population.

[037] Figure 4 A and B are a pair of scatter plots that demonstrate the correlation of the (A) pHluorin and (B) tdTomato signals with changes in cell area in single cells over the time course of insulin stimulation. Symbols represent measurements from single cells. The number in brackets denotes the correlation coefficient (r, Spearman correlation) . The dotted line is the linear regression line of the combined data.

[038] Figure 4 C is an intensity plot showing the individual cell responses traces over the time courses of insulin stimulation (100 nM) in 3T3-L1 adipocytes expressing rGLUTpHluor. Each row of pixels represents an individual cell and each column represents a point in time and the magnitude of the response is displayed as different intensities.

[039] Figure 5 is a series of graphs showing that the heterogeneity in the GLUT4 response is non-random. 3T3-L1 Adipocytes expressing rGLUTpHluor under constant perfusion were imaged by ratiometric epiM and subjected to multiple insulin stimulations as described.

[040] Figure 5A is a scatter plot representing the population response to a protocol of increasing doses of insulin (0.1, 1, 10 nM) interspersed by 2 h washouts (no insulin) for recovery. Data presented as the mean ± SEM of 26 cells.

[041] Figure 5 B is a plot of the steady state level reached by each cell in the protocol described above (A) . Matched symbols represent the responses to each dose of a single cell. Bar represents the population mean. Comparison between groups was made by Wilcoxon matched- pairs signed rank test (****p<0.0001, **p<0.01).

[042] Figure 5 C is a plot of the half-time (t 1/2) for each cell (as described above) to reach steady state at the doses indicated. Comparison between groups was made by Wilcoxon matched-pairs signed rank test (****p<0.0001, ***p<0.001) .

[043] Figure 5 D represents two single cell traces from (A) , displaying an example of a graded and a bimodal response.

[044] Figure 5 E shows the population response to a protocol of 2 x InM insulin stimulations interceded by a 2 h washout. Data presented as the mean + SEM of ~50 cells from one of two experiments (180 cells in total) .

[045] Figure 5 F is five separate traces reflecting the full dynamic range and reproducibility of responses presented in (E) .

[046] Figure 5 G is an intensity based plot showing the individual responses from (E) . Each row of pixels represents an individual cell and each column represents a point in time and the magnitude of the response is displayed as different intensities. [047] Figure 5 H shows the correlation between the first and second steady state level achieved in response to 1 nM insulin. The correlation was assessed by Pearson correlation.

[048] Figure 6 is a series of graph showing the calibration of rGLUTpHluor.

[049] Figure 6 A is a schematic representation of the experimental protocol for calculating the fractional amount of a pHluorin tagged protein at the cell surface, showing the pH and buffer changes.

[050] Figure 6 B is an example trace from a 3T3L-1 adipocyte expressing rGLUTpHluor subjected to the calibration protocol. NB: There is no second pH 5.5 wash and the cell was stimulated with 100 nM insulin for 40min, followed by a washout.

[051] Figure 6 C represents the fractional amount of rGLUTpHluor at the PM in 3T3-L1 adipocytes before and after stimulation with 100 nM insulin. Data are presented as the Mean ± SEM of cells (15≤n ≤50) . Three individual experiments .

[052] Figure 7 is a series of images and graphs demonstrating further dissection of GLUT4 trafficking - two-colour TIRFM imaging of rGLUTpHluor.

[053] Figure 7 A-D depicts a cartoon describing imaging of rGLUTphluor by Epi-M. In contrast to EpiM, TIRFM can deliver information on where a trafficking block occurs.

[054] Figure 7 A shows that under basal conditions, only a small amount of GLUT4 is on or near the surface of the cell and only the tdTomato molecules close to the PM are excited by TIRF. Only pHluorin molecules that have been inserted into the PM produce a significant signal.

[055] Figure 7 B represents the redistribution of

GLUT4 to the PM in the presence of insulin results in an increase in the amount of excitable tdTomato as more GLUT4 molecules enter the ^,TIRF-zone' and in the amount of excitable pHluorin as GSVs fuse with the PM.

[056] Figure 7 C shows that if fusion is blocked GLUT4 still traffics to the membrane and as such there is an increase in the tdTomato signal under these conditions. No pHluorin is exposed the extracellular environment, so there is no increase in pHluorin derived signal.

[057] Figure 7 D demonstrates that if transport is blocked, there is no change in either the pHluorin or tdTomato signals.

[058] Figure 7 E and F are a pair of scatter plots depicting single cell time course of insulin (100 nM) stimulated entry of rGLUTpHluor into the TIRF zone for the (E) pHluorin and (F) tdTomato fluorophores .

[059] Figure 7 G represents the half times of pHluorin and tdTomato responses from individual adipocytes expressing rGLUTpHluor stimulated with 100 nM insulin. Half-times for individual cells are shown as matched symbols. The line shows the grand mean ± SEM. The groups were compared by ilcoxon matched-pairs signed rank test and Spearman Correlation.

[060] Figure 7 H demonstrates the correlation between the half times of the pHluorin and tdTomato responses. Dotted line represents the line of equivalency (x =y) .

[061] Figures 7 I and J are a pair of scatter graphs plotting the time course of insulin stimulated changes in the (I) pHluorin signal (green) and the (J) tdTomato signal (red) treated with MK-2206 (10 μΜ) , Wortmannin (100 nM) , Latrunculin B (10 μ_.Μ) or control ^~(0.1% DMSO) . Data represents the mean ± SEM of 12 cells from three separate experiments .

[062] Figure 7 K is a comparison of the insulin response of the major constructs used to assess GLUT4 trafficking by live cell microscopy. Data represents the mean ± SEM of 12-25 cells from three experiments.

[063] Figure 8 represents the visualization, detection and quantitation of individual GLUT4 fusion events.

[064] Figure 8 A is a representative image series of high-frequency (HF) TIRFM images (10Hz) showing the fusion of an rGLUTpHluor containing vesicle with plasma membrane

(PM) . The numbers represent the time in seconds from when the event first becomes visible. Scale Bar = 1 itm.

[065] Figure 8 B is a scatter plot representing the fluorescence trace from A, with a 3x3 pixel area (0.23 μπι²) centered over the site of fusion showing a characteristic fusion signature.

[066] Figure 8 C is a scatter plot graph representing the time-lapse fluorescence trace (line) and identified fusion events (ellipses) from a single 3T3-L1 adipocyte expressing rGLUTpHluor.

[067] Figure 8 D is a scatter plot showing single cell traces over the time course of insulin stimulation (100 nM) in 3T3-L1 adipocytes expressing rGLUTpHluor imaged by high frequency TIRFM.

[068] Figure 9 represents the assessment of prefusion behaviour of GLUT4 vesicles by two colour High- Frequency TIRFM imaging of rGLUTpHluor. In this series of images, 3T3 -LI adipocytes expressing rGLUTpHluor were imaged by HF (10Hz) two-colour TIRFM

[069] Figures 9 A to C are a series of HF TIRFM imaging of rGLUTpHluor showing fusion event with a long (-1.5 min; A) , a medium (-30 s; B) , or a short (~3 sec; C) dwell time prior to fusion. Arrows denote the appearance of the vesicle. Dotted lines denote the midline of fusion. Scale bar = 2 μτ .

[070] Figures 9 D to F are a series of scatter graph showing the time- lapse fluorescence traces from within a 3x3 area centred over the site of fusion from events A, B and C of the pHluorin and the tdTomato signals .

[071] Figure 9 G is a graph representing the 2D path prescribed by a vesicle prior to fusion. The shadowing of the path represents the time (s) as described in the figure.

[072] Figure 9 H is a graph representing the instantaneous speed (solid line) and relative displacement, over the time course.

[073] Figure 9 I is a scatter plot showing the mean fluorescence within a 3x3 area centred over the site of fusion for both channels. Approach and attachment appear as increases in the red signal, while fusion appears as a characteristic increase in fluorescence in the green channel.

[074] . Figure 10 A is a scatter plot showing the correlation as assessed by Pearson correlation,- between expression level and magnitude of the response of the cells described above.

[075] Figure 10 B is a brightfield image of electroporated 3T3-L1 adipocytes prior to imaging by ratiometric widefield fluorescence. Scale bar=40 μπι.

[076] Figure 10 C is a fluorescence image (tdTomato, red) of (B) showing cells expressing rGLUTpHluor. The image has had the gamma adjusted to 0.5 in order to aid visualization (due to a 10 -fold range in expression levels) . Numbers represent the mean fluorescence of the adjacent cells. Intact circles denote cells that would be included in downstream analysis, whereas cells denoted by the dotted circles would be excluded based on lipid density.

[077] Figure 11 is a schematic representation of the proposed workflow for screening for molecules involved in GLUT4 trafficking. The workflow consists of a rapid ratiometric epifluorescence based screen that will identify if GLUT4 trafficking is effected by an intervention. Low frequency two-colour TIRFM can then be employed to dissect the site of involvement. Finally HF- TIRFM can be used to investigate at sub-cellular resolution.

DETAILED DESCRIPTION OF THE INVENTION [078] Insulin regulation of GLUT4 trafficking pathway plays a central role in peripheral actions of insulin. The stepwise molecular dissection of this process is essential in pinpointing key regulatory nodes and stimuli or conditions that contribute either to insulin regulation or to insulin resistance. There is a need to develop reagents and methods that can be used, for example, to dissect individual steps in this pathway.

[079] Therefore, the present invention refers to a modified protein that comprises GLUT . The modified protein can comprise at least one cytosolic fluorophore and optionally at least a lumenal pH- sensitive fluorophore. The modified protein as described herein is also referred to as GLUT4 probe, GLUT4 reporter, or GLUT4 construct. The modified protein is engineered in such a manner that it may be detected, quantified, visualized and localised.

[080] The modified protein may be built in such a manner that the function of GLUT4 is essentially retained. For example, one of the best indices of GLUT4 function is its high degree of insulin responsiveness. Hence, the modified GLUT4 may be designed to retain this insulin responsiveness.

[081] Based on the known characteristics of the endogenous GLUT4 and some desired advantages as described herein, the design of the modified protein is disclosed herein. In more details, insulin- stimulated glucose uptake in adipose and muscle tissues is achieved by the translocation of GLUT 4, from intracellular storage compartments to the cell surface.

[082] In the absence of insulin, GLUT4 is concentrated in the perinuclear region and in the tubule-vesicular storage compartments of cells; maintaining low levels at the cell surface. The sequestering of GLUT4 intracellularly in the absence of insulin is considered as fundamental for the regulation of glucose uptake. The absence of insulin is often referred to as the basal state or basal condition. Insulin stimulates the redistribution of GLUT4 to the plasma membrane (PM) through a well described signalling cascade involving the activation of class-1 phosphatidylinositol-3-kinase (PI3K) , Akt/PKB and subsequent deactivation of Rab-GAP AS160.

[083] Translocation of GLUT4 occurs through the exocytosis of specialized vesicles enriched with GLUT4 (GLUT4 storage vesicles or GSVs) , which traffic to and fuse with the PM. GLUT translocation involves a number of steps including sorting of GLUT4 ; biogenesis of GSVs ; movement of GSVs along cytoskeletal elements; attachment of GSVs to the PM and finally the fusion of the two lipid bilayers.

[084] Accordingly provided - herein are functional modified proteins whose amino acid sequences comprise sequences substantially identical to the amino acid sequence of GLUT4 (SEQ ID NO: 2). In an example, a modified protein.

[085] Accordingly, the modified protein is labelled with one or more fluorophores that may a) provide a measure of total construct expression levels, allow b) detection of fusion events and c) visualisation and quantification of prefusion behaviour. For example, it can comprise one or more fluorophore (s) on the endofacial side of GLUT4 and one or more fluorophore (s) on the exofacial side of GLUT4. The endofacial side of GLUT4 corresponds to the cytosolic side of the vesicle membrane, while the exofacial side of GLUT4 is on the lumenal (i.e. in the lumen) side of the vesicle membrane.

[086] To facilitate detection of the cytosolic fluorophore (s) and the lumenal fluorophore (s) , the fluorophore (s) are distinct. Having distinct fluorophores allow the separate detection/distinction from one another. In an example, the one or more exofacial fluorophore (s) are pH-sensitive fluorophores; that is, the fluorophores emit different signals upon change of pH.

[087] Preferably, the exofacial fluorophores may have a different signal at about pH 5.5 to pH 6.5 and about pH 7.4. A pH 5.5 to a pH 6.5 is substantially the pH of the lumen of an intracellular vesicle, whereas a pH 7.4 corresponds substantially to the pH of the extracellular space of the lumen when the vesicle is exocytosed, while the cytoplasm has a pH of about 7.2. The pH-sensitive fluorophore may be a fluorescent sensor that is differentially sensitive to protons for at least two excitation or emission wavelengths. Such a pH-sensitive fluorophore may be used for ratiometric . detection. For instance, for a suitable fluorescent dye, emission at one carefully chosen wavelength may be enhanced or diminished relative to the emission at another.

[088] In one example, the cytosolic fluorophore is a fluorophore and the lumenal fluorophore is a ^" pH-sensitive fluorophore. Advantageously, the cytosolic fluorophore and the lumenal pH- sensitive fluorophore are non- interfering. The term non- interfering as used herein refers to different/distinct/non-overlapping emission spectra of the two fluorophores ; that is, the two fluorophores are detectable at different wavelengths.

[089] The cytosolic fluorophore may be a fluorescent protein, or a peptide, polypeptide or protein that may bind a fluorescent probe. The cytosolic fluorophore should be able to provide a measure of total construct expression levels and to account for imaging artefact such as photobleaching, optical path length, local probe concentration, and leakage from cells.

