Direct Quantification of Fusion Rate Reveals a Distal Role for AS160 in Insulin-stimulated Fusion of GLUT4 Storage Vesicles
2007; Elsevier BV; Volume: 283; Issue: 13 Linguagem: Inglês
10.1074/jbc.m708688200
ISSN1083-351X
AutoresLi Jiang, Junmei Fan, Li Bai, Yan Wang, Yu Chen, Yang Lü, Liangyi Chen, Tao Xu,
Tópico(s)Diabetes and associated disorders
ResumoInsulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress toward elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway. Here, we have developed a novel dual colored probe to monitor the translocation process of GLUT4 based on dual color fluorescence measurement. We demonstrate that this probe is more than an order of magnitude more sensitive than the current technology for detecting fusion events from single GLUT4 storage vesicles (GSVs). A small fraction of fusion events were found to be of the "kiss-and-run" type. For the first time, we show that insulin stimulation evokes a ∼40-fold increase in the fusion of GSVs in 3T3-L1 adipocytes, compared with basal conditions. The probe can also be used to monitor the prefusion behavior of GSVs. By quantifying both the docking and fusion rates simultaneously, we demonstrate a proportional inhibition in both docking and fusion of GSVs by a dominant negative mutant of AS160, indicating a role for AS160 in the docking of GSVs but not in the regulation of GSV fusion after docking. Insulin-stimulated GLUT4 translocation to the plasma membrane constitutes a key process for blood glucose control. However, convenient and robust assays to monitor this dynamic process in real time are lacking, which hinders current progress toward elucidation of the underlying molecular events as well as screens for drugs targeting this particular pathway. Here, we have developed a novel dual colored probe to monitor the translocation process of GLUT4 based on dual color fluorescence measurement. We demonstrate that this probe is more than an order of magnitude more sensitive than the current technology for detecting fusion events from single GLUT4 storage vesicles (GSVs). A small fraction of fusion events were found to be of the "kiss-and-run" type. For the first time, we show that insulin stimulation evokes a ∼40-fold increase in the fusion of GSVs in 3T3-L1 adipocytes, compared with basal conditions. The probe can also be used to monitor the prefusion behavior of GSVs. By quantifying both the docking and fusion rates simultaneously, we demonstrate a proportional inhibition in both docking and fusion of GSVs by a dominant negative mutant of AS160, indicating a role for AS160 in the docking of GSVs but not in the regulation of GSV fusion after docking. Type II diabetes mellitus is a devastating metabolic disease characterized by insulin resistance and aberrant glucose metabolism. One of the major steps regulated by insulin is the removal of glucose from the blood stream into muscle and fat cells. This is mediated by redistribution of the insulin-responsive glucose transporter GLUT4 (1Birnbaum M.J. Cell. 1989; 57: 305-315Abstract Full Text PDF PubMed Scopus (469) Google Scholar, 2James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (472) Google Scholar) from intracellular GLUT4 storage vesicles (GSVs) 3The abbreviations used are:GSVGLUT4 storage vesiclePMplasma membraneTIRFMtotal internal reflection fluorescence microscopyGFPgreen fluorescent proteinEGFPenhanced GFPRITSratiometric-based IRAP translocation sensor. to the plasma membrane (PM) (3Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell Biol. 2002; 3: 267-277Crossref PubMed Scopus (940) Google Scholar). Reduced insulin-stimulated glucose transport has been proposed as one of the earliest metabolic abnormalities observed during the natural course of type 2 diabetes (4Henry R.R. Abrams L. Nikoulina S. Ciaraldi T.P. Diabetes. 1995; 44: 936-946Crossref PubMed Scopus (0) Google Scholar, 5Garvey W.T. Maianu L. Zhu J.H. Brechtel-Hook G. Wallace P. Baron A.D. J. Clin. Investig. 