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• W2007012258 abstract "Cell membranes are laterally organized into functionally discrete domains that include the cholesterol-dependent membrane “rafts.” However, how membrane domains are established and maintained remains unresolved and controversial but often requires the actin cytoskeleton. In this study, we used fluorescence resonance energy transfer to measure the role of the actin cytoskeleton in the co-clustering of membrane raft-associated fluorescent proteins (FPs) and FPs targeted to the nonraft membrane fraction. By fitting the fluorescence resonance energy transfer data to an isothermal binding equation, we observed a specific co-clustering of raft-associated donor and acceptor probes that was sensitive to latrunculin B (Lat B), which disrupts the actin cytoskeleton. Conversely, treating with jasplakinolide to enhance actin polymerization increased co-clustering of the raft-associated FPs over that of the nonraft probes. We also observed by immunoblotting experiments that the actin-dependent co-clustering coincided with regulation of the raft-associated Src family kinase Lck. Specifically, Lat B decreased the phosphorylation of the C-terminal regulatory tyrosine of Lck (Tyr505), and combining the Lat B with filipin further decreased the Tyr505 phosphorylation. Furthermore, the Lat B-dependent changes in Lck regulation required CD45 because no significant changes occurred in treated T cells lacking CD45 expression. These data define a role for the actin cytoskeleton in promoting co-clustering of raft-associated proteins and show that this property is important toward regulating raft-associated signaling proteins such as Lck. Cell membranes are laterally organized into functionally discrete domains that include the cholesterol-dependent membrane “rafts.” However, how membrane domains are established and maintained remains unresolved and controversial but often requires the actin cytoskeleton. In this study, we used fluorescence resonance energy transfer to measure the role of the actin cytoskeleton in the co-clustering of membrane raft-associated fluorescent proteins (FPs) and FPs targeted to the nonraft membrane fraction. By fitting the fluorescence resonance energy transfer data to an isothermal binding equation, we observed a specific co-clustering of raft-associated donor and acceptor probes that was sensitive to latrunculin B (Lat B), which disrupts the actin cytoskeleton. Conversely, treating with jasplakinolide to enhance actin polymerization increased co-clustering of the raft-associated FPs over that of the nonraft probes. We also observed by immunoblotting experiments that the actin-dependent co-clustering coincided with regulation of the raft-associated Src family kinase Lck. Specifically, Lat B decreased the phosphorylation of the C-terminal regulatory tyrosine of Lck (Tyr505), and combining the Lat B with filipin further decreased the Tyr505 phosphorylation. Furthermore, the Lat B-dependent changes in Lck regulation required CD45 because no significant changes occurred in treated T cells lacking CD45 expression. These data define a role for the actin cytoskeleton in promoting co-clustering of raft-associated proteins and show that this property is important toward regulating raft-associated signaling proteins such as Lck. Current models define cell membranes to be structurally heterogeneous in nature, composed of discrete domains with unique physical and biological properties. This lateral heterogeneity is essential for many membrane functions, including cell signaling (1Bray D. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 59-75Crossref PubMed Scopus (147) Google Scholar, 2Simons K. Toomre D. Nat. Mol. Cell Biol. Rev. 2000; 1: 31-39Crossref PubMed Scopus (5187) Google Scholar), cell motility (3Gomez-Mouton C. Abad J.L. Mira E. Lacalle R.A. Gallardo E. Jimenez-Baranda S. Illa I. Bernad A. Manes S. Martinez A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9642-9647Crossref PubMed Scopus (434) Google Scholar), protein and lipid uptake (4Pohl J. Ring A. Ehehalt R. Schulze-Bergkamen H. Schad A. Verkade P. Stremmel W. Biochemistry. 