[090] The fluorescent protein as described ^"above may be a red fluorescent protein, a UV or blue fluorescent protein, a cyan fluorescent protein, a green fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein or a far-red fluorescent protein. For example, the red fluorescent protein may comprise tdTomato, mRaspberry, mCherry, mStrawberry, mTangerine, dsRed, dsRed2 ("RFP"), TagRFP, TagRFP-T, AsRed2 , mRFPl , J- Red, R-phycoerythrin, HcRedl, mApple, mRuby, and mRuby2 ; a UV or blue fluorescent protein comprising TagBFP, mTagBFP2, Azurite, EBFP2 , mKalamal, Sirius, Sapphire and T-Sapphire; a cyan fluorescent protein comprising ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP and mTFPl; a green fluorescent protein comprising EGFP, Emerald, Superfolder GFP, monomeric Azami Green, TagGFP2, mUKG, m asabi and Clover; a yellow fluorescent protein comprising EYFP, Citrine, Venus, SYFP2, TagYFP; an orange fluorescent protein comprising Monomeric Kusbira Orange, mKOK, mK02 , mOrange, and mOrange2 and; a far-red fluorescent protein comprising mPlum, HcRed-Tandem, mKate2 (TagFP635) , TurboFP635, Katusha, m eptune and NirFP.

[091] As used herein the term "fluorescent protein" means a protein that is fluorescent; e.g., it may exhibit low, medium or intense fluorescence upon irradiation with light of the appropriate excitation wavelength. The fluorescent characteristic of fluorescent protein is one that arises from the fluorophore wherein the fluorophore results from autocatalytic cyclization of two or more amino acid residues in the polypeptide backbone. As such, the fluorescent proteins of the present invention do not include proteins that exhibit fluorescence only from residues that act by themselves as intrinsic fluors, i.e., tryptophan, tyrosine and phenylalanine.

[092] In many embodiments, the subject fluorescent proteins comprised in the modified protein have an absorbance maximum ranging from about 300 to 700 nm, usually from about 350 to 650 nm and more usually from about 400 to 600 nm. The subject proteins are fluorescent proteins, by which is meant that they can be excited at one wavelength of light following which they will emit light at another wavelength. The excitation spectrum of the subject fluoresecent proteins typically ranges from about 300 to 700 nm.

[093] The fluorescent proteins as described herein generally have a maximum extinction coefficient that ranges from about 25,000 to 150,000 and usually from about 45,000 to 129,000. The subject fluorescent proteins typically range in length from about 150 to 300 amino acids and usually from about 200 to 300 amino acid residues, and generally have a molecular weight ranging from about 15 to 35 kDa, usually from about 17.5 to 32.5 kDa.

[094] In certain examples, the fluorescent proteins as disclosed herein are bright, where by bright is meant that the protein fluorescence can be detected by common methods

(e.g., visual screening, spectrophotometry, spectrofluorometry, fluorescent microscopy, by FACS machines, etc.) Fluorescence brightness of particular fluorescent proteins is determined by its quantum yield multiplied by maximal extinction coefficient.

[095] As used herein, the term "GFP" refers to the green fluorescent protein from A. victoria, including prior art versions of GFP engineered to provide greater fluorescence or fluoresce in different colors.

[096] As used herein, "fluorescent property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy. A measurable difference can be determined as the amount of any quantitative fluorescent property, e.g., the amount of fluorescence at a particular wavelength, or the integral of fluorescence over the emission spectrum.

[097] The fluorescent probe may be a small fluorescent organic molecule, a nanoparticle or a quantum dot. The fluorophore may be attached to functional groups on proteins, such as amino groups, carboxyl groups, thiol or azide .

[098] The lumenal pH-sensitive fluorophore may comprise a pH-sensitive fluorescent protein, a peptide, polypeptide or protein capable of binding a pH-sensitive probe. The pH- sensitive protein as described above may comprise super-ecliptic pHluorin, pHluorin, and mNectarine. Any mutant of an existing fluorescent protein that is sensitive to pH variation may be used. For example, the emission at one carefully chosen wavelength may be enhanced or diminished when the pH varies from a value to another. In another example, the emission wavelength may be enhanced or diminished at a carefully chosen excitation wavelength upon change of pH; that is the emission may switch from red to green upon variation of the pH.

[099] The cytosolic fluorophore and the pH-sensitive lumenal fluorophore must be non-interfering. In other words, the cytosolic fluorophore must be detectable at a wavelength different from that of the lumenal fluorophore. Thus, in an assay by means of fluorescence, the exofacial fluorophore and the endofacial fluorophore of the modified protei may be both detectable independently without any overlap of the emission spectra. For example, the cytosolic fluorophore may be tandem Tomato (tdTomato) , a red shifted derivative of eGFP that emits at 568 nm and is significantly brighter than other red fluorescent proteins. To complement the use of tdTomato, the lumenal pH-sensitive fluorophore may be a super ecliptic pHluorin, which emits at 507 nm.

[100] The cytosolic fluorophore may be bound to any endofacial domain of GLUT . Both the carboxy- (C) and amino- (N) termini of GLUT4 are cytosolic. Moreover, GLUT4 has five endofacial loops. However, attachment of the cytosolic fluorophore should not interfere with the function of GLUT . For example, the cytosolic fluorophore may be attached to the C-terminus of GLUT4. The cytosolic fluorophore may allow detection and quantification of the modified protein.

[101] The cytosolic fluorophore may be engineered to be comprised in a nucleic acid (i.e. a polynucleotide (DNA or RNA) ) that may be expressed in the cells to give the modified protein as described herein. Alternatively, the cytosolic fluorophore may be chemically or enzymatically attached to GLUT4. For example, click chemistry and bioorthogonal labelling may be carried out to attach a fluorophore to GLUT4. The alcohols on the amino acids serine, threonine and tyrosine may be modified. The modification may be the selective oxidation of N-terminal serine and threonine residues by periodate to an aldehyde group. Any reactive group in the amino acids constituting the sequence of the full length GLUT4 of SEQ ID NO: 2 may be used to attach a cytosolic fluorophore, with the proviso that the cytosolic fluorophore does not interfere with the function and localisation of the modified GLUT4, when compared with that of the endogenous GLUT4.

[102] The cytosolic fluorophore may provide a measure of the total amount of the modified protein expression; that is the total cellular GLUT4. Its fluorescence should not be altered by physiologic conditions or changes in conditions (pH, ionic concentrations, reactive oxygen species...) in the cells. In addition the cytosolic fluorophore may account for imaging artefacts such as photobleaching and light source fluctuations.

[103] The lumenal pH- sensitive fluorophore may be as indicated above a fluorescent protein. The fluorescently labelled modified protein may be engineered as a polynucleotide capable of expression in cells of interest. The lumenal pH-sensitive fluorophore may be for example, attached on the first exofacial loop of GLUT4. The labelling of the modified protein should not affect the function of GLUT4. In instances, the fluorescent lumenal protein may be inserted between amino acid 55 and amino acid 66 of full length GLUT4 of SEQ ID NO: 2. In some instances, pH-sensitive fluorophores may be attached to the GLUT4 protein by methods described above [Dear Inventors, thank you for the sequences.]

[104] The modified protein may comprise additional fluorophores or any detectable labels on the cytosolic and lumenal domain of GLUT , with the proviso that the modified protein may be detected and quantified and that the labels should not interfere with one another when detecting the modified GLUT4 , which should have a conserved activity, function and localisation in the cell.

[105] One example of the modified protein as described herein is the protein of SEQ ID NO: 8, comprising a tdTomato protein of SEQ ID NO: 4 attached to the C- terminus of GLUT4 , the GLUT4 protein and a super-ecliptic pHluorin of SEQ ID NO: 6 inserted in the first exofacial loop, between amino acid 55 and amino acid 66 of the full length GLUT4 of SEQ ID NO: 2.

[106] Other examples of the modified protein may include modified proteins with about 85% to about 99% amino acid sequence identity with the modified protein of SEQ ID NO: 8. In some variations, the modified proteins that are substantially similar or substantially identical to the specific amino acid sequences of the subject invention, i.e., SEQ ID NO: 8 are also provided. Sequence identity is calculated based on a reference sequence as determined using MegAlign, DNAstar clustal algorithm as described in D.G. Higgins and P.M. Sharp, "Fast and Sensitive multiple Sequence Alignments on a Microcomputer," CABIOS, 5 pp. 151-3 (1989) (using parameters ktuple 1, gap penalty 3, window 5 and diagonals saved 5) . In many examples, amino acid sequences of interest have much higher sequence identity e.g., 93%, 95%, 97%, 99%, 100%, particularly for the sequence of the amino acids that provide the functional regions of the protein.

[107] Proteins that are mutants of the above-described proteins are also provided. Mutants may retain biological properties of the source proteins, or may have biological properties which differ from the wild type proteins. The term "biological property" of the proteins of the present invention refers to, but is not limited to, fluorescent properties; biochemical properties, such as in vivo and/or in vitro stability (e.g., half-life); maturation speed, aggregation tendency and oligomerization tendency and other such properties. Mutations include single amino acid changes, deletions or insertions of one or more amino acids, N-terminal truncations or extensions, C-terminal truncations or extensions and the like. Mutants can be generated using standard techniques of molecular biology as described in details below.

[108] Given the guidance provided in the Examples, and using standard techniques, those skilled in the art can readily generate a wide variety of additional mutants and test whether a biological (e.g. biochemical, spectral, etc.) property has been altered. For example, fluorescence intensity can be measured using a spectrophotometer at various excitation wavelengths.

[109] The modified proteins as described herein may be present in the isolated form, by which is meant that the protein is substantially free of other proteins and other naturally-occurring biological molecules, such as oligosaccharides, nucleic acids and fragments thereof, and the like, where the term "substantially free" in this instance means that less than 70%, usually less than 60% and more usually less than 50% of the composition containing the isolated protein is some other natural occurring biological molecule. In certain examples, the proteins are present in substantially purified form, where by "substantially purified form" means at least 95%, usually at least 97% and more usually at least 99% pure.

[110] In a preferred embodiment, the subject proteins are synthetically produced, e. g. by expressing a recombinant nucleic acid coding sequence encoding the protein of interest in a suitable host, as described above. Any convenient protein purification procedures may be employed, where suitable protein purification methodologies are known to the person skilled in the art. For example, a lysate may be prepared from the original source and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, and the like.

[Ill] Also provided are fusion proteins comprising a protein of the present invention, or functional fragments thereof, fused, for example, to a degradation sequence, a sequence of subcellular localization {e.g. nuclear localization signal, peroximal targeting signal, Golgi apparatus targeting sequence, mitochondrial targeting sequence, etc.), a signal peptide, or any protein or polypeptide of interest. Fusion proteins may comprise for example, a fluorescent protein of subject invention and a second polypeptide ("the fusion partner") fused in-frame at the N-terminus and/or C-terminus of the fluorescent protein. Fusion partners include, but are not limited to, polypeptides that can bind antibodies specific to the fusion partner {e.g., epitope tags), antibodies or binding fragments thereof, polypeptides that provide a catalytic function or induce a cellular response, ligands or receptors or mimetics thereof, and the like.

[112] In yet another example, the modified protein comprises GLUT4 comprising the amino acid sequence shown in SEQ ID NO: 2. In some variations, the modified protein comprises GLUT4 comprising the amino acid sequence shown in SEQ ID NO: 2 in which one or a few amino acid residues are replaced, deleted, inserted and/or added, the protein having the GLUT4 activity. In some variations, the modified protein comprises GLUT4 comprising a protein comprising an amino acid sequence having about 86.0% or greater of homology with the amino acid sequence shown in SEQ ID NO: 2, the protein having the GLUT4 activity.

[113] The modified protein may be encoded by an isolated nucleic acid. As used herein the term "isolated" means a molecule or a cell that is an environment different from that in which the molecule or the cell naturally occurs .

[114] Reference to a nucleotide sequence "encoding" a polypeptide means that the sequence, upon transcription and translation of mRNA, produces the polypeptide. This includes both the coding strand, whose nucleotide sequence is identical to mRNA and whose sequence is usually provided in the sequence listing, as well as its complementary strand, which is used as the template for transcription. As any person skilled in the art recognizes, this also includes all degenerate nucleotide sequences encoding the same amino acid sequence. Nucleotide sequences encoding a polypeptide include sequences containing introns .

[115] As used herein the term "mutant" refers to a protein disclosed in the present invention, in which one or more amino acids are added and/or substituted and/or deleted and/or inserted at the N-terminus, and/or the C- terminus, and/or within the native amino acid sequences of the proteins of the present invention. As used herein the term "mutant" refers to a nucleic acid molecule that encodes a mutant protein. Moreover, the term "mutant" refers to any shorter or longer version of the protein or nucleic acid herein.

[116] As used herein, "homologue or homology" is a term used in the art to describe the relatedness of a nucleotide or peptide sequence to another nucleotide or peptide sequence, which is determined by the degree of identity and/or similarity between said sequences compared.

[117] As used herein, an amino acid sequence or a nucleotide sequence is "substantially identical" to a reference sequence if the amino . acid sequence or nucleotide sequence has at least 95% sequence identity {e.g. 95%, 97%, 98%, 99%, or 100% sequence identity) with the reference sequence over a given comparison window. As used herein, an amino acid sequence or a nucleotide sequence is "substantially similar" to a reference sequence if the amino acid sequence or nucleotide sequence has at least 80% sequence identity (e.g. 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity) with the reference sequence over a given comparison window. Sequence identity is calculated based on a reference sequence. Algorithms for sequence analysis are known in the art, such as BLAST.

[118] A nucleic acid molecule as used herein is a DNA molecule, such as genomic DNA molecules or cDNA molecules, or an RNA molecule, such as mKNA molecules.