1998; 101: 2377-2386Crossref PubMed Scopus (348) Google Scholar). Despite extensive efforts, the mechanism by which insulin signaling stimulates the translocation of GLUT4 remains elusive. This is not only due to the complexity of both the insulin signaling and GLUT4 trafficking pathways but also to the lack of robust, quantitative, and easy-to-use assays to monitor the in vivo GLUT4 translocation process in real time. GLUT4 storage vesicle plasma membrane total internal reflection fluorescence microscopy green fluorescent protein enhanced GFP ratiometric-based IRAP translocation sensor. Conventional methods used to study GLUT4 distribution include using membrane fractionation and immunoblotting to quantify GLUT4 content in different fractions (6Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar). Alternatively, by inserting an epitope (e.g. hemagglutinin tag) into the extracellular domain of GLUT4, one can visualize the membrane distribution of GLUT4 by anti-hemagglutinin antibody staining employing immunofluorescence microscopy (7Czech M.P. Chawla A. Woon C.W. Buxton J. Armoni M. Tang W. Joly M. Corvera S. J. Cell Biol. 1993; 123: 127-135Crossref PubMed Scopus (70) Google Scholar). Although samples can be prepared at different time points after insulin stimulation, allowing for some time resolution, these methods are generally tedious to perform, hard to quantify/compare, and not in real time. Recently, total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate GFP-labeled GLUT4 translocation (8Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (146) Google Scholar, 9Tengholm A. Meyer T. Curr. Biol. 2002; 12: 1871-1876Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 10Zeigerer A. McBrayer M.K. McGraw T.E. Mol. Biol. Cell. 2004; 15: 4406-4415Crossref PubMed Scopus (189) Google Scholar). The evanescent field generated from a TIRFM selectively illuminates GLUT4-EGFP within a few hundreds of nanometers beneath the PM (11Lang T. Wacker I. Steyer J. Kaether C. Wunderlich I. Soldati T. Gerdes H.H. Almers W. Neuron. 1997; 18: 857-863Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and thus images those GLUT4-EGFP molecules in the PM or in vesicles very close to the PM. The translocation of GLUT4-EGFP into the PM will result in an increase in the total fluorescence under TIRFM. However, it is not clear whether the fluorescence increase in the total internal reflection fluorescence zone is due to an increase in the insertion of GLUT4 in the PM or to more docked/recruited vesicles close to the PM. To solve this problem, time-resolved TIRFM has been employed to track and analyze the dynamics of single GSVs (8Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (146) Google Scholar, 12Li C.H. Bai L. Li D.D. Xia S. Xu T. Cell Res. 2004; 14: 480-486Crossref PubMed Scopus (50) Google Scholar). It has been demonstrated that fusion of GSVs can be monitored by scrutinizing the radial diffusion pattern of fluorescence. Additionally, the docking/tethering of GSVs can be inferred by analyzing the mobility of vesicles (8Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (146) Google Scholar, 13Bai L. Wang Y. Fan J. Chen Y. Ji W. Qu A. Xu P. James D.E. Xu T. Cell Metab. 2007; 5: 47-57Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). However, these methods are not straightforward and require extensive training in TIRFM imaging and image analysis. In conclusion, what is needed is a robust, easy-to-apply real time method that allows the dynamics of GLUT4 translocation to be visualized in their natural context. AS160 has recently been identified as a substrate of Akt that functions in GLUT4 trafficking (14Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar). AS160 possesses a Rab GTPase-activating protein domain, so it may regulate the activity of a Rab protein that is involved in GLUT4 trafficking. AS160 is phosphorylated at four separate sites by Akt. It has previously been shown that overexpression of an AS160 mutant (AS160-4P) in which each of these phosphorylation sites has been mutated inhibits insulin-stimulated GLUT4 translocation in adipocytes (14Sano H. Kane S. Sano E. Miinea C.P. Asara J.M. Lane W.S. Garner C.W. Lienhard G.E. J. Biol. Chem. 2003; 278: 14599-14602Abstract Full Text Full Text PDF PubMed Scopus (732) Google Scholar). However, the exact site of action of AS160 along the GLUT4 trafficking pathway remains to be identified. Previously, we showed that overexpression of AS160-4P blocked the docking of GSVs to the plasma membrane (13Bai L. Wang Y. Fan J. Chen Y. Ji W. Qu A. Xu P. James D.E. Xu T. Cell Metab. 2007; 5: 47-57Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). However, it is not clear whether insulin-induced phosphorylation of AS160 participates in the insulin-regulated later steps after docking. Without a reliable fusion assay for GSVs, we were not able to address this question at that time. In the current study, we developed a probe by attaching the pH-sensitive fluorescence protein pHluorin (15Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1999) Google Scholar) to the luminal terminus of IRAP and the red fluorescence protein Tdimer2 (16Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (2015) Google Scholar) to the cytosolic end of IRAP. The resultant dual colored probe (TDimer2-IRAP-pHluorin) co-localizes with GLUT4-EGFP and allows for easy identification of the fusion of single GSVs, as well as their prefusion history. By quantifying both the docking and fusion rates simultaneously, we demonstrate a proportional inhibition in both the docking and fusion of GSVs by AS160-4P, indicating a role for AS160 in the docking of GSVs but not in the control of GSV fusion after docking. Moreover, we demonstrate that this probe can be used to monitor GLUT4 translocation in real time from live cells simply by ratiometric fluorescence measurement. DNA Construction—To generate the pHluorin-N1 vector, the pH-sensitive fluorescence protein pHluorin (kindly provided by Dr. James Rothman) was used to substitute for EGFP in pEGFP-N1 (Clontech Laboratories, Palo Alto, CA). The pHluorin primers used were: forward, 5′-GATCGGATCCCACCATGAGTAAAGGAGAAGAAC-3′, and reverse, 5′-GATCGCGGCCGCTTATTTGTATAGTTCATCCATG-3′. Plasmid GLUT4-EGFP was constructed as previously described (12Li C.H. Bai L. Li D.D. Xia S. Xu T. Cell Res. 2004; 14: 480-486Crossref PubMed Scopus (50) Google Scholar). Total 3T3-L1 RNA was extracted with the RNeasy mini kit (Qiagen) following the manufacturer's instructions, and RNA integrity was identified by formaldehyde electrophoresis. The IRAP cDNA was generated using reverse transcriptase. The complete coding sequence of IRAP was obtained by high fidelity PCR amplification with Pfu DNA polymerase (Stratagene, La Jolla, CA). The specific forward and reverse primers used in the experiment were 5′-GATCCTCGAGCATGGAGTCCTTTACCAATGATCGGCTTCAG-3′ (forward) and 5′-GCAAGGATCCTTCAGCCACTGGGAGAGCGTTTTCAGATTC-3′ (reverse). The N-terminal 393-bp fragment of IRAP was then amplified with Pfu DNA polymerase (Stratagen, La Jolla, CA). The primers used were: forward, 5′-GATCCTCGAGCCACCACCATGGAGTCCTTTACCAATGATCGG-3′, and reverse, 5′-GCAAGGATCCGGCAGTAGATAAATCACCATGATTACAGAGACC-3′. For construction of the IRAP-pHluorin fusion protein, the N-terminal 393-bp PCR fragment of IRAP was digested with XhoI and BamHI restriction enzymes and ligated into the subclone vector pHluorin-N1. To generate the IRAP-Tdimer2 fusion protein, the coding sequence for the red fluorescence protein Tdimer2 was digested from the subclone vector pcDNA3.1-TDimer2 (16Campbell R.E. Tour O. Palmer A.E. Steinbach P.A. Baird G.S. Zacharias D.A. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7877-7882Crossref PubMed Scopus (2015) Google Scholar, 17Yang X. Xu P. Xu T. Biochem. Biophys. Res. Commun. 