2004; 43: 4179-4187Crossref PubMed Scopus (83) Google Scholar, 5Pelkmans L. Burli T. Zerial M. Helenius A. Cell. 2004; 118: 767-780Abstract Full Text Full Text PDF PubMed Scopus (420) Google Scholar), and even the uptake and assembly of infectious agents (6Manes S. del Real G. Martinez A.C. Nat. Rev. Immunol. 2003; 3: 557-568Crossref PubMed Scopus (401) Google Scholar, 7Marsh M. Helenius A. Cell. 2006; 124: 729-740Abstract Full Text Full Text PDF PubMed Scopus (911) Google Scholar). However, studies of membrane structure and function remain challenging, because functionally discrete domains are often below the resolution of light microscopy. Accordingly, properties of cell membranes and membrane domains remain unclear and even controversial (8Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1333) Google Scholar). An important example of domains in biological membranes is the cholesterol-dependent membrane rafts. In the membrane raft model, rafts form through associations between cholesterol and other membrane lipids, giving rise to a discrete lipid phase with which specific membrane proteins associate (9Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8157) Google Scholar). Many studies have characterized raft domains using membrane fractionation, where it is postulated that rafts are represented by a nonionic detergent-resistant, glycolipid-enriched membrane fraction that is present in cell lysates (10Brown D.A. Rose J.K. Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2618) Google Scholar). Notably, however, some investigators contend that this fraction is an artifact of detergent lysis, and the membrane raft model is inaccurate (8Munro S. Cell. 2003; 115: 377-388Abstract Full Text Full Text PDF PubMed Scopus (1333) Google Scholar). Supporting the notion that the detergent-resistant glycolipid-enriched membrane fraction is representative of membrane domains in intact cells, separate studies have demonstrated a protein and lipid clustering that is specific to glycolipid-enriched membrane-associated molecules (11Friedrichson T. Kurzchalia T.V. Nature. 1998; 394: 802-805Crossref PubMed Scopus (480) Google Scholar, 12Varma R. Mayor S. Nature. 1998; 394: 798-801Crossref PubMed Scopus (1031) Google Scholar, 13Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1803) Google Scholar, 14Sharma P. Varma R. Sarasij R.C. Ira Gousset K. Krishnamoorthy G. Rao M. Mayor S. Cell. 2004; 116: 577-589Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar, 15Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (697) Google Scholar, 16Rodgers W. Zavzavadjian J. Exp. Cell Res. 2001; 267: 173-183Crossref PubMed Scopus (59) Google Scholar, 17Sohn H.W. Tolar P. Jin T. Pierce S.K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8143-8148Crossref PubMed Scopus (94) Google Scholar). Imaging studies of raft-associated probes suggest that the rafts are heterogeneous in nature (18Jacobson K. Mouritsen O.G. Anderson R.G. Nat. Cell Biol. 2007; 9: 7-14Crossref PubMed Scopus (910) Google Scholar), ranging in size from nanoclusters that are ∼5 nm in diameter and containing no more than a few protein molecules (14Sharma P. Varma R. Sarasij R.C. Ira Gousset K. Krishnamoorthy G. Rao M. Mayor S. Cell. 2004; 116: 577-589Abstract Full Text Full Text PDF PubMed Scopus (713) Google Scholar), to larger nanodomains that are ∼25-100 nm in size (12Varma R. Mayor S. Nature. 1998; 394: 798-801Crossref PubMed Scopus (1031) Google Scholar, 19Pralle A. Keller P. Florin E.L. Simons K. Horber J.K. J. Cell Biol. 2000; 148: 997-1008Crossref PubMed Scopus (847) Google Scholar, 20Prior I.A. Muncke C. Parton R.G. Hancock J.F. J. Cell Biol. 2003; 160: 165-170Crossref PubMed Scopus (612) Google Scholar). Finally, fluorescence imaging experiments have demonstrated micron-size raft macrodomains that are detergent-resistant and enriched with raft-associated molecules (3Gomez-Mouton C. Abad J.L. Mira E. Lacalle R.A. Gallardo E. Jimenez-Baranda S. Illa I. Bernad A. Manes S. Martinez A.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9642-9647Crossref PubMed Scopus (434) Google Scholar, 15Janes P.W. Ley S.C. Magee A.I. J. Cell Biol. 1999; 147: 447-461Crossref PubMed Scopus (697) Google Scholar, 16Rodgers W. Zavzavadjian J. Exp. Cell Res. 2001; 267: 173-183Crossref PubMed Scopus (59) Google Scholar, 21Viola A. Schroeder S. Sakakibara Y. Lanzavecchia A. Science. 