[119] In particular, said nucleic acid molecules are

DNA molecules comprising an open reading , frame that encodes a modified protein of the invention. The subject nucleic acids are present in an environment other than their natural environment; e.g., they are isolated, present in enriched amounts, or are present or expressed in vitro or in a cell or organism other than their naturally occurring environment. In one example, nucleic acid molecules of the present invention are engineered, i.e. obtained from a naturally occurring protein, e.g. wild type GLUT , by means of modifications.

[120] The modifications, as well as additions or deletions can be introduced by any method known in the art including error prone PCR, shuffling, oligonucleotide- directed mutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site directed mutagenesis, random mutagenesis, gene reassembly, gene site saturated mutagenesis (GSSM) , synthetic ligation reassembly (SLR) , or a combination thereof. The modifications, additions or deletions may be also introduced by a method comprising recombination, recursive sequence recombination, phosphothioate- modified DNA mutagenesis, uracil- containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair- deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction selection mutagenesis, restriction-purification mutagenesis, artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acid multimer creation or a combination thereof .

[121] Also provided are nucleic acids that hybridize to the above-described nucleic acids under stringent conditions, preferably under high stringency conditions (i.e., complements of the previously-described nucleic acids) . An example of stringent conditions is hybridization at 50°C or higher and O.lxSSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of high stringency hybridization conditions is overnight incubation at 42 °C in a solution of 50% formamide, 5xSSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 x Denhardt ' s solution, 10% destran sulfate, and 20 μς/πιΐ denatured, sheared salmon sperm DNA, followed by washing in O.lxSSC at about 65°C. Other high stringency hybridization conditions are known in the art and may also be used to identify nucleic acids described herein. [122] In addition, degenerate variants of the nucleic acids that encode the modified proteins as disclosed herein are also provided. Degenerate variants of nucleic acids comprise replacements of the codons of the nucleic acid with other codons encoding the same amino acids. In particular, degenerate variants of the nucleic acids are generated to increase its expression in a host cell. In this embodiment, codons of the nucleic acid that are non- preferred or a less preferred in genes in the host cell are replaced with the codons overrepresented in coding sequences in genes in the host cell, wherein said replaced codons encode the same amino acid. In a preferred embodiment, nucleic acids of the present invention are humanized. As used herein, the term "humanized" refers to changes made to the nucleic acid sequence to optimize the codons for expression of the protein in mammalian (human) cells.

[123] In one example, the modified protein is encoded by the nucleic acid of SEQ ID NO.:7. Other examples of modified GLUT4 protein of the invention may include modified protein encoded by a nucleic acid comprising the nucleotide sequence shown in SEQ ID NO. : 7 in which 1 to 30 nucleotides are replaced, deleted, inserted and/or added, the nucleic acid encoding for the protein having the GLUT4 activity. In another example, the modified GLUT4 protein may comprise a nucleic acid encoding for the protein of SEQ ID NO.: 8. In yet another example, the modified GLUT4 protein is a protein that may be encoded by a nucleic acid that hybridizes, under a stringent condition as described above, with a polynucleotide comprising a nucleotide sequence complementary to that of any one of the nucleic acids as defined above, the nucleic acid encoding for a protein having the GLUT4 activity. [124] The nucleic acids as described herein, the corresponding cDNAs, full length genes and constructs can be generated synthetically by a number of different protocols known to those of skill in the art. Appropriate nucleic acid constructs are purified using standard recombinant DNA techniques as described in the art .

[125] The modified GLUT4 may be produced by a variety of methods, such as recombinant DNA methods, enzymatic modification of GLUT4 or chemical modification of the protein. The recombinant method may comprise methods to produce DNA or RNA encoding all or a portion of the modified protein, followed by expression by an appropriate recombinant vector or system, and if appropriate or necessary further modification or joining of portions/components, such as the cytosolic and lumenal fluorophores as described herein.

[126] It has been found that fluorescent proteins can be genetically fused to other target proteins and used as markers to identify the location and amount of the target protein produced. Accordingly, this invention provides nucleic acids encoding the modified protein that comprise GLUT4 , a cytosolic fluorescent protein, a lumenal pH- sensitive fluorescent protein and additional amino acid sequences. For example, described herein is the nucleotide sequence of SEQ ID NO: 1 encoding the GLUT4 protein, SEQ ID NO: 3 encoding the tdTomato protein, SEQ ID NO: 5 encoding the pHluorin protein and SEQ ID NO: 7 encoding the modified protein described herein. Such sequences can be, for example, up to about 15, up to about 100, up to about 200 or up to about 1000 amino acids long. The fusion proteins possess the ability to fluoresce that is determined by a fluorescent protein portion.

[127] Also provided are vector and other nucleic acid constructs comprising the subject nucleic acids. Suitable vectors include viral and non-viral vectors, plasmids, cosmids, phages, etc., preferably plasmids, and used for cloning, amplifying, expressing, transferring etc. of the nucleic acid sequence of the present invention in the appropriate host.

[128] In one example, the viral vector may comprise any viral vector that comprises a nucleic acid encoding for the modified GLUT4 when introduced into a cell. In another example, the vector may comprise a retroviral vector, an adenoviral vector, an adeno-associated virus, a hepatitis virus, a herpes virus, a lentivirus, a retrovirus, a baculovirus, a vaccinia virus or other eukaryotic expression vectors such as replication- deficient forms of the viruses.

[129] The choice of appropriate vector is well within the skill of the art, and many such vectors are available commercially. To prepare the constructs, the partial or full-length nucleic acid is inserted into a vector typically by means of DNA ligase attachment to a cleaved restriction enzyme site in the vector. Alternatively, the desired nucleotide sequence can be inserted by homologous recombination in vivo, typically by attaching regions of homology to the vector on the flanks of the desired nucleotide sequence. Regions of homology are added by ligation of oligonucleotides, or by polymerase chain, reaction using primers comprising both the region of homology and a portion of the desired nucleotide sequence, for example .

[130] Also provided are expression cassettes or systems used inter alia, for the production of the subject fluorescent proteins or fusion proteins thereof or for replication of the subject nucleic acid molecules. The expression cassette may exist as an extra chromosomal element or may be integrated into the genome of the cell as a result of introduction of said expression cassette into the cell. For expression, the gene product encoded by the nucleic acid of the invention is expressed in any convenient expression system, including, for example, bacterial, yeast, insect, amphibian, or mammalian systems. In the expression vector, a subject nucleic acid is operably linked to a regulatory sequence that can include promoters, enhancers, terminators, operators, repressors and inducers. Methods for preparing expression cassettes or systems capable of expressing the desired product are known for a person skilled in the art.

[131] Cell lines, which stably express the proteins of present invention, can be selected by the methods known in the art (e.g. the co-transfection with a selectable marker such as dhfr, gpt, neomycin, or hygromycin allows the identification and isolation of the transfected cells that contain the gene integrated into a genome) .

[132] The above-described expression systems may be used in prokaryotic or eukaryotic hosts. Host-cells such as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of a higher organism such as vertebrates, e.g., COS 7 cells, HEK 293, CHO, Xenopus oocytes, etc., may be used for production of the protein.

[133] In some examples, the host cells comprise mammalian cells in which GLUT4 exocytosis is stimulated by insulin. Cells can be differentiated or undifferentiated and in some examples, are adipocytes, fibroblasts or muscle cells, such as 3T3-L1 cells or Chinese Hamster Ovary cells.

[134] When any of the above-referenced host cells, or other appropriate host cells or organisms are used to replicate and/or express the nucleic acids of the invention, the resulting replicated nucleic acid, expressed protein or polypeptide is within the scope of the invention as a product of the host cell or organism. The product may be recovered by an appropriate means known in the art .

[135] The nucleic acids of the present invention can be used to generate transgenic organisms or site-specific gene modifications in cell lines.

[136] Transgenic cells of the subject invention include one or more nucleic acids according to the subject invention present as a transgene. For the purposes of the invention any suitable host cell may be used including prokaryotic (e.g. Escherichia coli, Streptomyces sp., Bacillus subtilis, Lactobacillus acidophilus, etc) or eukaryotic host-cells. Transgenic organisms of the subject invention can be prokaryotic or eukaryotic organisms including bacteria, cyanobacteria, fungi, plants and animals, in which one or more of the cells of the organism contain heterologous nucleic acid of subject invention introduced by way of human intervention, . such as by transgenic techniques well known in the art.

[137] The isolated nucleic acid of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the nucleic acid molecules (i.e. DNA) into such organisms are widely known and provided in the art .

[138] In one example, the transgenic organism can be a prokaryotic organism. Methods on the transformation of prokaryotic hosts are well documented in the art.

[139] In another embodiment, the transgenic organism can be a fungus, for example yeast. Yeast is widely used as a vehicle for heterologous gene expression) . Several types of yeast vectors are available, including integrative vectors, which require recombination with the host genome for their maintenance, and autonomously- replicating plasmid vectors.

[140] Another host organism is an animal. Transgenic animals can be obtained by transgenic techniques well known in the art. For example, transgenic animals can be obtained through homologous recombination, where the endogenous locus is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retroviruses and other animal viruses, YACs, and the like.

[141] The nucleic acid can be introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus or with a recombinant viral vector and the like. The term genetic manipulation does not include classical cross-breeding or in vitro fertilization, but rather is directed to the introduction of a recombinant nucleic acid molecule. This nucleic acid molecule may be integrated within a chromosome, or it may be extrachromosomally replicating DNA.

[142] DNA constructs for homologous recombination will comprise at least a portion of a nucleic acid described herein, wherein the gene has the desired genetic modification (s) , and includes regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and negative selection may be included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art.

[143] For embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, such as a mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast- feeder layer or grown in the presence of leukaemia inhibiting factor (LIF) . Transformed ES or embryonic cells may be used to produce transgenic animals using the appropriate technique described in the art .

[144] The transgenic animals may be any non-human animals including nonhuman mammal (e.g. mouse, rat), a bird or an amphibian, etc., and used in functional studies, drug screening and the like.

[145] The modified proteins as described (as well as other components of the subject invention described above) find use in a variety of different applications. Representative uses for each of these types of proteins will be described below, where the uses described herein are merely exemplary, and are in no way meant to limit the use of the modified proteins of the present invention to those described.

[146] In one example, the modified proteins as described find use as in vivo labels (or reporter molecules) in cell and molecular biology assays. The assays of interest include but are not limited to assays for gene expression, protein localization and co- localization, protein-protein interactions, high- throughput assessment of GLUT4 exocytosis in multiple cells, GLUT4 trafficking assessment in multiple cells, assessment of individual GLUT4 trafficking steps (fusion and prefusion) , screening for drugs affecting GLUT4 exocytosis and trafficking etc.

[147] For example, the modified protein may be used in screening compounds that can promote GLUT4 translocation and membrane insertion, for understanding the mechanism of action of existing/candidate pharmaceutical compositions, and investigating the biological basis of GLUT4 trafficking and its regulation. The modified proteins as described herein find use as a measure of total construct expression levels, as a marker for imaging artefacts as described above, and as real-time probes for fusion events and prefusion events such as approach and attachment in living cells and fixed cells.

[148] The subject proteins may find use for identifying and/or measuring the expression of a protein or polypeptide of interest in biological material. This method may include, but is not limited to the following steps: i) introducing into a cell a nucleic acid molecule comprising a nucleotide sequence encoding the modified protein as described herein wherein said nucleic acid molecule is operatively linked to and under the control of an expression control sequence which controls expression of the protein or polypeptide of interest; ii) expression of said nucleic acid under suitable conditions; and iii) detecting the fluorescence emission of the cytosolic fluorescent protein as a means of measuring the expression of the modified GLUT4.

[149] Also, the subject proteins find use for localization of the modified GLUT4 in biological material. This method may include, but is not limited to the following steps: i) introducing into a cell a nucleic acid molecule comprising a nucleotide sequence encoding a cytosolic fluorescent protein as described herein wherein said nucleic acid molecule is fused with a sequence encoding GLUT4 and operatively linked to and under the control of an suitable expression control sequence; ii) culturing the cell under conditions suitable for the expression of the modified GLUT4 ; and iii) detecting the fluorescence emission of the fluorescent protein as a means of measuring the localization of the modified GLUT4.

[150] Additionally, provided herein is a method for detecting fusion events of the modified GLUT4. This method may include but is not limited to the following steps: i) introducing into a cell a nucleic acid molecule comprising a nucleotide sequence encoding a lumenal pH-sensitive fluorescent protein as described herein wherein said nucleic acid molecule is fused with a sequence encoding GLUT4 and operatively linked to and under the control of an suitable expression control sequence; ii) culturing the cell under conditions suitable for the expression of the modified GLUT4 ; and iii) detecting the fluorescence emission of the fluorescent protein as a means of measuring the fusion of the modified GLUT4 to the plasma membrane .

[151] In one example, provided herein is a method for assessing GLUT4 exocytosis in response to a condition, such as the presence of insulin, another hormone, decreased glucose concentration or conditions which mimic insulin resistance, such as high glucose levels or increased tumour necrosis factor-a concentration, high non-esterified fatty acid concentration and other conditions or a stimulus, such addition of an agent in multiple individual cells.

[152] The method may provide important biological information that is masked by population-averaged assays. As used herein, the term exocytosis includes both movement from an intracellular location, such as GLUT4 storage vesicles (GSVs) to the plasma membrane, and movement from the plasma membrane to an intracellular location. GLUT4 translocation has been described above. It includes sorting of GLUT4 , biogenesis of GSVs, movement of GSVs along cytoskeletal elements, attachment of GSVs to the PM and finally the fusion of the two lipid bilayers.