2005; 330: 914-920Crossref PubMed Scopus (19) Google Scholar) and used to substitute for pHluorin in the N1-IRAP-pHluorin vector. For construction of Tdimer2-IRAP-pHluorin, the Tdimer2 coding sequence was amplified and cloned into N1-IRAP-pHluorin. The primers used were as follows: forward, 5′-GAAGCTAGCGACCATGGTGGCCTCCTCCGAGGACG-3′, and reverse, 5′-GCTGGATATCTGCAAGATCTCAGGAACAGGTGGTGG-3′. Construct integrity was verified using DNA sequencing analysis provided by Invitrogen. Cell Culture and Transfection—3T3-L1 cells were cultured in high glucose Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% newborn calf serum (Invitrogen) at 37 °C and 5% CO2. One day after confluence, the cells were switched into differentiation medium containing 10% fetal bovine serum (Invitrogen), 1 μm bovine insulin, 0.5 mm 3-isobutyl-1-methylxanthine, and 0.25 m dexamethasone. Two days later, the medium was replaced with 10% fetal bovine serum and 1 μm insulin for another 2 days. The cells were then maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Seven days after differentiation, 3T3-L1 adipocytes were treated with 0.05% trypsin-EDTA (Invitrogen) and washed twice with OPTI-MEM (Invitrogen) by centrifugation at 1500 rpm at room temperature. The cells were resuspended in OPTI-MEM and 30 μg of IRAP-pHluorin or TDimer2-IRAP-pHluorin, or 30 μg of TDimer2-IRAP-pHluorin plus 60 μg AS160-4P plasmid were added to a final volume of 800 μl. Electroporation was performed at 360 V for 10 ms using a BTX 830 electroporator (Biocompare, South San Francisco, CA), and the cells were plated on coverslips coated with poly-l-lysine. The experiments were performed 2 days after transfection in KRBB solution containing 129 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 5 mm NaHCO3, 10 mm Hepes, 3 mm glucose, 2.5 mm CaCl2, 1.2 mm MgCl2, 0.1% bovine serum albumin (pH 7.2). Prior to imaging experiments, adipocytes were serum-starved for at least 2 h and transferred to a home-made closed perfusion chamber. Insulin stimulation was applied at a concentration of 100 nm throughout the study. Unless otherwise stated, all of the drugs were purchased from Sigma. Western Blotting—Cell extracts were prepared by washing the cells with phosphate-buffered saline and then extracting proteins with lysis buffer (10 mm Tris, 3 mm CaCl2, 2 mm MgCl2, 2.5% Nonidet P-40, pH 7.5). Samples from these cell lysates were denatured and subjected to SDS-PAGE using a 12% (w/v) running gel and analyzed by standard Western blotting techniques. The expression of endogenous IRAP and IRAP-pHluorin were identified and quantified using an anti-IRAP polyclonal antibody (1:1500) (gift from Dr. David James). Fluorescence Imaging—The cells were viewed with an Olympus FV500 confocal laser scanning biological microscope with a 60× (NA = 1.40) oil objective after transfection. pHluorin and TDimer2 fluorescence were both excited with a 488-nm argon laser. The images were acquired and analyzed using FLUOVIEW (Olympus Optical Co., Tokyo, Japan) and Photoshop 6.0. The TIRFM setup was constructed using an Olympus IX71 microscope based on the prism-less and through-the-lens configuration, as previously described (12Li C.H. Bai L. Li D.D. Xia S. Xu T. Cell Res. 2004; 14: 480-486Crossref PubMed Scopus (50) Google Scholar). Dual color images were collected at 5 Hz by a PCO EMCCD (PCO, Kelheim, Germany) at the left lateral port of the microscope after using a GFP/DsRed dual view microimager (Optical Insights, Tucson, AZ). The penetration depth of the evanescent field was estimated to be 113 nm by measuring the incidence angle of the 488-nm laser beam with a prism (n = 1.5218). For ratiometric fluorescence measurement under epi-fluorescence illumination, excitation was selected at 480 nm from a TILL monochromator (Polychrome V, TILL Photonics, GmbH). Calculation of Surface Fraction—We designed experiments to quantify the relative amount of TDimer2-IRAP-pHluorin on the cell surface compared with the total amount. 