1999; 283: 680-682Crossref PubMed Scopus (842) Google Scholar, 22Villalba M. Bi K. Rodriguez F. Tanaka Y. Schoenberger S. Altman A. J. Cell Biol. 2001; 155: 331-338Crossref PubMed Scopus (196) Google Scholar, 23Jordan S. Rodgers W. J. Immunol. 2003; 171: 78-87Crossref PubMed Scopus (43) Google Scholar). One example of the macrodomains is the immunological synapse in stimulated lymphocytes (24Monks C.R. Freiberg B.A. Kupfer H. Sciaky N. Kupfer A. Nature. 1998; 395: 82-86Crossref PubMed Scopus (1984) Google Scholar, 25Grakoui A. Bromley S.K. Sumen C. Davis M.M. Shaw A.S. Allen P.M. Dustin M.L. Science. 1999; 285: 221-227Crossref PubMed Scopus (2563) Google Scholar), which forms where lymphocytes contact a cell containing antigen. In T cells and other lymphocytes, evidence of membrane rafts also comes from studies of signaling proteins in the outer membrane. For example, enriched in the membrane raft fraction are proteins that participate in signaling from the surface antigen receptors, and their targeting to the raft fraction is often necessary for efficient cell activation (26Cherukuri A. Dykstra M. Pierce S.K. Immunity. 2001; 14: 657-660Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). One such enzyme is the Src family kinase Lck, which functions in signaling from the T cell receptor following binding to major histocompatibility complex-peptide complexes. Disruption of Lck targeting to rafts attenuates T cell activation (27Kabouridis P.S. Magee A.I. Ley S.C. EMBO J. 1997; 16: 4983-4998Crossref PubMed Scopus (318) Google Scholar), and in resting T cells, membrane rafts function in regulating Lck (28Rodgers W. Rose J.K. J. Cell Biol. 1996; 135: 1515-1523Crossref PubMed Scopus (286) Google Scholar, 29Kabouridis P.S. Janzen J. Magee A.L. Ley S.C. Eur. J. Immunol. 2000; 30: 954-963Crossref PubMed Scopus (292) Google Scholar). The factors that govern formation of membrane domains, including membrane rafts, continue to be elucidated. One such factor is the actin cytoskeleton, which can affect the lateral distribution and mobility of membrane proteins (30Rodgers W. Glaser M. Biochemistry. 1993; 32: 12591-12598Crossref PubMed Scopus (73) Google Scholar, 31Lenne P.F. Wawrezinieck L. Conchonaud F. Wurtz O. Boned A. Guo X.J. Rigneault H. He H.T. Marguet D. EMBO J. 2006; 25: 3245-3256Crossref PubMed Scopus (392) Google Scholar). In relation to rafts, actin occurs in the detergent-resistant raft fraction (23Jordan S. Rodgers W. J. Immunol. 2003; 171: 78-87Crossref PubMed Scopus (43) Google Scholar), and actin-filaments co-associate with known membrane raft markers (16Rodgers W. Zavzavadjian J. Exp. Cell Res. 2001; 267: 173-183Crossref PubMed Scopus (59) Google Scholar, 23Jordan S. Rodgers W. J. Immunol. 2003; 171: 78-87Crossref PubMed Scopus (43) Google Scholar). The actin cytoskeleton is also necessary for formation of some types of raft macrodomains in lymphocytes (16Rodgers W. Zavzavadjian J. Exp. Cell Res. 2001; 267: 173-183Crossref PubMed Scopus (59) Google Scholar, 23Jordan S. Rodgers W. J. Immunol. 2003; 171: 78-87Crossref PubMed Scopus (43) Google Scholar, 32Rodgers W. Farris D. Mishra S. Trends Immunol. 2005; 26: 97-103Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). However, the degree to which the actin cytoskeleton participates in establishing membrane rafts, such as its contribution toward protein clustering relative to that of the cholesterol-dependent lipid ordering, is not established. Importantly, the actin cytoskeleton is a dynamic structure that changes in response to extracellular signals, and it may therefore represent one mechanism for governing the size and distribution of membrane rafts in the plasma membrane. By measuring fluorescence resonance energy transfer (FRET) 2The abbreviations used are:FRETfluorescence resonance energy transferFPfluorescent proteinCFPcyan fluorescent proteinYFPyellow fluorescent proteinLATlinker for activation in T cellsLat Blatrunculin BTXSTriton X-100 solublePIP2phosphatidylinositol 4,5-biphosphate. between separate membrane-anchored fluorescent proteins, we show here a specific clustering of raft-associated membrane probes by the actin cytoskeleton. By immunoblotting, we also show the actin cytoskeleton is necessary for effective regulation of Lck. Similar effects on protein co-clustering and Lck regulation were observed in cells