[153] The method may comprise i) culturing mammalian cells expressing the modified protein as described herein under a condition or stimulus to be assessed for its effect on exocytosis of modified GLUT4 , wherein the cells are referred to as test cells. In the absence of insulin or the condition to be tested the cells are under basal conditions. Intensity of both the cytosolic fluorophore and the lumenal fluorophore are determined. The modified protein may be detected using apparatus that include but are not limited to fluorescence microscopes, confocal microscopes, and other more advanced/specialized microscopes (such as total internal reflection microscopes) , super resolution microscopes, and high- throughput fluorescence systems.

[154] In a further example, the intensity may be determined by subcellular fractionation, microscopy, such as confocal microscopy, widefield epifluorescence microscopy (epi-M) , or Total Internal Fluorescence Microscopy (TIRFM) , a powerful application of microscopy that illuminates less than 200 nm into the basal surface of the cell. When combined with the modified protein, this technique allows individual trafficking events to be resolved, notably fusion and prefusion behaviour. The method can further comprise ii) a time-resolved detection of a signal from the cytosolic fluorophore and a signal from the luminal pH-sensitive fluorophore.

[155] Preferably, two sets of values are obtained, each corresponding to fluorescence intensities at two distinct wavelengths, one each for the basal intensity (in the absence of insulin) for example, in the red channel (corresponding to the cytosolic fluorophore) and one in the green channel (corresponding for example, to the lumenal fluorophore). The values are then obtained upon a time course after stimulation of the cells for example, with insulin. Thus, a ratio of the signal from the cytosolic fluorophore and the signal from the pH-sensitive fluorophore of the modified protein in the test cells of ii) as defined above can be determined. A test value is thereby produced, which is indicative of the plasma membrane localisation of the modified protein of interest normalised to said protein total expression level; that is the value is indicative of the extent to which translocation of the protein has occurred.

[156] The following example makes specific reference to GLUT4, but the same procedures, calculations and analyses can be applied to any protein of interest whose translocation is to be assessed. The two wavelengths, F_L and F_c, correspond respectively to the peak of emission of the lumenal pH-sensitive fluorophore, and the cytosolic fluorophore in the cell. As mentioned above, cell surface GLUT4 can be measured by pH- sensitive super ecliptic pHluorin. The two sets of values are as follows: 1. Basal fluorescences of the cells used in the presence of the modified protein as described herein. Here, F_L (represented by A in the formula below) can be the fluorescence of cells expressing the modified GLUT4 protein on the green channel and F_c (represented in the formula by B) is the fluorescence of control cells that express modified GLUT4 on the red channel, in the absence of insulin (i.e. basal state); and 2 . fluorescence intensities (F_L and F_c, represented, respectively, in the formula by C and D) of stimulated cells (cells cultured in the presence of/ exposed to conditions to be assessed for their effects on exocytosis) which express the cytosolic fluorescent protein and lumenal pH- sensitive fluorescent protein. C is the total fluorescence of the lumenal protein at that wavelength and D is the total fluorescence due to the endofacial label at that wavelength. The next step iii) is the determination of a ratio of the signals C/A and D/B, thus providing a time-resolved trace fluorescence of the green and red signal over the course of a stimulation with a condition or a substance of interest, for example insulin. The last steps iy) is a comparison of the test value with the control value, where the control value corresponds to the ratio of A/B; that is the value of the green signal over the value of the red signal in the absence of a condition that activates exocytosis (for example, pHluorin: tdTomato ratio). This value can then be compared to the time-resolved values obtained in the exocytosis conditions to be assessed. In other words, the ratio C/D is calculated over the time course of the stimulation.

[157] The test ratio is then compared with the control

(basal) ratio to give a value R. If R=l, then the condition to which the test cells (also referred to as stimulated cells) were subjected (e.g., stimulation by insulin) caused no change in the proportion of total GLUT4 at the cell surface. If R=3.5 (as in example 1 below), then the condition (e.g., stimulation by insulin) caused a 3.5-fold increase in the fraction of total GLUT4 present at the cell surface. Thus, the A/B and C/D ratio provide an accurate indication of surface (i.e. plasma membrane) GLUT4 levels relative to total GLUT4. For example, in single cells, the tdTomato signal was insensitive to insulin stimulation, thus B/D=l. As described in example 2 below, the above can be used to calculate the pHluorin: tdTomato ratio, thus providing a high-throughput ratio-metric imaging of the modified protein as described herein. In one example, the detection of the fluorescence intensity is carried out by two-colour epi-M.

[158] This example of the present method of determining or assessing exocytosis of GLUT4 may comprise: i) culturing cells expressing modified GLUT4 under conditions to be assessed for their effects on translocation of GLUT , wherein modified GLUT4 is GLUT4 as described herein and wherein the cells are referred to as test cells; ii) determining the proportion of modified GLUT4 at the cell membrane to total modified GLUT4 in the test cells, thereby producing a test value; iii) comparing the test value with a control value, wherein the control value is the proportion of modified GLUT4at the cell membrane to total modified GLUT4 in control cells, wherein the control cells are the same cells as are cultured in i) and are cultured under the same conditions as in i) , except that the control cells are not cultured under the condition or stimulus to be assessed. If the test value is greater than the control value, then there is a greater proportion of GLUT4 at the cell membrane of the test cells than at the cell membrane of control cells. In particular examples, modified GLUT4 at the cell membrane is assessed (quantified or detected) by means of super-ecliptic pHluorin detectable at a wavelength different from the wavelength at which the cytosolic tdTomato is detected.

[159] As discussed above, in one embodiment, the test cells are assessed and the change in the proportion of GLUT4 at the cell surface is determined as follows: The fluorescence intensity at the cell surface (F_L) is determined, as described herein, thus providing a measure of GLUT4 at the cell surface of test cells. The fluorescence - intensity of the cytosolic fluorescent protein is determined, thus providing a measure of total GLUT4 in test cells. This value is designated B in the formula. The fluorescence intensity at the cell surface and the fluorescence intensity of the cytosolic protein are determined for control (or reference) cells, referred to as basal unstimulated control cells thus providing a measure of GLUT4 at the cell surface and a measure of total GLUT4 for both types of cells. [160] Background cells are the same type of cells as the test cells and are cultured under the same conditions as the conditions under which test cells are cultured, except that they are not subjected to the conditions to which the test cells are subjected in order to alter translocation of GLUT4. (For convenience, cells that are not subjected to the conditions or stimuli to which test cells are subjected in order to alter translocation of GLUT4 are referred to as "unstimulated or basal cells").

[161] In the formula presented above, F_L and F_c are represented, respectively by A and B, as described. Basal control cells are also the same type of cells as the test cells and, like the test cells, express modified GLUT . Unlike the test cells, basal cells are not subjected to conditions to alter translocation. For example, if test cells are treated with insulin, in order to enhance translocation of GLUT4 , unstimulated control cells are cultured under the same conditions except in the absence of insulin. Fluorescence intensity at the surface of unstimulated control cells (F_L) is assessed, as described herein (e.g., by means of the lumenal pH-sensitive fluorophore) , thus providing a measure of GLUT4 at the cell surface.

[162] The fluorescence intensity of the cytosolic fluorescent protein (F_c) is also determined, thus providing a measure of total GLUT4 in the basal control cells. The intensity of F_L is designated A and the intensity of F_c is designated B for basal control cells. The change in the proportion (or ratio) of GLUT4 at the cell surface of test cells (R) is assessed/calculated as follows in this embodiment: R= [ (C) / (D) ] / [ (A) / (B) ] where (C) / (D) is proportional to the fraction of GLUT4 at the cell surface in test cells and (A) / (B) is proportional to the fraction of GLUT4 at the cell surface of basal (unstimulated) control cells.

[163] If R=l, the condition to which test cells were subjected (e.g., insulin stimulation, low glucose concentration, high glucose concentration) caused no change in the proportion of GLUT4 at the cell surface. If R is greater than 1, the condition to which test cells were subjected caused a change in the proportion of GLUT4 at the cell surface. For example, if R=5, the condition caused a 5-fold increase in the fraction of total GLUT4 at the cell surface. The test cell values (fluorescence at the cell membrane and fluorescence of the intracellular tag or reporter protein) can be compared with control cell values (e.g., unstimulated control cell values) which are obtained through assessments carried out at the same time that test cells are assessed or obtained through assessments carried out prior or subsequent to assessment of test cells.

[164] In the latter instances, respectively, the test values are compared with a previously-established set of control values (a previously-established reference) or a subsequently-established set of control values (a subsequently established reference) .

[165] The present invention also relates to a method of identifying or screening a drug or agent that alters GLUT4 translocation from an intracellular location to the cell (plasma) membrane. In the method, cells in which modified GLUT4 is expressed are cultured in a condition that promotes exocytosis of the modified protein as described herein. The cells are combined with a candidate drug (a drug to be assessed for its ability to alter GLUT4 translocation) and the proportion of modified GLUT4 at the plasma membrane (relative to total GLUT4 in the cells) is determined and compared with the proportion of modified GLUT4 at the plasma membrane in control cells, which are cells of the same type as the test cells cultured under the same conditions as the test cells, but in the absence of the candidate drug. Example 2 below gives some specific examples of drugs that interfere or inhibit the insulin- related translocation of a modified protein as described herein.

[166] In one example of the present method by which a drug that enhances GLUT4 translocation to the cell membrane is identified, cells that express modified GLUT4 protein are cultured in the absence or presence of a candidate drug, for sufficient time for the effect (if any) of the candidate drug to be assessed. A candidate drug is one (a compound or molecule) whose effects are being assessed.

[167] In each case, the effect of insulin to cause an increase in the proportion of modified GLUT4 at the plasma membrane (relative to the total GLUT4 content of the cells) is determined and compared with the proportion of modified GLUT4 at the plasma membrane in an appropriate control or controls. For example, mammalian cells (which can be differentiated or undifferentiated) expressing modified GLUT4 protein are cultured with or without a candidate drug, as well as with or without conditions that stimulate GLUT4 translocation (e.g., the presence of insulin) and changes in the proportion of plasma membrane GLUT4 to total cell GLUT4 are determined for each set of conditions. Cells cultured in the presence of the drug are referred to as test cells and the resulting proportion of GLUT4 at the cell surface in the absence or presence of insulin (or other stimulator) is referred to as test values. The proportions of GLUT4 at the cell membrane in the test cells are compared with the proportions in control cells, which are cells of the same type as the test cells that are cultured in the same manner as are the test cells, except in the absence of the candidate drug.

[168] The proportion of GLUT4 at the cell surface in the presence, or absence of insulin, or in the absence of the drug is referred to as control values. If the test values are greater than the control values, the candidate drug is a drug that enhances GLUT4 translocation to the cell membrane. In certain cases, the method measures actual proportions, while in other cases relative proportions are measured. An insulin sensitizing drug may enhance the ability of insulin to cause GLUT4 translocation to the cell surface.

[169] In the present method of identifying/screening a drug or agent that enhances or inhibits GLUT4 translocation to the cell membrane, an appropriate population of cells (such as adipocytes, an adipocyte cell line, muscle cells, a muscle cell line or any other cell type in which GLUT4 exocytosis is stimulated by insulin) in which modified GLUT4 is expressed (referred to as test cells) is combined with a drug to be assessed for its effects on GLUT4 translocation. Modified GLUT4 is expressed from a vector present in the test cells or is stably incorporated into the host cell DNA and expressed. Prior to stimulating cells for GLUT4 translocation, cells can be preconditioned by subjecting them to conditions which mimic insulin resistance.

[170] The resulting combination is maintained under appropriate conditions and for sufficient time for the drug to have its effect on the cells, which are referred to as treated cells. The treated cells are exposed to or contacted with a substance, such as insulin, which induces GLUT4 translocation. This results in stimulation of GLUT4 translocation in the treated cells. GLUT4 translocation is assessed by determining the extent to which the pH- sensitive lumenal protein occurs extracellularly, normalized to the total amount of the GLUT4 reporter present, as described above. This is done using known methods, such as by an immunoassay or by measuring fluorescence at the membrane. If there is greater proportion of total GLUT4 at the plasma membrane in test cells than in untreated cells (such as cells of the same type as the test cells which have not been treated with the drug but are otherwise maintained under the same conditions) , the drug or agent is one which enhances GLUT4 translocation.

[171] In one example of the method to determine whether a drug enhances GLUT4 translocation, eight types of cells can be assessed: 1) cells which express modified GLUT4 and are stimulated by insulin or subjected to conditions which mimic insulin resistance; 2) cells which express modified GLUT4 and are not stimulated by insulin or subjected to conditions which mimic insulin resistance; 3) cells which do not express modified GLUT4 and are stimulated by insulin or subjected to conditions which mimic insulin resistance; 4) cells which do not express modified GLUT4 and are not stimulated by insulin or subjected to conditions which mimic insulin resistance and; 5) -8) cells treated the same as cells in l)-4), but in the presence of the drug. Cells which do not express modified GLUT4 are an additional control, in that they indicate the basal fluorescence.

[172] In some examples, the method may be used to dissect the steps of the modified protein trafficking. As described above the steps of trafficking include approach, attachment and fusion. It was previously noted that a modified protein comprising only one eGFP fused with a protein of interest cannot be used to distinguish between a protein in vesicles near the plasma membrane or in the plasma membrane itself by TIRF; all the molecules within the TIRF zone, a region of about 200 nm adjacent to the coverslip, are detected. Therefore, the modified protein as described herein may allow detection of the different steps for the following reasons. The cytosolic fluorescent protein, when imaged by TIRFM, would reflect the total amount of protein of interest entering the TIRF zone while the lumenal pH-sensitive fluorescent protein would provide a measure of the protein of interest only on the PM.