3T3-L1 adipocytes were first incubated in nonpermeating normal wash solution (pH 7.4) after 48 h of transfection. The external solution was then changed sequentially to nonpermeating pH 5.5 solution, permeating pH 7.4 NH4Cl solution and normal wash solution. The surface fraction of TDimer2-IRAP-pHluorin and the pH of the intracellular compartment (pHi) were determined as previous described (18Yang X. Xu P. Xiao Y. Xiong X. Xu T. J. Biol. Chem. 2006; 281: 15457-15463Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Image Analysis—Image pretreatment and analysis were based on the protocol described previously (13Bai L. Wang Y. Fan J. Chen Y. Ji W. Qu A. Xu P. James D.E. Xu T. Cell Metab. 2007; 5: 47-57Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). A fusion event was defined when the fluorescence increase exceeded five times the standard deviation of the background fluorescence (see Fig. 3d). We then counted the number of fusion events that occurred every 20 s and derived the fusion rate. For analyzing the docking state prior to fusion using TDimer2-IRAP-pHluorin, we first identified the fusion events according to the green fluorescence. Subsequently, we tracked back to the first frame when the TDimer2 fluorescence was stabilized. This time was taken as the initial docking time (Fig. 4b). Another criteria imposed as a requirement for docking was that during the interval between initial docking and fusion, the vesicle should remain immobilized with a three-dimensional displacement < 0.067 μm, as previously described (13Bai L. Wang Y. Fan J. Chen Y. Ji W. Qu A. Xu P. James D.E. Xu T. Cell Metab. 2007; 5: 47-57Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar).FIGURE 4Visualizing prefusion history using the dual colored marker, TDimer2-IRAP-pHluorin (RITS). a, sequential images of a single GSV labeled with TDimer2-IRAP-pHluorin undergoing insulin-stimulated exocytosis. The time indicated is relative to the onset of fusion. Bar, 1 μm. b, the fluorescence intensities of the green and red channels were averaged from 1-μm-diameter circles enclosing the vesicle. The second vertical dashed line marks the time of fusion, and the duration between two vertical dashed lines measures the dwell time in the docking/priming stage prior to fusion. c, histogram distribution of the latencies between docking and fusion for insulin-stimulated fusion events. Superimposed is the exponential fit with a time constant of 4.67s (n = 129 vesicles from 12 cells). The cumulative distribution of the fusion latency is displayed in the inset.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For normally distributed data, population averages are given as the means ± S.E., and statistical significance was tested with Student's t test. Rationale of the New Fusion Probe—EGFP-labeled GLUT4 (GLUT4-EGFP) has been employed to monitor the movement of single GLUT4-containing vesicles under TIRFM (8Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (146) Google Scholar, 12Li C.H. Bai L. Li D.D. Xia S. Xu T. Cell Res. 2004; 14: 480-486Crossref PubMed Scopus (50) Google Scholar). Although fusion of GLUT4 vesicles can be identified by monitoring the lateral diffusion of GLUT4-EGFP in the PM (8Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (146) Google Scholar), this method is extremely time-consuming and requires extensive training. Moreover, it has been suggested that monitoring fusion events with a sole EGFP reporter protein tends to under-estimate the fusion rate because of the complex diffusion kinetics of released and membrane-bound EGFP following fusion (19Taraska J.W. Perrais D. Ohara-Imaizumi M. Nagamatsu S. Almers W. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2070-2075Crossref PubMed Scopus (303) Google Scholar, 20Tsuboi T. Rutter G.A. Curr. Biol. 2003; 13: 563-567Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Thus, it is desirable to develop a rather straightforward and easy-to-apply methodology to reliably detect fusion events at the single vesicle level. We thus turned to the ecliptic pHluorin, which displays high contrast fluorescence changes upon environmental pH changes (15Miesenbock G. De Angelis D.A. Rothman J.E. Nature. 