where cholesterol was sequestered using filipin. We conclude that both cholesterol and the actin cytoskeleton are necessary in establishing a membrane environment that provides for efficient regulation of raft-associated enzymes such as Lck. fluorescence resonance energy transfer fluorescent protein cyan fluorescent protein yellow fluorescent protein linker for activation in T cells latrunculin B Triton X-100 soluble phosphatidylinositol 4,5-biphosphate Gene Construction—Generation of CFP and YFP fusion proteins containing either the first 10 amino acids of Lck (L10) or the first 15 residues of Src (S15) was performed as described previously (33Rodgers W. BioTechniques. 2002; 32: 1044-1051Crossref PubMed Scopus (44) Google Scholar). The sense and antisense oligonucleotides (33Rodgers W. BioTechniques. 2002; 32: 1044-1051Crossref PubMed Scopus (44) Google Scholar) for the L10 and S15 sequences were annealed and subcloned into the SmaI site of pWay20 (34Lo W. Rodgers W. Hughes T. BioTechniques. 1998; 25 (98): 94-96Crossref PubMed Scopus (14) Google Scholar) containing CFP or YFP in place of GFP. A fragment encoding the first 36 residues of linker for activation of T cells (LAT36) was amplified from the sequence of the full-length protein using the following primers: 5′-CATCATCTAGAATGGAGGAGGCCATCCTGG-3′ (coding); and 5′-TCTAGTGAATTCGTTGTTGTTGTTGTTGTTGTCGTAGGAGCCTGGTC-3′ (noncoding). The underlined nucleotides represent restriction sites for XbaI and EcoRI in the coding and noncoding primers, respectively. The amplified product was digested and subcloned into the XbaI and EcoRI sites of pcDNA 3.1 (Invitrogen), upstream and in-frame to YFP. Lyn-CFP-YFP and CFP-T2DN-YFP have been described previously (17Sohn H.W. Tolar P. Jin T. Pierce S.K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8143-8148Crossref PubMed Scopus (94) Google Scholar) and were generously provided by Dr. S. Pierce (National Institutes of Health). Cell Culture and Transfection—Jurkat T cells (clone E6-1) were maintained in medium containing RPMI 1640 supplemented with antibiotics and 10% fetal bovine serum at 37 °C in the presence of 5% CO2. For gene expression, the cells were transfected by electroporation (Gene Pulser II; Bio-Rad) as described (23Jordan S. Rodgers W. J. Immunol. 2003; 171: 78-87Crossref PubMed Scopus (43) Google Scholar). For transfection, 107 cells were suspended in 0.5 ml of RPMI and containing 25 μg of plasmid DNA. Settings of 330 V and 960 μF were used for electroporation. 48 h post-transfection, viable cells were separated by centrifuging the samples over Cellgro™ (MediaTech, Inc., Herndon, VA). Stable clones expressing CFP fusion proteins were selected by limiting dilution using medium containing G418 (Invitrogen) at a concentration of 1.0 mg/ml. Following drug selection, the clones were enriched for protein expression by flow cytometry and maintained in medium containing 500 μg/ml G418. Cell Lysis, Equilibrium Centrifugation, and Immunoblotting—For sucrose gradient equilibrium centrifugation experiments, 107 cells were lysed with 1.0 ml of a 1% Triton X-100 solution in 10 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 5 mm EDTA (TNE). For lysis, the samples were incubated with the detergent solution for 20 min at 4 °C and then processed with a Dounce homogenizer (8 strokes). Intact nuclei and large cell debris were removed by centrifuging (Eppendorf model 5417C; Brinkman Instruments, Westbury, NY) at 4000 rpm at 4 °C for 5 min. The supernatant was collected and diluted with an equal volume of an 85% sucrose solution in TNE. After gently mixing, the sample was overlaid in a SW50.1 rotor tube with 3.0 ml of a 30% and 1.5 ml of a 5% solution, each in TNE. The samples were centrifuged for 16-18 h at 200,000 × g. Following centrifugation, the gradients were harvested by fractionating from the top. Approximately 10% of each fraction was separated by gel electrophoresis. The fluorescent probes were detected by immunoblotting using a monoclonal antibody specific to GFP that recognizes YFP and CFP (Covance Research Products, Richmond, CA). Assays of Lck regulation began by lysing 106 cells in 100 μl of Laemmeli sample buffer containing 1% 2-mercaptoethanol, followed by incubation at 100 °C for 5 min. After lysis, equivalent amounts of the samples