[173] Therefore, the method as described above may comprise: culturing multiple cells expressing the modified protein under basal conditions, imaging the cells by low frequency TIRFM, determining the intensity of the fluorescence at two distinct wavelength allowing detection of the two fluorescent proteins, providing the cells with the conditions or stimuli to be studied for their effects on specific steps of the protein of interest trafficking, measuring/determining the fluorescence intensity and localisation of the modified protein under the same two wavelengths as previously used, and comparing the ratio of fluorescence between the two fluorescent proteins and between the two conditions over time (i.e. presence/absence of the studied compound) .

[174] The cells will express varying amounts of the construct and this can be monitored by assessing fluorescence using the cytosolic fluorescent protein. No change in green fluorescence in the cell population indicates that the treatment has not caused an increase in extracellular levels by affecting the amount of the protein present, since if the treatment increased the amount of the protein present, there would also be an increase in red fluorescence due to the pH-sensitive lumenal fluorescent protein (e.g., green fluorescence) to red fluorescence indicates where the modified GLUT4 is located in the cell (inside or outside the cell) .

[175] In one example, there is provided a method of assessing individual modified protein vesicle trafficking to explore fusion and prefusion event. Advantageously, the pH-sensitive lumenal fluorescent protein may be used to detect fusion events. The detection of fusion events can be achieve because of the sensitivity of the lumenal fluorescent protein to change in pH from the inside of the lumen (pH<6.5) to the extracellular environment (pH 7.4). Accordingly, the delivery of the modified protein to the plasma membrane can be visualized.

[176] In one specific example, high-frequency TIRF (10

Hz; HF-TIRFM) may be used to capture images of a single adipocyte expressing the modified protein as defined herein. More specific details are given in example 4 below.

[177] The fusion events detected by the method described above may advantageously be detected by automated fusion event detection algorithms. These algorithms are enabled by the distinct profile characteristic of the fusion event. This profile is characterized by a rapid increase in intensity of the signal from the lumenal protein followed by a radial dispersal of the signal and a decrease in intensity as the protein diffuses in the plasma membrane away from the site of fusion.

[178] As defined herein, the fusion event rate (F_ER)is defined as the amount of fusion per unit time per cell area. The F_ER enables to quantify the total number of fusion events. The method as described herein also provides for comparative analysis to enable correlating the F_ER with the total fluorescence observed in the TIRF- zone . [179] The method may comprise culturing the cells expressing the modified protein under conditions or stimulus to be assessed that affect fusion events. Under basal conditions (see supra) , the rate of fusion is estimated over time and can be plotted as a cumulative fusion over time. If the rate of fusion is constant under basal conditions, it means that the amount of the modified protein in the plasma membrane is constant. Advantageously, this data can be used to calculate the rate of endocytosis of the modified protein. The rate of endocytosis in this case is identical to the rate of exocytosis and is a reflection of the overall signal from the lumenal pH- sensitive protein. The basal rate of endocytosis can then be used to correct the cumulative fusion for rate of endocytosis. The F_ER may be used in test cells to investigate whether conditions or stimuli as described herein affect fusion events of the modified proteins in single cells and in multiple cells. The modified protein may therefore advantageously be used to image individual GSV fusion events. More advantageously, the modified protein as described herein can be used to measure the F_ER at any point of time . Even more advantageously, the modified protein can be used to derive the rate of endocytosis of the protein in live single cells when combining the F_ER with the mean TIRFM signal as indicated above .

[180] Provided herein is a method to assess individual prefusion events of the modified protein as described herein. The method is based on the retrospective study of the behaviour of each event prior to fusion as described above. The method may comprise quantitatively determining behaviour of the modified protein containing vesicles just prior to fusion. Further the method may comprise determining the presence of a signal from the cytosolic fluorescent protein prior to the fusion event.

[181] The modified proteins as disclosed herein also find use in applications involving the automated screening of arrays of cells expressing fluorescent reporting groups by using microscopic imaging and electronic analysis.

[182] Screening can be used for drug discovery and in the field of functional genomics where the modified proteins are used as markers of individual cells to detect changes in localization and activity of GLUT4.

[183] The modified proteins as described herein also can be used in high content screening to detect co- localization of other fluorescent fusion proteins with localization markers as indicators of movements of intracellular fluorescent proteins/peptides or as markers alone .

[184] Unless specified otherwise, the terms

"comprising" and "comprise", and grammatical variants thereof, are intended to represent "open" or "inclusive" language such that they include recited elements but also permit inclusion of additional, unrecited elements.

[185] As used herein, the term "about", in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/ - 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value. EXAMPLES

[186] Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

[187] EXAMPLE 1

[188] Generation and Validation of a Novel GLUT4 Construct

[189] A dual-colour GLUT4 probe hereafter called rGLUTpHluor was engineered to be capable among other functions of revealing aspects of GLUT4 exocytosis. This probe comprises a pH-sensitive fluorophore (ecliptic- pHluorin) on the lumenal/extracellular side of the membrane and a second, spectrally distinct (i.e. non- interfering) fluorophore located in the cytosol (tdTomato; Figure 1A, B) .

[190] Given that both the amino and carboxy termini of GLUT4 are located on the cytosolic side, it became necessary to engineer a fluorophore into one of the loops exposed on the lumenal face. Since other epitopes have been successfully engineered into the first exofacial loop of GLUT4 without any apparent loss of function, a similar strategy was pursued here, inserting super ecliptic pHluorin between amino acids 55 and 66 of full length GLUT4.

[191] To complement the use of pHluorin, which emits at 507nm, tandem Tomato (tdTomato) , a red shifted derivative of the enhanced Green Fluorescent Protein

(eGFP) that emits at 568nm and is significantly brighter than other red fluorescent proteins was employed. tdTomato was attached to the C-terminal end of GLUT4, since N- terminal tagging of GLUT4 may result in an aberrant localisation of the protein.

[192] The dtTomato, if imaged by widefield epifluorescence microscopy (epiM) may advantageously provide a measure of the total construct expression levels and account for imaging artifacts such as bleaching and light source fluctuations. By TIRFM, fusion events may be easily detected with the pHluorin, whilst the tdTomato may allow visualizing and quantifying prefusion behaviour.

[193] When expressed 3T3-L1 adipocytes, rGLUTpHluor generated a protein product of ~140 kDa which was of the expected size (Figure 2A) . The protein was detected -by western blotting with antibodies against either GFP (recognizing epHluorin and tdTomato) or GLUT4 (Figure IB) . The pattern of migration of this band was similar to that observed for endogenous GLUT4, a phenomenon attributed to glycolysation.

[194] By microscopy, the tdTomato signal was detected in the perinuclear region and in peripheral puncta and tubules (Figure 2B; tdTomato) similar to endogenous GLUT4. A relatively weak pHluorin signal of similar distribution to the red signal (tdTomato) was also detected (Figure 2B; pHluorin) consistent with quenching of this label by the acidic lumenal environment. Incubation of cells with 50mM ammonium chloride to neutralize the intracellular pH resulted in a marked increase in the pHluorin signal

(Figure 2C) , which was highly colocalized with tdTomato

(Figure 2D) .

[195] To further confirm appropriate targeting of rGLUTpHluor, its localization to endogenous IRAP was compared, because the two proteins are highly colocalized in adipocytes. It was found that 72+3% of rGLUTpHluor positive internal structures also contained IRAP under basal conditions (Figure 2C, D) . Similar results were obtained in cells coexpressing the fusion proteins IRAP-pH and GLUT4mK0 (Figure 2 F) .

[196] One of the best indices of GLUT4 function is its high degree of insulin responsiveness. Using subcellular fractionation (Figure 2E) and microscopy (Figure 2F, G) , a robust insulin-dependent movement of rGLUTpHluor to the PM was observed analogous to that observed for both endogenous GLUT4 (Figure 2E) and GLUT4 -eGFP (Figure 2F, G) . In order to substantiate this observation, cells electroporated with either GLUT4 -eGFP or rGLUTpHluor were mixed and simultaneously imaged by TIRFM.

[197] Very little change in signal over time was detected for either construct under basal conditions. As noted above, only a weak signal was detected from pHluorin (Figure 1H) . Time-resolved fluorescence measurements show that the magnitude of the insulin-dependent increase of fluorescence over basal was the same for cells expressing either construct (eGFP vs. tdTomato), averaging -3.5 fold and the rate of change was indistinguishable (Figure II) .

[198] These results confirm that the novel two-colour construct localizes and responds to insulin in a manner comparable to endogenous GLUT4 and other fluorescently tagged GLUT4 fusion proteins when assessed by fractionation, intracellular localization and live cell imaging. As such this novel two-colour construct should not only be ideal for measuring GLUT4 responses in multiple cells but also for dissecting the multiple steps required for delivering GLUT4 to the PM and the molecules involved in these processes. [199] EXAMPLE 2

[200] Assessing GLUT4 exocytosis in Multiple Cells;

High-Throughput Ratio-metric Imaging of rGLUTpHluor.

[201] The use of rGLUTpHluor in a high throughput single cell assay for GLUT4 exocytosis was next sought. The signal from tdTomato when imaged by eplM is insensitive to changes in GLUT4 localisation and provides a direct measure of total construct levels per cells. This is in contrast to its behaviour when imaged by TIRFM where the signal intensity reflects the amount of GLUT4 on or near the PM. Conversely, the signal from pHluorin is sensitive to changes in GLUT4 localisation.

[202] When imaged by widefield epifluorescence microscopy (epiM) , the pHluorin signal is proportional to GLUT4 inserted into the PM, as it is dependent on the relative amount of GLUT4 residing in either intracellular compartments where pHluorin is exposed to the acidified environment within the lumen of vesicles or at the PM where pHluorin is exposed to the neutral pH of the extracellular medium.

[203] In contrast, the tdTomato signal is insensitive to changes in localization, and as such provides a measure of total expression levels and accounts for changes in cell size and imaging artefacts (Figures 3A-F) . Thus, the pHluorin: tdTomato ratio provides a reliable measure of plasma membrane (PM) localized GLUT4 relative to total (Figure 3G) . This method also has the advantage of controlling for artefacts such as intensity fluctuations and photobleaching. When imaging GLUT4 responses in this manner, additional cellular responses to insulin such as changes in cell shape can also be assessed (Figure 3G) . Such multivariate analyses are necessary to properly dissect complex heterogeneity and potentially reveal coupling between distinct cellular outputs.

[204] To further validate this approach, GLUT4 adipocytes expressing rGLUTpHluor were imaged under conditions where insulin action is compromised. When up- scaled to facilitate the interrogation of multiple single cells, the expected sensitivity was observed to known inhibitors of the insulin signalling pathway, such as the PI3K inhibitor Wortmannin (100 nM) and the Akt inhibitor MK-2206 (10 μΜ; Figure 3H) . In control cells, insulin caused a robust increase in the pHluorin : tdTomato ratio whereas pretreatment with Wortmannin (100 nM) and MK-2206 (10 μΜ) resulted in a marked attenuation of insulin- stimulated GLUT4 exocytosis, with the steady state level reduced by 99% and 85%, respectively (Figure 3H) .

[205] These results indicate that two-colour ratiometric imaging of rGLUTpHluor provides a real-time measurement of surface GLUT4 levels capable of detecting defects in insulin-stimulated GLUT4 exocytosis and thus provides an ideal platform for a high throughput assay

(HTA) . This is the first description of a live cell based high throughput assay that provides a direct measure of GLUT4 insertion into the PM.

[206] As noted above, insulin has a robust effect on cell size and shape (Figure 3G) due to its well-documented effects on the cytoskeleton. In our system, we observed a strong negative correlation between the cell area and tdTomato signal across multiple cells (Figure 4A; -0.99<r <-0.72) implying that changes in cell size have a substantial effect on the measured fluorescence signal. Thus, integrated intensity (signal x area) will give a more accurate measurement of fluorescent signals when imaging live cells by epiM. The ratiometric approach we have applied to rGLUTpHluor negates this requirement, thereby simplifying the analysis of complex cell mixtures. This also implies the tdTomato signal can be used to estimate changes in cell size. While surface GLUT4 levels and cell area respond in a positive manner to insulin stimulation they are only weakly correlated (Figure 4B; - 0.43<r <0.93). This suggests that these are indeed independent measures of insulin action. These data also demonstrate that the response of both parameters is highly heterogeneous at the single cell level, as clearly observed when the GLUT4 response of multiple cells are viewed simultaneously (Figure 4C) . [207] Further investigation of this heterogeneity- revealed that the population responded in a dose-dependent manner (Figure 5A) , whereas significant intercellular heterogeneity was observed in both the response magnitude (Figure 5B) and response half-time (Figure 5C) . Within this data set, multiple distinct response profiles were observed. For example, at the lowest dose (0.1 nM) , only 30% of cells had a detectable response. Further, some cells displayed a graded response to increasing input, whereas others displayed a bimodal response (Figure 5D) . One possible reason for this heterogeneity is that the GLUT4 response is stochastic, and that an individual cell will display a highly varied response when challenged with repeated stimulations with the same insulin dose. To investigate this, we analysed cellular responses to repeated insulinstimulations . This revealed that intracellular responsiveness was highly reproducible (Figure 5E-H) . These data imply that the cellular response to a specific dose of insulin is an intrinsic property of each cell. Based on these data the intercellular heterogeneity combined with the intracellular graded response both contribute to the shape of the population dose response curve . [208] EXAMPLE 3

[209] pH-based calibration of rGLUTpHluor

[210] More precise measurement of surface GLUT4 levels can be made by calibrating the system with a series of buffer exchanges, designed to modulate the pH of the extracellular and intracellular environments. This was achieved using a modification of the protocol described elsewhere, and involved a number of buffer exchanges to modulate the pH of the extracellular medium and/or the internal cellular compartments (Figure 6A,B). [211] This calibration was performed over multiple experiments and it was found that the response to insulin stimulation is highly reproducible across populations of cells (Figure 6C) . Under basal conditions, there was 8% of total rGLUTpHluor on the cell surface and this was increased to ~40% in the presence of a maximum insulin stimulus. These values are in close alignment with estimates made from surface labelling techniques known in the art .