1998; 394: 192-195Crossref PubMed Scopus (1999) Google Scholar). pHluorin is brightly fluorescent at pH 7.4 and is essentially nonfluorescent at an approximate pH of <6.0. Because both the N and C termini of GLUT4 face the cytosol, attaching pHluorin to either end of GLUT4 will not result in a pH-induced fluorescence change. The insulin-regulated aminopeptidase (IRAP) has been identified as a major protein that co-localizes with GLUT4 in insulin-responsive GSVs (21Ross S.A. Scott H.M. Morris N.J. Leung W.Y. Mao F. Lienhard G.E. Keller S.R. J. Biol. Chem. 1996; 271: 3328-3332Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 22Abel E.D. Graveleau C. 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Cell Sci. 2000; 113: 4065-4076Crossref PubMed Google Scholar, 29Subtil A. Lampson M.A. Keller S.R. McGraw T.E. J. Biol. Chem. 2000; 275: 4787-4795Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). IRAP is a single transmembrane protein with its C terminus facing the vesicle lumen. We thus attached pHluorin to the C terminus of IRAP (IRAP-pHluorin) in the hope that IRAP-pHluorin would label insulin-responsive GSVs and could be used as a reporter for fusion. The idea is that pHluorin will be located in the lumen of GSVs (which is acidic) prior to fusion and will be exposed to the extracellular medium (which is neutral) upon fusion. Hence, we could identify a fusion event simply by abrupt high contrast brightening of pHluorin fluorescence. To ensure that pHluorin-labeled IRAP was correctly sorted, we examined the co-localization of IRAP-pHluorin with GLUT4-containing vesicles. Because a static co-localization assay by confocal microscopy is often masked by large fractions of co-localization in the endoplasmic reticulum and Golgi networks, we employed TIRFM to visualize single IRAP-pHluorin-labeled vesicles and validated whether these vesicles co-localize with GSVs labeled with GLUT4-EGFP. As shown in the snapshots of TIRFM images in Fig. 1, both IRAP-pHluorin and IRAP-TDimer2 co-localize very well with GLUT4-EGFP-labeled vesicles. The percentage of co-localization was estimated to be 99.2% for IRAP-Tdimer2 and GLUT4-EGFP, 98.1% for IRAP-pHluorin and IRAP-Tdimer2, and 99.1% for GLUT4-EGFP and the dual colored probe TDimer2-IRAP-pHluorin. The expression level of exogenous IRAP-pHluorin was estimated by Western blotting using anti-IRAP antibody, as shown in Fig. 1d. By normalizing the transfection efficiency for each batch of cells used for Western blotting, we estimated that the IRAP-pHluorin probe was ∼24 ± 6-fold greater than native IRAP proteins. IRAP-pHluorin, a Robust and Reliable Probe for Fusion Detection—Having determined the localization of IRAP-pHluorin within GLUT4-containing vesicles, we next examined whether IRAP-pHluorin can be used to detect fusion. By time lapse TIRFM imaging, adipocytes transfected with IRAP-pHluorin normally display weak immobilized fluorescence dotted with a few moving particles (Fig. 2a). The immobilized fluorescence was distributed and largely quenched by extracellular perfusion of a pH 5.5 solution, suggesting it represents fluorescence from IRAP-pHluorin already in the PM. The moving particles were weak and rarely observed, which is in stark contrast to the bright and numerous GLUT4-EGFP-labeled vesicles (8Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (146) Google Scholar, 12Li C.H. Bai L. Li D.D. Xia S. Xu T. Cell Res. 2004; 14: 480-486Crossref PubMed Scopus (50) Google Scholar), indicating that most IRAP-pHluorin is quenched inside the acidic vesicle lumen. Occasionally, we observed a sudden brightening of fluorescent spots. These brightening events increased dramatically after insulin treatment and diminished completely during extracellular perfusion with a pH 5.5 solution (Fig. 2), suggesting that they represent fusion of IRAP-pHluorin-containing vesicles. An example fusion event is shown in Fig. 3a. To quantify the