were separated by gel electrophoresis and immunoblotted using monoclonal antibody to Lck (clone 2B;; BD Biosciences, San Jose, CA) or rabbit antibody to Tyr(P)505 of Lck (Cell Signaling Technology; Danvers, MA). All of the immunoblots were developed by ECL (Amersham Biosciences) and detected using a LumiImager work station (Roche Applied Science). Fluorescence Microscopy and Image Analysis—The samples were prepared for microscopy by seeding ∼106 cells onto a poly-l-lysine (Sigma)-coated coverslip and then fixed by incubating in a 2% solution of paraformaldehyde for 30 min at room temperature. For the drug treatments, seeded coverslips were washed with RPMI containing 50 mm HEPES (pH 7.4) (RPMIHEPES). Next, the medium was replaced with RMPI-HEPES containing either 1% Me2SO (Sigma) alone, or drug diluted from a 100× stock solution in Me2SO. Final drug concentrations were 5 μm latrunculin B (Lat B) (Calbiochem, La Jolla, CA), 0.5 μm jasplakinolide (Invitrogen), and 5 μg/ml filipin (Cayman Chemicals, Ann Arbor, MI). All of the incubations were for 30 min at 37 °C. Microscopy was performed using a Zeiss LSM 510 META confocal microscope. CFP was excited at 458 nm, and emission was detected between 473 and 505 nm. YFP was excited at 514 nm, and emission was detected from 530 to 600 nm. The images were collected using a 63× oil objective (NA 1.2), and recorded in 12-bit mode at a scan rate of 2.56 μs/pixel. All of the image processing and quantitation was performed using IPLab (BD Biosciences). FRET was measured based on the increase in the CFP signal following photobleaching of the YFP. Photobleaching of YFP was restricted to a region of interest 8 × 5 pixels (2.1 × 1.3 μm) in size and was performed by illuminating the sample for 750 iterations (∼45 s) at 514 nm with the laser at full power. Four images were acquired for each region of interest: 1) an image of YFP fluorescence before bleach (YFPpre), 2) an image of CFP fluorescence before bleach (CFPpre), 3) an image of YFP fluorescence after bleach (YFPpost), and 4) an image of CFP fluorescence after bleach (CFPpost). In addition, to correct for bleaching of CFP during YFP bleaching (CFPYFP bleach), cells expressing CFP alone were taken through all the steps of FRET measurement. Cells expressing only YFP were used to determine the YFP bleed-through into the CFP channel (CFPYFP). Fluorescence intensities of each region of interest were measured on a pixel-by-pixel basis, and measurement of YFPpre - YFPpost showed the photobleaching extinguished >95% of the YFP fluorescence. CFPpre was corrected for YFP bleed-through into the CFP channel using the following operation. CFPpre,corrected=CFPpre-CFPYFP(Eq. 1) CFPpost bleach was corrected for both bleaching of CFP during YFP bleaching and YFP bleed-through by the following equation. CFPpost,corrected=(CFPpost-CFPYFP)+CFPYFPbleach(Eq. 2) From the corrected values, FRET efficiency (E%) was calculated as follows. E&x0025;=(CFPpost,corrected-CFPpre,corrected)CFPpost,corrected×100(Eq. 3) FRET was measured in 75-100 separate cells, restricted to those cells where the expression of the donor and acceptor were approximately equal. Co-clustering was quantitated by fitting plots of E% versus acceptor intensity to Equation 4 (below). Curve fitting and all of the statistical analysis were performed using Igor Pro (WaveMetrics, Lake Oswego, OR). Standard error was determined as described (35Cumming G. Fidler F. Vaux D.L. J. Cell Biol. 2007; 177: 7-11Crossref PubMed Scopus (609) Google Scholar) using standard deviations from the curve fitting. The statistical significance of the differences in K values was determined using a two-tailed Student's t test for distributions of unequal variance (Equations 14.2.3 and 14.2.4 in Ref. 36Press W.H. Teukolsky S.A. Vetterling W.T. Flannery B.P. Numerical Recipes: The Art of Scientific Computing. 3rd Ed. Cambridge University Press, New York2007: 720-772Google Scholar). The K values determined to be significantly different are indicted by asterisks, which represent a probability greater than 99.99% that the two values are different. Targeting of Fluorescent Proteins to the Raft and Nonraft Membrane Fractions—The membrane-targeted FPs used in this study are illustrated in Fig. 1A. Specifically, CFP and YFP served as the FRET donor and acceptor, respectively. Each was targeted to the membrane fraction of Jurkat T cells by either the N-terminal 10 residues of Lck (L10), the N-terminal 15 residues of Src (S15), or the N-terminal 36 residues of linker for activation of T cells (LAT36). We have described the L10 and S15 membrane targeting signals previously (33Rodgers W. BioTechniques. 