[212] The above approach can also be used to measure the pH of the intracellular compartment in which the construct is located as described in Supporting Information. Using this approach we found the pH of the intracellular rGLUTpHluor compartment was 6.1±0.1. This is the first description of a simple, high throughput live cell assay that provides a direct measure of GLUT4 insertion into the PM.

[213] EXAMPLE 4

[214] Dissecting GLUT4 trafficking; TIRFM imaging of rGLUTpHluor

[215] It was next sought to determine whether this construct could be used to dissect individual steps (such as approach, attachment and fusion) in the GLUT4 trafficking pathway. Previous observations have indicated that the levels of GLUT4 -eGFP detected by TIRFM do not necessarily equate to the amount of GLUT4 that fuses with the PM. This is because GLUT4 -eGFP when imaged by TIRFM is unable to differentiate between GLUT4 in vesicles near the PM or in the PM itself; all molecules within the TIRF- zone, a region of about 200 nm adjacent to the coverslip, are detected.

[216] The inventors hypothesized that rGLUTpHluor in combination with total internal reflection fluorescence microscopy (TIRFM) , an application of fluorescence imaging that allows the selective imaging of only those fluorophores that are on or close to the PM, would facilitate a detailed dissection of GLUT4 trafficking (Figure 7A-D) . In this context, the tdTomato signal is sensitive to its cellular localization and is a measure of GLUT4 recruitment to the PM (Figure 7E) , while the pHluorin signal is a marker of fusion (Figure 7F) . The ability to distinguish between GSVs moving to the membrane from those undergoing fusion with the PM is a significant advantage over previous GLUT4 constructs. Interestingly, following insulin stimulation the half-time of the response was faster for tdTomato than pHluorin, likely reflecting the time required to engage the fusion machinery prior to insertion into the PM (Figure 7G, t 1/2 =5.494±0.37 min versus 5.929+0.41 min, p=0.048). As with epiM, there was significant intercellular heterogeneity in response to insulin (Figure 7E,F). However, within a cell, the response of each fluorophore was highly correlated, highlighting the intimate connection between transport, attachment and fusion (Figure 7H) .

[217] Inhibition of PI3K with Wortmannin almost completely inhibited the insulin-dependent increase in the pHluorin and tdTomato. signals (Figure 71, J) confirming PI3K as the primary node in the insulin signalling network. The Akt inhibitor MK-2206 had a more significant inhibitory effect on the pHluorin signal (65%; Figure 71) compared to tdTomato (50%; Figure 7J) , consistent with data suggesting that Akt has a more dominant role in regulating steps at the PM than in movement of GLUT4 vesicles into the TIRF zone. Disruption of the cortical actin cytoskeleton with Latrunculin B inhibited the pHluorin signal by 65% (Figure 71) while the tdTomato signal was inhibited by only 24% (Figure 7J) , consistent with a role for actin in docking/fusion but not in GSV trafficking to the PM .

[218] rGLUTpHluor was compared to other constructs such as GLUT4 -EGFP and surrogate markers of GLUT4 , such as IRAP-pH and VAMP2-pH that have been imaged by LF-TIRFM to quantify GLUT4 translocation. The kinetics of rGLUTpHluor translocation (tdTomato) was indistinguishable from GLUT4- eGFP (Figure 7K) . In contrast, the responses of IRAP-pH and VAMP2-pH were 2.3 and 3.2 times lower than rGLUTpHluor (pHluorin) , respectively. This suggests that rGLUTpHluor has trafficking kinetics that mimic GLUT4 -eGFP and that it is superior in performance to either IRAP-pH or VAMP2-pH. Moreover, the dual output provides information about both fusion and translocation, not revealed by imaging constructs with a single fluorescent tag.

[219] These data validate rGLUTpHluor combined with

LFTIRFM imaging as a powerful methodology for dissecting the precise role of molecules that participate in regulated GLUT4 trafficking.

[220] EXAMPLE 5

[221] Quantification of individual GSV trafficking events

[222] Further dissection of GLUT4 trafficking steps can be achieved by imaging single cells at high spatiotemporal resolution. A single adipocyte was imaged by high frequency TIRFM for 5 min prior to, and 20 min after stimulation with 100 nM insulin. Multiple fusion events, identified by their characteristic fusion profile, were evident (Figure 8A,B). ^TIRF Explorer' was used to quantify fusion events (Figure 8C) . Cumulative fusion (integrated fusion events) was well correlated with the total fluorescence increase, suggesting that the number of events detected was sufficient to account for the change in PM fluorescence (Figure 8D) .

Table 1: Comparison of constructs that have been used to analyse GLUT4 exocytosis by TIRFM

F_B basal F_BR insulin

Construct ( events /min/ym²) ( events/min/μιη²)

GLUT4-GFP 0. .001±0. .000 0. .004±0. .002

IRAP-pH 0. .008±0. .002 0 , .034+0. .005

VAMP2-pH 0. .042+0. .001 0. .210±0. .054 rGLUTpHluor 0. .009±0. .002 0. .041±0. .005

Summary of the key variables measured during HF-TIRFM imaging of adipocytes expressing rGLUTpHluor, GLUT4-eGFP, IRAP-pH and VAMP2-pH. Basal FER measurements were derived the average over a 5 min period. Insulin FER were calculated over the 2 minute period (4-6min post- insulin) . Data represents the mean + SEM of eight cells (IRAP-pH GLUT4-eGFP) , nine cells (rGLUTpHluor) and three cells (VAMP2-pH) .

[223] The kinetics of GSV fusion has been the subject of numerous studies. To validate the performance of rGLUTpHluor in this context, we imaged multiple cells expressing either rGLUTphluor, GLUT4 -eGFP , IRAP-pH or VAMP2-pH in the absence or presence of insulin and quantified the rate of fusion, referred to here as the fusion event rate (FER =fusion/unit time/cell area) . rGLUTpHluor had a basal rate FER of 0.009 events/min/ m2 , increasing 4.6 -fold in the presence of insulin to 0.0041 events/miη/μιη² (Table 1). Relatively few fusion events were detected in cells expressing GLUT4-eGFP. Indeed, in ~50% of the cells imaged no fusion events were detected at all. The FER for GLUT4 -eGFP was 10 -fold lower (0.001 events/min/pm²) than for rGLUTpHluor, increasing to 0.004 events/πιίη/μιη² in the presence of insulin (Table 1) . This is consistent with previous observations that fusion is difficult to detect using GLUT4 -eGFP . This led to the development of surrogate GLUT4 markers lumenally tagged with pHluorin, such as IRAP-pH.

[224] In the inventor' s hands, the FER of IRAP was similar to rGLUTpHLuor. Insulin induced a robust 4.3-fold increase from 0.008 to 0.034 events/min/ m². Conversely, the rate of fusion was approximately five -fold higher for VAMP2-pH (0.042 events /πιίη/μπι² ) increasing five-fold in the presence of insulin to a peak of 0.210 events /min/pm , suggesting that VAMP2 is not specific to GSVs, in contrast to previous studies. The behaviour of vesicles prior to fusion was next studied using the tdTomato output. By imaging 3T3-L1 adipocytes expressing rGLUTpHluor using two-colour HF-TIRFM, the inventors observed that 88±2% of fusion events were associated with a red structure, present prior to or at the time of fusion. This appeared unchanged across the time course of insulin stimulation. Multiple pre- fusion behaviours were observed including highly variable docking or dwell times, ranging from 0.5s to >2min. Examples of long (Figure 9 A,D) ; medium (Figure 9B,E) and short dwell times (Figure 9C,F) are shown. Other behaviours such as long-range movement and vesicle budding were also observed. In one example, a vesicle appeared and displayed highly restricted motion before undergoing a more mobile phase, moving ~17 μιη over the next 6 s with a peak velocity of 3 μπι/s (Figure 9G-I) , consistent with kinesin-directed movement along microtubules. At the site of fusion, vesicle movement was restricted and the red signal increased, consistent with attachment and/or docking with the PM (Figure 9H,I), before undergoing fusion (Figure 91) . Similar behaviours have been described in other exocytotic systems. [225] Thus, for the first time the inventors have been able to detect a large subset of fusion events from GLUT4 containing vesicles and retrospectively study the behaviour of each event prior to fusion. The use of HF- TIRFM coupled to an automated analysis system will allow detailed dissection of GSV behaviours at the PM, both at and prior to fusion, permitting interrogation of molecules that may be involved in these steps.

[226] Here we describe a novel construct and methodology for studying multiple facets of insulin action and GLUT4 exocytosis. The principles underpinning the design of both the construct and method are applicable to other cellular perturbations (e.g. disease states) and to numerous recycling proteins.

[227] The data described herein demonstrate that rGLUTpHluor behaves in a manner consistent with that reported for endogenous GLUT4 (Figure 1) . Significantly, being on the same molecule, the ratio between pHluorin (surface GLUT4) and tdTomato (total GLUT4) enables a rapid simple assessment of GLUT4 delivery to the PM using epifluorescence microscopy, a technique that should be readily available to all researchers, requiring little in the way of sophisticated expertise. This output can be translated into the actual percentage of total GLUT4 levels found at the PM by using pH-based calibration, which also facilitates the measurement of the pH of the internal GLUT4 compartment. Under basal conditions, there was 8% of total rGLUTpHluor on the cell surface and this was increased to ~40% in the presence of a maximum insulin stimulus, consistent with previous estimates made using immuno-electron microscopy, subcellular fractionation and surface labelling. This sensitive, high-throughput approach forms the basis of our workflow for assessing GLUT4 trafficking (Figure 10) . [228] Further phenotypic dissection can be achieved using low frequency two-colour TIRFM to analyse the movement of vesicles from inside the cell toward the PM. In this context, rGLUTpH1uor behaved identically to GLUT4- eGFP, while providing an additional layer of information using the pHluorin. rGLUTpHluor was significantly more sensitive than either IRAP-pH or VAMP2-pH and it displayed a dynamic response to insulin that was inhibited both by PI3 and Akt specific small molecule inhibitors ( ortmannin and MK2206) . rGLUTpHluor could also be used to distinguish different steps in the GLUT4 trafficking pathway, as indicated with the use of the actin depolymerising agent Latrunculin B (Figure 3) .

[229] High frequency two-colour TIRFM enables detailed analysis of individual vesicle docking and fusion events in single cells in a manner similar to that described for TDimer2-IRAP-pHluorin, with the added advantage that tdTomato is the brightest fluorescent protein described to date, facilitating high frequency imaging with ultra-low intensity radiation for over 40min without noticeable cytotoxicity.

[230] Throughout this study, we have compared rGLUTpHluor to other constructs that have been used to study GLUT4 trafficking in live cells. We found that despite similarity in the rates of fusion between IRAP-pH and rGLUTpHluor, there was a significantly lower fold increase of IRAP at the PM as measured by LF-TIRFm, possibility indicating differences in trafficking between these two proteins. Consistent with this, quantitative colocalization of these proteins in adipocytes indicates an overlap on the order of 75% (Figure 1) , implying 25% of these proteins are in discrete locations.

[231] VAMP2 has been suggested to be a selective marker of GSVs. VAMP2-pH (synapto-pHluorin) was developed to image vesicle fusion events in neurons and has since been used as a surrogate marker for GLUT4 in adipocytes . In contrast to the other constructs, we observed only a weak increase (1.6±0.4 fold) in mean fluorescence in response to insulin stimulation. Notably, the basal FER was five times that observed for rGLUTpHluor. The high fusion rate is consistent with VAMP2 being a general marker of exocytic vesicles as opposed to one that is specific for GSVs . In further support of this, we have recently demonstrated considerable redundancy in the requirement for SNARE isoforms in GLUT4 exocytosis and only ~20% of isolated GSVs were positive for VAMP2.

[232] As a combined workflow (Figure 10) , multiple parameters that depict important attributes of cell behaviour, including changes in cell size, movement of exocytic vesicles toward the membrane, vesicle docking and membrane fusion, can be investigated. This represents the first description of a live cell GLUT4 reporter that provides a powerful, quantitative, functional screen of the individual steps in the GLUT4 trafficking pathway.

[233] The high degree of heterogeneity in insulin action between individual adipocytes was surprising and potentially explains previous controversies in the field that have utilized single cell assays to interrogate hormone action. Such response heterogeneity in many cases has been considered to reflect the intrinsic stochastic or random nature of biological processes. However, we observed this not to be the case for insulin regulation of GLUT4 trafficking as the response of individual cells was highly reproducible (Figure 5E-H) . Response heterogeneity was manifest both in response to submaximal and maximal doses. This was not related to variance in GLUT4 expression levels (Figure SI) . It is also unlikely to be related to variance in insulin receptor levels as previous studies have shown that changes in insulin receptor number can change the sensitivity but not the maximum responsiveness of insulin action.

[234] Significant intercellular heterogeneity has been observed in the PI3K/Akt/PKBAkt signalling pathway inMCFlOA cells, which is of interest in light of the key role of this pathway as a major node in insulin regulated GLUT4 translocation. Functional heterogeneity has been observed during the differentiation of 3T3-L1 adipocytes with low PI3K activity correlating with low levels of lipid accumulation. One could argue that the heterogeneity that is observed reflects poor differentiation efficiency as this can vary between batches of cells. However, the inventors optimized differentiation efficiency and assembled bright field images of all cells that were used for detailed analysis. This indicates that all cells used were similarly differentiated based upon lipid droplet density (Figure S2) .