change in fluorescence, we placed two concentric circles centered at the fusion site with inner and outer diameters of ∼0.9 and ∼1.2 μm, respectively. As demonstrated in Fig. 3d, the fluorescence from the inner circle exhibited an abrupt increase. The diffusion of IRAP-pHluorin could be observed as a significant increase in fluorescence followed by an exponential decay within the annulus (Fig. 3d). Occasionally, we observed a transient fluorescence increase in the inner circle without apparent diffusion into the annulus (Fig. 3, b and e). This is analogous to the so-called "kiss-and-run" mode of fusion found during synaptic vesicle fusion (30Sudhof T.C. Neuron. 2000; 28: 317-320Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The kiss-and-run fusion of GSVs constitutes only a small fraction (∼15%) of the total fusion events. The high contrast fluorescence change of IRAP-pHluorin during fusion makes it much easier to detect fusion events even by eye (supplemental Movie S1). As a comparison, we show a fusion event detected by GLUT4-EGFP in Fig. 3c. Fusion was defined by monitoring the radial diffusion of GLUT4-EGFP fluorescence. We considered a fusion event to have occurred when the fluorescence in the annulus between the two concentric circles increased significantly above the background fluorescence (13Bai L. Wang Y. Fan J. Chen Y. Ji W. Qu A. Xu P. James D.E. Xu T. Cell Metab. 2007; 5: 47-57Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 20Tsuboi T. Rutter G.A. Curr. Biol. 2003; 13: 563-567Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Because of the bright fluorescence of the vesicle prior to fusion (Fig. 3c), the fluorescence change during fusion was normally not obvious and was followed by a fast decay after fusion, as shown in Fig. 3f. It was usually not easy to distinguish a fusion and undocking event simply based on the fluorescence change profile. Hence, when using GLUT4-EGFP to detect fusion, one must scrutinize every vesicle based on radial diffusion analysis. This is not only extremely time-consuming but also prone to influence by the local fluorescence change adjacent to the vesicle being analyzed, which probably explains the low detection of fusion using the EGFP probe (20Tsuboi T. Rutter G.A. Curr. Biol. 2003; 13: 563-567Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). We now provide a solution to this problem by defining GSV fusion based on a simple parameter, fluorescence change. In Fig. 3g, we compare the averaged fluorescence change (normalized to the prefusion value) of IRAP-pHluorin and GLUT4-EGFP fluorescence during fusion. Whereas no obvious fluorescence increase could be observed for GLUT4-EGFP, there was a ∼20-fold increase in fluorescence for IRAP-pHluorin upon fusion. Hence, we were able to improve the signal-to-noise ratio by more than an order of magnitude by using this new probe for the detection of GSV fusion. The diffusion of IRAP-pHluorin, which is indicated by the decay of fluorescence after fusion, is considerably slower than that of GLUT4-EGFP. We estimated the averaged diffusion coefficient for IRAP-pHluorin to be 0.018 μm2/s, which is much slower than that of GLUT4-EGFP (0.093 μm2/s) (13Bai L. Wang Y. Fan J. Chen Y. Ji W. Qu A. Xu P. James D.E. Xu T. Cell Metab. 2007; 5: 47-57Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). It is not clear why IRAP-pHluorin diffuses much more slowly than GLUT4-EGFP despite its smaller size (42 kDa for IRAP-pHluorin and 82 kDa for GLUT4-EGFP). Nevertheless, the slow diffusion of IRAP-pHluorin makes it advantageous for fusion detection because more frames will be captured during one fusion event. Quantifying the Fusion Rate in Adipocytes—Although previous studies have monitored fusion events using GLUT4-EGFP, the fusion rate has not been determined (8Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (146) Google Scholar). This is partly due to the difficulty in assessin
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