2002; 32: 1044-1051Crossref PubMed Scopus (44) Google Scholar), where it was shown the L10 sequence targets FPs to the detergent-resistant membrane raft fraction and the S15 signal limits FPs to the Triton X-100-soluble (TXS), nonraft membrane fraction. LAT36 contains the transmembrane domain of LAT but lacks its cytoplasmic domain with associated linker functions (37Samelson L.E. Annu. Rev. Immunol. 2002; 20: 371-394Crossref PubMed Scopus (470) Google Scholar). Furthermore, the LAT36 sequence targets fusion proteins to the raft fraction (38Shogomori H. Hammond A.T. Ostermeyer-Fay A.G. Barr D.J. Feigenson G.W. London E. Brown D.A. J. Biol. Chem. 2005; 280: 18931-18942Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) because of palmitoylation of a set of membrane-proximal cysteine residues (39Zhang W. Trible R.P. Samelson L.E. Immunity. 1998; 9: 239-246Abstract Full Text Full Text PDF PubMed Scopus (754) Google Scholar). In Fig. 1B are fractionation data showing the membrane targeting features of the L10, S15, and LAT36 sequences. In addition, in Fig. 1B are confocal images showing each membrane anchor efficiently targeted the FPs to the plasma membrane. Visualization of FRET by Acceptor Photobleaching—To establish detection of FRET, we first measured energy transfer in T cells expressing a control peptide consisting of CFP and YFP separated by a two-amino acid linker and containing the membrane-anchoring signal of Lyn (Lyn-CFP-YFP). The energy transfer efficiency (E%) was measured based on the increase in donor fluorescence following photobleaching of the acceptor (40Pentcheva T. Edidin M. J. Immunol. 2001; 166: 6625-6632Crossref PubMed Scopus (68) Google Scholar). Furthermore, to show that observed changes in CFP fluorescence following YFP photobleaching were specific to FRET, measurements were made using cells expressing a second control peptide containing CFP and YFP separated by a 236-amino acid linker (CFP-T2DN-YFP). Examples of FRET measurements of the CFP-YFP fusion peptides are shown in Fig. 2A. The yellow box in each image represents the region where the acceptor was photobleached. Accompanying the images are plots of the fluorescence intensity values of the outer membrane in the CFP and YFP channels before (red) and after (green) photobleaching. The vertical lines in the plots mark the boundaries of the bleached regions and show the photobleaching extinguished ∼95% of the YFP fluorescence. For the Lyn-CFP-YFP, the photobleaching was accompanied by an approximately 2-fold increase in the CFP signal. In contrast, photobleaching YFP of the CFP-T2DN-YFP resulted in no detectable change in its CFP fluorescence. Thus, the changes in CFP fluorescence following photobleaching of YFP were specific to FRET. Resolution of Protein Co-clustering by FRET—Earlier studies showed that co-association of FRET donor and acceptor pairs with membrane domains is reflected in plots of E% versus acceptor concentration (13Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1803) Google Scholar, 17Sohn H.W. Tolar P. Jin T. Pierce S.K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8143-8148Crossref PubMed Scopus (94) Google Scholar, 40Pentcheva T. Edidin M. J. Immunol. 2001; 166: 6625-6632Crossref PubMed Scopus (68) Google Scholar, 41Kenworthy A.K. Edidin M. J. Cell Biol. 1998; 142: 69-84Crossref PubMed Scopus (404) Google Scholar, 42Tolar P. Sohn H.W. Pierce S.K. Nat. Immunol. 