[235] Intriguingly, previous studies observed that relatively few adipocytes exhibited simultaneous expression of adipocyte differentiation markers (PPARy, CEBPa, adiponectin) and lipid droplets. In fact, subpopulations of cells were identified that exhibited a counterintuitive negative correlation between these markers of adipogenesis . One possibility is that this represents discrete subpopulations of adipocytes that possess intrinsic differences in insulin sensitivity and responsiveness.

[236] This observation has significant implications. First, many studies of GLUT4 trafficking use single cell assays to describe the molecular regulation of this process which may be difficult to interpret in lieu of the heterogeneity described here. Second, changes in adipose function are usually studied at the whole organ or population level and it is naturally assumed that any defect will be due to a similar disruption in all cells. An alternate interpretation is that changes in adipose function may rather depict changes in the relative abundance of subpopulations of adipocytes that comprise the organ. The high degree of heterogeneity in insulin action between individual adipocytes was surprising and potentially explains previous controversies in the field that have utilized single, cell assays to interrogate hormone action. Finally, it has recently been shown that there are at least three different kinds of adipocytes (white, beige and brown) that can be easily segregated on the basis of their lipid droplet morphology and thermogenic potential .

[237] It is conceivable that as is the- case for many other cell types such as hematopoietic cells that adipocytes may represent a complex array of cell types one of the distinguishing features of which could be their intrinsic insulin sensitivity. The inventors propose that this method will overcome these limitations providing investigators with a novel tool for studies of this kind. Our approach provides a much needed platform for high- throughput single cell analysis of insulin action that is required to understand the nature of the heterogeneity that underpins insulin responses across populations.

[238] EXPERIMENTAL PROCEDURES

[239] Reagents

[240] DMEM cell culture medium, antibiotics, newborn calf serum, fluorescent antibodies and Matrigel were from Invitrogen (Carlsbad, CA) . Foetal calf serum was from ThermoTrace (Melbourne, Australia). All chemicals were obtained from Sigma Chemical Co. (St Louis, MO) . Bovine serum albumin (BSA) was from Bovogen (Essendon, Australia) . Bicinchoninic acid reagent and SuperSignai West Pico chemiluminescent substrate were from Pierce .(Rockford, IL) . Protease inhibitor cocktail tablets were from Roche Applied Science (Indianapolis, IN) . The Akt inhibitor, M -2206, was generously provided by Professor Dario Alessi (University of Dundee, Dundee, UK) . Paraformaldehyde was from ProSciTech (Thuringowa, Australia) .

[241] Cloning

[242] To create rGLUTpHluor a silent Xmal site was engineered between the codons P65 and G66, in the first exofacial loop of GLUT4 -eGFP (a generous gift from Dr Xu Tao, Institute of Biophysics of the Chinese Academy of Sciences, Beijing, China) by site directed mutagenesis using the primers 5 ' -GTA GGC AAG GTC CCG GGG GAC CGG A-3' (SEQ ID NO: 9) and 5 ' -TCC GGT CCC CCG GGA CCT TGC CTA C-3' (SEQ ID NO: 10) into this site, we then inserted a pair of annealed oligos (5'-CGC GTA CCG GTG TCG ACG-3 ' (SEQ ID NO: 11) and 5' -CGC GCG TCG ACA CCG GTA- 3' (SEQ ID NO: 12)) to add Mlul and Sail sites. Complementary sites where engineered onto Super-ecliptic pHluorin (epHluorin) by PCR using 5'-C GCG TCG ACC GTA AGT AAA GGA GAA GAA CTT TTC-3 ' (SEQ ID NO: 13) and 5'-GC CAC GCG TAG TTC ATC CAT GCC ATG TG-3¹ (SEQ ID NO: 14). The epHluorin was then was subsequently subcloned into the Mlul and Sail sites of GLUT4 -eGFP . Finally, the eGFP was replaced with tdTomato.

[243] Preparation of Matrigel -coated coverslips

[244] Glass coverslips (#1.5) were incubated at room temperature for 120 min with a 1:50 dilution of Matrigel in ice cold PBS. Coverslips were subsequently washed twice with PBS prior to use.

[245] Cell Culture and Electroporation

[246] 3T3-L1 fibroblasts obtained from the Howard

Green Laboratory (Boston, MA) were differentiated into adipocytes as described. 7-9 days post-differentiation, adipocytes were trypsinised with 5x Trypsin/EDTA for 5-10 min at 31^°C, washed twice with PBS and resuspended in Electroporation Solution (20mM Hepes, 135mM KC1, 2mM MgCl₂, 0.5% Ficol 400, 1% DMSO, 2 mM ATP and 5 mM Glutathione, pH7.6) and 5-20 μg of plasmid DNA. Cells were electroporated at 200mV for 20ms using an ECM 830 Square Wave Electroporation System, (BTX Molecular Delivery Systems, Massachusetts, USA) and seeded onto matrigel coated coverslips. Adipocytes were maintained in DMEM supplemented with 10% FCS until required.

[247] Subcellular Fractionation

[248] 3T3 -LI adipocytes were washed with ice-cold PBS and harvested in ice-cold HES buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 250 mM sucrose) containing Complete protease inhibitor mixture and phosphatase inhibitors (2 mM sodium orthovanadate , 1 mM sodium pyrophosphate, 10 mM sodium fluoride) . The cells were lysed with 12 passes through a 22~gauge needle and 6 passes through a 27-gauge needle. Cell lysates were then centrifuged at 500 x g for 10 min at 4°C to remove unbroken cells.

[249] The supernatant was centrifuged at 10,080 x g for 20 min at 4°C to yield the following two fractions : the pellet fraction consisting of PM and mitochondria/nuclei, and the supernatant fraction consisting of cytosol, and internal membranes (IM) . The supernatant was again centrifuged at 175,000 * g for 75 min at 4°C to obtain the cytosol fraction (supernatant) and the IM fraction (pellet). To obtain the PM fraction, pellet from the first ultracentrifuge spin was resuspended in HES buffer containing phosphatase and protease inhibitors and layered over high sucrose HES buffer (20 mm HEPES, pH 7.4, 1 mm EDTA, 1.12 m sucrose) and centrifuged at 78,925 x g for 60 min at 4°C. The PM fraction was collected above the sucrose layer, and the pellet was the mitochondria/nuclei fraction. Alt the fractions were resuspended in HES buffer containing phosphatase and protease inhibitors. Protein concentration for each fraction was performed using BCA assay. Samples were made up in SDS sample buffer and then kept at -20 °C.

[250] Western Blotting Analysis

[251] Cells were washed twice with ice-cold PBS and solubiiized in 2% SDS in PBS containing phosphatase inhibitors (1 mM sodium pyrophosphate, 2 ttiM sodium vanadate, 10 mM sodium fluoride) and complete protease inhibitor mixture. Insoluble material was removed by centrifugation at 18,000 x g for 10 min. Protein concentration was measured using the bicinchoninic acid method. Proteins were separated by SDS-PAGE for immunoblot analysis. After transferring proteins to polyvinylidene difluoride membranes, membranes were incubated in blocking buffer containing 5% skim milk in Tris-buffered saline and immunoblotted with the relevant antibodies overnight at 4 °C in blocking buffer containing 5% BSA, 0.1% Tween in Tris-buffered saline. After incubation, membranes were washed and incubated with horseradish peroxidase-labeled secondary antibodies and then detected by SuperSignal West Pico chemiluminescent substrate.

[252] Confocal Imaging and Colocalisation analysis

[253] 3T3-L1 adipocytes were electroporated (as described above) and seeded onto Matrigel-coated glass coverslips. Two days post-electroporation, the cells were serum-starved for 2 h at 37 °C, then incubated in the absence or presence of 100 nM insulin for 20min. Cells were washed twice with PBS and then fixed with 3.7% paraformaldehyde in PBS. For immunofluoresence , cells were permeabilized with 0.1% (w/v) saponin in PBS, and blocked with EnhanceFX. Antibody staining was carried out in 2% (w/v) BSA in PBS. Coverslips were mounted on glass slides with ImmunO™ (MP Biomedicals, LLC) .

[254] Suitably electroporated adipocytes were imaged using a Leica laser scanning confocal microscope (TCS SP2 AOBS with DM IRE2 ; Leica Microsystems), with a lOOx 1.4 oil immersion objective. Z- stacks were acquired at the optimal sampling density as defined by the Nyquist frequency. Prior to analysis, images of adipocytes were deconvolved using Huygens Essential Software (Scientific Volume Imaging, Hilversum, and The Netherlands) .

[255] Colocalisation was assessed in using a 3D structure based approach. Briefly, the vesicles and/or structures were identified in 3D space for each channel of interest using the "identify spot" function in IMARIS x64 V7.22 (Bitplane, Zurich, Switzerland). This process identifies the centre of mass in 3D space for each identified object. The closest spot in the alternate channel was identified and the distance between the two spots was measured. Vesicles were only considered colocalised if their centres were less than 50 nm apart in the xy plane and 150nM in the z plane.

[256] Live cell Microscopy

[257] Coverslips were mounted in a perfusion open/closed chamber (POC) containing modified KRP buffer (120 mM NaCl, 0.6 mM Na₂HP04, 0.4 mM NaH₂P0₄, 6 mM KC1, 1.2 mM MgS0₄, 12.5 mM HEPES, 1 mM CaCl₂, lOmM Glucose, lx MEM Amino Acids Solution, 20 mM GlutaMAX, 0.2% (w/v) BSA, pH7.4) and placed in a heated stage microscope insert 'P'

(Pecon) on an Axiovert 200M (Zeiss) equipped with a large incubator (XL, Pecon) maintained at 37 °C.

[258] Wide field epifluorescence imaging of rGLUTpHluor in live cells

[259] Healthy and suitably transfected cells were identified by brightfield and fluorescence using an appropriate objective (typically a Zeiss A-Plan 20x/0.45; Figure 10) . TdTomato and pHluorin were simultaneously excited using a 488/485nm bandpass filter. Emitted fluorescence was filtered by a 500nm LP filter and then split (568nm dichroic with 525/525nm and 607/670nm bandpass filters) onto two halves of an iXon DU-888D EMCCD camera (Andor) using a custom configured optosplit II (Cairn Research) . In this configuration, bleed through from green: red was measured at <3% and as such was considered negligible. All images were acquired using Manager.

[260] Calibration

[261] Calibration of rGLUTpHluor was achieved using a modification of the protocol described elsewhere. This involves a number of buffer exchanges and as such is best achieved using perfusion (Figure 6a) . Prior to experimental manipulation, the pH is changed to pH 5.5 in a MES buffered KRP (120mM NaCl, 0.6mM Na2HP04 , 0.4mM NaH2P04, 6mM KCl, 1.2mM MgS04 , 12.5mM MES, ImM CaC12, lOmM glucose, 0.2% (w/v) BSA, pH 5.5). This quenches the surface-derived pHluorin fluorescence, leaving the signal derived from the internal fluorescence (a) . This can be repeated any number of times during the experiment if required (a' ) . At the end of the experiment the pH is increased to 9.0 in modified KRP (70mM NaCl, 50mM NH4C1, 0.6mM Na2HP04, 0.4mM NaH2P04, 6mM KCl, 1.2mM MgS04 , 12.5mM Bicine, 0.2% (w/v) BSA, pH 9.0). This gives the total fluorescence of the pHluorin as the NH4C1 will effectively equilibrate the pH across all cellular compartments. The pH is then dropped to 5.0 by perfusion of modified KRP (70mM NaCl, 50mM NH4C1, 0.6mM Na2HP04 , 0.4mM NaH2P04, 6mM KCl, 1.2mM MgS04, 12.5mM MES, 0.2% (w/v) BSA, pH 5.0). This quenches all fluorescence of the pHluorin. [262] A transient change to pH 5.5 quenches the surface pHluorin signal (it was found that lower pH buffers significantly decreased cytosolic and endosomal pH) . The remaining signal (Int) is a combination of fluorescence from the pHluorin in internal compartments, autofluorescence and background fluorescence. At the termination of the experiment, the pH was increased to pH 9.0 and then dropped to 5.0 in the presence of 50mM NH4C1. This modulates the pH of both the extracellular and intracellular compartments and thus provides a measure of the maximum (Max) and minimum (Min) fluorescence of the pHluorin. The amount of protein (P) on the surface at time n (relative to the total), can be derived by equation (lj

Psurface =-— x 100 (1)

' (Max-Min)xF7A '

[263] where F₇.₄ is the fraction of pHluorin molecules fluorescing at pH 7.4 (the pH of the external media) . This can be determined using the Henderson-Hasselbalch equation in the form F(pH) = : (2)

^ ^J l-10 pKapHluorin-p ) '

[264] An example of a trace from a single 3T3-L1 adipocyte expressing rGLUTpHluor during such an experiment is shown in Figure 6B .

[265] Assessing the pH of the intracellular compartment

[266] The pH of the internal compartment can be calculated at any time that a pH 5.5 buffer exchange is performed. From equation (1) we can calculate the fraction of internal protein (P_Int) as

■Pint - 1 -^Surface [267] The maximum possible fluorescence of this GLUT4 subpool (Max_P_Int) is then given by

Max_P_Int = P_Int x Max

[268] So the fraction of the fluorescence of the GLUT4 subpool is

Int— Bkg

FP int =

Max GAlnt [269] The pH Of the internal GLUT4 compartment can then be derived by reorganizing equation (2) to give

Mt0"0— (log 10(^ -1) -pJt«)

[270] High speed single cell imaging

[271] Healthy and suitably transfected cells were identified by brightfield and fluorescence using a lOOx objective (NA 1.45 alpha-Plan-Fluar, Carl Zeiss) and total internal reflection fluorescence microscopy (TIRFM) was performed using a 488nm laser introduced into the excitation light path (488/485 nm) through the TIRF-slider (Carl Zeiss) and appropriately angled to image ~100-200nm into cells as previously described.

[272] Fluorescence (525/525 nm) was detected using an iXon DU-888D EMCCD camera (Andor) and images acquired at ~10 Hz. The open-source software package μΜΑ ΑΘΕΚ (University of California) was used for all microscope control and image acquisition.

[273] Image analysis

[274] Image analysis was performed using ImageJ (NIH,

Bethesda, USA) , fvlatlab (Mathworks, Massachusetts, USA) , Cell Profiler 2.0 (Broad Institute, Massachusetts, USA and Imaris (Bitplane, Zurich, Switzerland) .

[275] Statistical Analyses

[276] Statistical analyses were performed with the use of statistical software package GraphPad Prism 4.03 or STATA. Data is presented as the mean + SEM unless otherwise stated. Comparisons between groups were performed using appropriate tests as noted in the figure legends. Half-life (T_1/2) and plateaus for time course data were calculated by fit of asymmetric five parameter curves .

Claims

Claims

1. A modified protein comprising a) GLUT4 or a protein having at least 95% sequence identity to GLUT4 , b) at least one cytosolic fluorophore comprised in the GLUT4 sequence defined under a) and c) at least one lumenal pH- sensitive fluorophore comprised in the GLUT4 sequence defined under a) .

2. The modified protein according to claim 1, wherein the at least one cytosolic fluorophore and the at least one lumenal pH-sensitive fluorophore are non-interfering.

3. The modified protein according to claim 1 or 2, wherein the at least one cytosolic fluorophore is selected from the group consisting of a red fluorescent protein comprising tdTomato, mRaspberry, mCherry, mStrawberry, mTangerine, dsRed, TagRFP, TagRFP-T, mApple, mRuby, and mRuby2; a UV or blue fluorescent protein comprising TagBFP, mTagBFP2 , Azurite, EBFP2 , mKalamal, Sirius, Sapphire and T-Sapphire; a cyan fluorescent protein comprising ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, monomeric Midoriishi-Cyan, TagCFP and mTFPl; a green fluorescent protein comprising EGFP, Emerald, Superfolder GFP, monomeric Azami Green, TagGFP2 , mUKG, mWasabi and Clover; a yellow fluorescent protein comprising EYFP, Citrine, Venus, SYFP2 , TagYFP; an orange fluorescent protein comprising Monomeric Kusbira Orange, τηΚΟκ, mK02, mOrange, and mOrange2 and; a far-red fluorescent protein comprising mPlum, HcRed-Tandem, mKate2 , mNeptune and NirFP .

4. The modified protein according to any one of claims 1 to 3, wherein the at least one cytosolic fluorophore is a red fluorescent protein.

5. The modified protein according to any one of claims 1 -to 4, wherein the at least one cytosolic fluorophore is tdTomato .

6. The modified protein according to any one of claims 1 to 5, wherein the at least one cytosolic fluorophore is at the carboxy-terminus of GLUT .

7. The modified protein according to any one of claims 1 to 5, wherein the at least one lumenal pH-sensitive fluorophore is selected from the group consisting of super-ecliptic pHluorin, pHluorin and mNectarine .

8. The modified protein according to any one of claims 1 to 7, wherein the lumenal pH-sensitive fluorophore is on an exofacial loop of the modified GLUT4.

9. The modified protein according to any one of claims 1 to 8, wherein the lumenal pH-sensitive fluorophore is on the first exofacial loop of the modified GLUT4.

10. The modified protein according to any one of claims 1 to 9, wherein the cytosolic fluorophore is tdTomato and the lumenal pH-sensitive fluorophore is super ecliptic pHluorin.

11. The modified protein according to any one of claims 1 to 9, wherein the protein comprises the amino acid sequence of SEQ ID No.2 [GLUT4] , the amino acid sequence of SEQ ID No.4 [tdTomato] and the amino acid sequence of SEQ ID No.6 [super ecliptic pHluorin] .

12. The modified protein according to any one of claims 1 to 9, the protein being any one of proteins (a) through

(c) , below:

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 8;

(b) a protein comprising the amino acid sequence shown in SEQ ID NO: 4 in which 1 to 6 amino acid residues are replaced, deleted, inserted and/or added, the protein having the GLUT4 activity;

(c) a protein comprising an amino acid sequence having 86.0% or greater of homology with the amino acid sequence shown in SEQ ID N0:4, the protein having the GLUT4 activity.

13. An isolated nucleic acid molecule encoding the modified protein according to any of the preceding claims.

14. A recombinant vector comprising a nucleic acid molecule according to claim 15.

15. A host cell, wherein the host cell is capable of expressing the modified protein of any one of claims 1 to 13 when comprising the vector of any one of claims 16 to 18.

16. A method for screening a compound that affects exocytosis of a protein of interest in a plurality of individual mammalian cells, comprising the use of a modified protein of interest, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH-sensitive fluorophore and at least one cytosolic fluorophore.^"

17. A method of assessing whether a condition or a stimulus affect exocytosis of a protein of interest in a plurality of individual mammalian cells, comprising:

(a) culturing mammalian cells expressing a modified protein of interest under a condition or stimulus to be assessed for its effect on exocytosis of said protein, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH-sensitive fluorophore and at least one cytosolic fluorophore, wherein the cells are referred to as test cells;

(b) detecting a signal from the cytosolic fluorophore, which is indicative of total modified protein of interest in the cells and a signal from the luminal pH-sensitive fluorophore, which is indicative of fusion events from an intracellular compartment to an extracellular environment of the test cells;

(c) determining a ratio of the signal from the cytosolic fluorophore and the signal from the pH-sensitive fluorophore of the modified protein of interest in the test cells of (b) , thereby producing a test value, which is indicative of the plasma membrane localisation of the modified protein of interest normalised to said protein total expression level;

(d) comparing the test value with a control value, wherein the control value corresponds to a ratio of modified protein of interest at the surface of the cell in control cells to total modified protein of interest in control cells, and the control value is determined from control cells which are the same cell as cultured in (a), and which are cultured under the same conditions as in (a) , expect that the control cells are not cultured under the condition or stimulus to be assessed;

wherein if the test value is greater than the control value, then the condition or stimulus affects exocytosis of the modified protein of interest in the cells.

18. A method of assessing the steps affected in the trafficking pathway of a modified protein of interest by a condition or a stimulus in a plurality of individual mammalian cells, comprising:

(a) culturing mammalian cells expressing a modified protein of interest under a condition or stimulus that activate translocation of said protein from an intracellular location to the plasma membrane of the cells, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH- sensitive fluorophore and at least one cytosolic fluorophore thereof, wherein the cells are referred to as control cells;

(b) detecting a first signal from the cytosolic fluorophore, which is indicative of the total amount of modified protein of interest in the cells and a second signal from the lumenal pH-sensitive fluorophore, which is indicative of the presence of the modified protein of interest in the plasma membrane of the control cells, thereby producing a first control value from the cytosolic fluorophore and a second control value from the pH-sensitive fluorophore;

(c) providing the control cells with the condition or stimulus that affect the trafficking of the modified protein of interest, thereby producing test cells;

(d) detecting a third signal from the cytosolic fluorophore, which is indicative of the total amount of modified protein of interest in the cells and a fourth signal from the pH-sensitive fluorophore, which is indicative of the presence of the modified protein of interest in the plasma membrane of the test cells, thereby producing a first test value from the cytosolic fluorophore and a second test value from the pH-sensitive fluorophore;

(e) determining a first proportion of the first test value and the first control value and a second proportion of the second test value and the second control value;

(f) comparing the first proportion with the second proportion;

wherein if the first proportion is less than 1, the intracellular movement of the modified protein of interest is inhibited, and if the second proportion is less than 1, the fusion of the modified protein of interest is inhibited and wherein if the first proportion is greater than the second proportion, then the condition or stimulus mainly effects the fusion of the modified protein of interest to the plasma membrane in the cells.

19. A method of assessing a fusion event of a modified protein of interest affected by a condition or a stimulus in at least one mammalian cell, comprising:

(a) culturing mammalian cells expressing a modified protein of interest, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH-sensitive fluorophore and at least one cytosolic fluorophore thereof;

(b) detecting a signal from the lumenal pH-sensitive fluorophore, which is indicative of the presence of the modified protein of interest in the plasma membrane of the at least one mammalian cell, thereby localizing the site of fusion from the presence of the pH-sensitive fluorophore;

(c) capturing multiple images of the at least one cell over a period of time prior to providing the cell with the condition or stimulus that affects fusion, the multiple images comprising the signal from thereby- producing a basal imaging period;

(d) providing the at least one cell with the condition or stimulus that affect the fusion event;

(e) capturing multiple images of the at least one cell over a period of time after providing the cell with the condition or stimulus as in (d) ;

(f) analysing the images obtained in (c) and (e) , thereby quantifying the total number of fusion events and generating a fusion event rate ( F_ER) ;

(g) comparing the F_ER during the basal imaging period and after providing the at. least one cell with the condition or stimulus;

wherein if the F_ER is increased after providing the at least one cell with the condition or stimulus, then the condition or stimulus activate the fusion of the modified protein of interest to the plasma membrane in the cells.

20. A method of assessing a pre-fusion event of a modified protein of interest affected by a condition or a stimulus in at least one mammalian cell, comprising:

(a) culturing mammalian cells expressing a modified protein of interest under a condition or stimulus to be assessed for its effect on exocytosis of said protein, wherein the modified protein of interest comprises the protein of interest and at least one lumenal pH- sensitive fluorophore and at least one cytosolic fluorophore;

(b) detecting a signal from the cytosolic fluorophore, which is indicative of total modified protein of interest in the cells and a signal from the luminal pH-sensitive fluorophore, which is indicative of fusion events from an intracellular compartment to an extracellular environment of the cells;

(c) capturing multiple images of the at least one cell over a period of time prior and after providing the cell with the condition or stimulus that affects fusion, wherein the images comprising a first signal from the cytosolic fluorophore, and a second signal from the lumenal pH-sensitive fluorophore;

(d) determining the time of presence of the first signal or absence thereof of the first signal at the site of fusion, thereby providing a dwell-time;

wherein the dwell-time is representative of whether the condition or stimulus affect the pre-fusion events of the modified protein of interest in the at least one cell.

21. The method of any one of claims 16 to 20, wherein the stimulus is an agent suspected to be capable of affecting exocytosis.

22. The method according to any one of claims 16 to 21, wherein the protein of interest is GLUT4.

23. A method of assessing whether a condition or a stimulus affect exocytosis of GLUT 4 in a plurality of individual mammalian cells, comprising:

(a) Culturing mammalian cells expressing the modified protein according to claim 1 under a condition or stimulus to be assessed for its effect on exocytosis of GLUT , wherein the cells are referred to as test cells;

(b) detecting a signal from the cytosolic fluorophore, which is indicative of total modified GLUT4 in the cells and a signal from and a signal from the lumenal pH- sensitive fluorophore, which is indicative of fusion events from an intracellular compartment to an extracellular environment of the test cells;

(c) determining a ratio of the signal from the cytosolic fluorophore and the signal from the lumenal pH-sensitive fluorophore of the modified protein in the test cells of (b) , thereby producing a test value;

(d) comparing the test value with a control value, wherein the control value corresponds to a ratio of the modified protein at the surface of the cell in control cells to the total modified protein in control cells, and the control value is determined from control cells which are the same cells as cultured in (a) , and which are cultured under the same conditions as in (a) , expect that the control cells are not cultured under the condition or stimulus to be assessed;

wherein if the test value is greater than the control value, then the condition or stimulus causes exocytosis of the modified GLUT4 in the cells.

24. A method of assessing the steps affected in the trafficking pathway of the modified protein of claim 1 by a condition or a stimulus in a plurality of individual mammalian cells, comprising:

(a) culturing mammalian cells expressing the modified protein under a condition or stimulus that activate translocation of GLUT4 from an intracellular location to the plasma membrane of the cells, wherein the cells are referred to as control cells;

(b) detecting a first signal from the cytosolic fluorophore, which is indicative of the total amount of modified protein in the cells and a second signal from the lumenal pH-sensitive fluorophore in the lumenal, which is indicative of the presence of the modified protein in the plasma membrane of the control cells, thereby producing a first control value from the cytosolic fluorophore and a second control value from the lumenal pH- sensitive fluorophore ;

(c) providing the control cells with the condition or stimulus that affect the trafficking of the modified protein, thereby producing test cells;

(d) detecting a third signal from the cytosolic fluorophore, which is indicative of the total amount of modified protein in the cells and a fourth signal from the lumenal pH-sensitive fluorophore in the lumenal, which is indicative of the presence of the modified protein in the plasma membrane of the test cells, thereby producing a first test value from the cytosolic fluorophore and a second test value from the lumenal pH- sensitive fluorophore ;

(e) determining a first proportion of the first test value and the first control value and a second proportion of the second test value and the second control value; (f) comparing the first proportion with the second proportion,

wherein if the first proportion is less than 1, the intracellular movement of the modified protein is inhibited, and if the second proportion is less than 1, the fusion of the modified protein of interest is inhibited and wherein if the first proportion is greater than the second proportion, then the condition or stimulus mainly effects the fusion of the modified protein of interest to the plasma membrane in the cells .

25. The method of any one of claims 16 to 24, wherein the cells are cultured in the presence of insulin, thereby activating - exocytosis .

26. The method of any one of claims 16 to 25, wherein the modified protein is as described in claim 1.