2005; 6: 1168-1176Crossref PubMed Scopus (180) Google Scholar). The principal behind this property is illustrated in Fig. 2B. First, in the absence of co-clustering, E% increases linearly with increasing acceptor as the distance between the donor and acceptor decreases proportionally because of molecular crowding. In contrast, when the donor and acceptor co-cluster, such as through co-association with membrane domains, the distance between the donor-acceptor pair reaches a minimal distance, after which E% no longer increases with increasing acceptor concentration. Tsien and co-workers (13Zacharias D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1803) Google Scholar) showed the associative properties of the donor and acceptor pair such as that illustrated in Fig. 2B can be discriminated by the parameter K in the following isothermal binding equation, E&x0025;=E&x0025;maxF(F+K)(Eq. 4) where F represents the fluorescence intensity of the acceptor, which we defined as the prebleach intensity of the YFP fluorescence. Based on Equation 4, K is analogous to a dissociation constant of the donor and acceptor pair, approaching zero as the degree of co-clustering of the fluorescent proteins increases. In Fig. 2C are examples of K representing the relative co-clustering of donor and acceptor pairs using FRET data from the Lyn-CFP-YFP and CFP-T2DN-YFP fusion proteins. In the experiment with Lyn-CFP-YFP, K approached zero (K = 12), reflecting an apparent co-clustering that is due to an adjacent donor and acceptor in the fusion protein. In contrast, for CFP-T2DN-YFP, where we predicted the apparent co-clustering to be minimal because of a large separation of the donor and acceptor pair, K is 2 orders of magnitude larger (K = 1710) than that of the Lyn-CFP-YFP. Note also for each fit that the residual differences between the experimental and predicated values for E% are minimal and distributed randomly about zero, thus indicating a favorable fit of the experimental data to Equation 4. In contrast, fitting the FRET data from Lyn-CFP-YFP to Equation 4 but constrained by the K determined for CFP-T2DN-YFP resulted in a large deviation of the residual differences from zero (supplemental Fig. S1, top row). Thus, application of Equation 4 reliably discriminates the co-clustering of the separate donor-acceptor pairs. As an additional control, we determined the effect of protein aggregation on the apparent co-clustering of the L10-anchored probes (Fig. 2C). Specifically, we measured the FRET between L10-CFP and L10-YFP in cells treated with methyl-β-cyclodextrin and sphingomylinase, which represent conditions where raft-associated proteins aggregate (43Nishimura S.Y. Vrljic M. Klein L.O. McConnell H.M. Moerner W.E. Biophys. J. 2006; 90: 927-938Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 44Hao M. Mukherjee S. Maxfield F.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13072-13077Crossref PubMed Scopus (251) Google Scholar). K for the co-treated samples approached that of the Lyn-CFP-YFP (K = 68), and this value is approximately one-fifth that determined for L10-CFP and L10-YFP in untreated cells (Fig. 3). These data therefore further validate the approach of using K to distinguish the relative co-clustering of separate membrane proteins. Cholesterol-dependent Co-clustering of Membrane-anchored Fluorescent Proteins—To determine the relative co-clustering of the membrane-anchored FPs, we fitted FRET data to Equation 4 from cells co-expressing L10-or S15-CFP with L10-, LAT36-, or S15-YFP (Fig. 3; see also Fig. 5). Consistent with the membrane raft model, these data show a specific and cholesterol-dependent co-clustering of probes that associate with the detergent-resistant raft fraction. For example, the lowest values for K occurred in samples double-labeled with L10-CFP and either L10-YFP or LAT36-YFP (K = 263 and 154, respectively), and these values were up to 11-fold lower than K determined for cells co-expressing an S15-anchored probe with the L10- or LAT36-FPs (1100 < K < 1700) (Fig. 3). Furthermore, cells co-expressing L10-CFP with either L10- or LAT36-YFP and treated with filipin to sequester cholesterol (45Bolard J. Biochim. Biophys. Acta. 1986; 864: 257-304Crossref PubMed Scopus (690) Google Scholar) and disrupt the membrane raft fraction (Fig. 4) exhibited up to a 10-fold increase in K. However, disruption of protein co-clustering by filipin was specific to donor and acceptor" @default.
• W2007012258 created "2016-06-24" @default.
• W2007012258 creator A5059663610 @default.
• W2007012258 creator A5065317972 @default.
• W2007012258 date "2007-12-01" @default.
• W2007012258 modified "2023-10-17" @default.
• W2007012258 title "Clustering of Membrane Raft Proteins by the Actin Cytoskeleton" @default.
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