Clustering of Membrane Raft Proteins by the Actin Cytoskeleton

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. 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However, the to which the actin cytoskeleton in membrane such as toward protein clustering to that of the cholesterol-dependent lipid is the actin cytoskeleton is a structure that changes in to and it for the size and of membrane rafts in the By fluorescence resonance energy transfer used resonance energy fluorescent fluorescent for in T between separate fluorescent we show a specific clustering of raft-associated membrane probes by the actin cytoskeleton. By we also show the actin cytoskeleton is for regulation of Lck. protein co-clustering and Lck regulation observed in cells where cholesterol was using We that cholesterol and the actin cytoskeleton are in a membrane that for regulation of raft-associated such as Lck. fluorescence resonance energy transfer fluorescent protein fluorescent protein fluorescent protein for in T cells latrunculin B of and proteins containing the of Lck the of Src was as W. 2002; PubMed Scopus Google Scholar). and W. 2002; PubMed Scopus Google for the and and into the of W. Rodgers W. T. 1998; PubMed Scopus Google containing in of the of for of T cells was from the of the protein using the and for and in the and was and into the and of and to and have H.W. Tolar P. Jin T. Pierce S.K. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8143-8148Crossref PubMed Scopus (94) Google and by S. Pierce of Cell and T cells maintained in containing with and in the of the cells by as S. Rodgers W. J. Immunol. 2003; 171: 78-87Crossref PubMed Scopus (43) Google Scholar). cells in of and containing of of and used for cells by the over proteins by using containing a of the enriched for protein by and maintained in containing Cell and cells with of a in and lysis, the with the detergent for and with a and cell by model for was and with an of an in the was in a with of a and of a in for the by from the of fraction was by fluorescent probes by immunoblotting using a specific to that and of Lck regulation by cells in of containing by for lysis, of the by and using to Lck to of Lck of the by and using a and for by cells a and by in a of for the with containing the was with containing from a in latrunculin B (Lat jasplakinolide and filipin of the for was using a was and was between and was and was from to using a and in a of of the and was using was the in the of the of was to a of in size and was by the for nm with the for of an of fluorescence an of fluorescence an of fluorescence and an of fluorescence In to for of cells through the of used to the into the of of a and of the of the was for into the using the was for of and by the the was as was in separate cells, to cells where the of the donor and acceptor was by fitting of acceptor to fitting and of the using was as G. F. J. Cell Biol. 2007; PubMed Scopus Google using from the of the in was using a for of and in of Scholar). to be are by which a than that the are of to the and FPs used in this are in Specifically, and as the donor and was targeted to the membrane fraction of T cells by the of Lck the of Src the of for of T cells We have the and membrane W. 2002; PubMed Scopus Google Scholar), where it was the FPs to the detergent-resistant membrane raft fraction and the FPs to the nonraft membrane fraction. the of but with Annu. Rev. Immunol. 2002; PubMed Scopus Google Scholar). Furthermore, the proteins to the raft fraction H. E. D.A. J. Biol. Full Text Full Text PDF PubMed Scopus Google because of of a of W. 1998; 9: Full Text Full Text PDF PubMed Scopus Google Scholar). In are data the membrane of the and In in are membrane targeted the FPs to the of by of we energy transfer in T cells a of and by a and containing the of energy transfer was the in donor fluorescence of the acceptor T. M. J. 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Science. 2002; 296: 913-916Crossref PubMed Scopus (1803) Google the properties of the donor and acceptor such as that in be by the in the isothermal binding equation, where the fluorescence of the which we as the of the is to a of the donor and acceptor as the of co-clustering of the fluorescent proteins In are of the co-clustering of donor and acceptor using data from the and In the with an co-clustering that is to an donor and acceptor in the In for where we the co-clustering to be because of a of the donor and acceptor is of larger than that of the also for that the between the and for are and a of the data to In fitting the data from to but by the for in a of the from of the co-clustering of the separate an we the of protein the co-clustering of the probes Specifically, we the between and in cells treated with and which where raft-associated proteins M. Biophys. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar). for the that of the and this is that for and in cells These data further the of using to the co-clustering of separate membrane of the co-clustering of the we data to from cells with also with the membrane raft model, data show a specific and cholesterol-dependent co-clustering of probes that associate with the detergent-resistant raft fraction. the for occurred in with and and and to than for cells an with the Furthermore, cells with and treated with filipin to cholesterol J. Biophys. PubMed Scopus Google and the membrane raft fraction to a in K. However, of protein co-clustering by filipin was specific to donor and acceptor where with the raft because cells and a in in and observed in co-clustering of and in the of a cholesterol-dependent of probes. that in cells and that for with the raft-associated probes co-clustering of the proteins in of the of Lat and T raft and membrane of T cells by of cell to lysis, the with Lat B filipin is in and the fraction of protein in the raft fraction of is are of in cells in the for the we also the of the observed fitting by a using data from with and data to using from cells and in a significant in the and from a the separate between the with and and with in the a that the for are in of raft such as the immunological in T cells, associate with actin W. Zavzavadjian J. Exp. Cell Res. 2001; 267: 173-183Crossref PubMed Scopus (59) Google Scholar). the actin cytoskeleton is for of membrane raft W. D. S. Immunol. Full Text Full Text PDF PubMed Scopus Google Scholar). the properties of the actin cytoskeleton and membrane rafts an actin-dependent clustering of raft we co-clustering of the with other FPs was Accordingly, we treated cells and with Lat B to the actin cytoskeleton. the Lat B no of the probes with the membrane raft fraction it co-clustering of with and for the Lat and increased over that of cells and B), and a in occurred in with and and In co-clustering of the and increased in because decreased from for the to for that the Lat B and the of protein co-clustering by Lat B was specific to where the donor and acceptor the from with filipin Lat B in that in some the actin cytoskeleton was more significant than cholesterol toward promoting co-clustering of the raft-associated probes. in the cells with and for the treated with filipin was that for that the Lat B and B), and this was significant Furthermore, the in the notion that separate to Finally, and also that membrane rafts and the actin cytoskeleton are to for co-clustering of with Specifically, with Lat B and filipin in for that the acceptor and this is of a of donor and acceptor molecules D.A. Violin J.D. Newton A.C. Tsien R.Y. Science. 2002; 296: 913-916Crossref PubMed Scopus (1803) Google Scholar). co-clustering of the raft-associated probes was we that the actin cytoskeleton using the jasplakinolide L. F. A. H. D. J. 2006; PubMed Scopus Google enhance the observed in are data using T cells actin and with jasplakinolide in the of the cytoskeleton we observed that treating T cells with jasplakinolide a cholesterol-dependent in the co-clustering of and B and for the was than that of cells However, the with filipin and jasplakinolide increased to a than that of the treated with jasplakinolide of donor and acceptor co-clustering by jasplakinolide was specific to the and because the also increased the co-clustering of and However, the in co-clustering of and was because the with filipin and jasplakinolide in a to that of the cells treated with jasplakinolide and We also observed that jasplakinolide the co-clustering of and to that of cells, and we this as of an co-clustering of the probes that is to changes in the actin cytoskeleton. Finally, was for the with and and as as the filipin and jasplakinolide cells and These data suggest a co-clustering of the probes in the membrane and the donor and acceptor associate with membrane rafts. and of Lck in T and have the raft-associated of Lck is to that in the nonraft membrane represented by phosphorylation of regulatory Tyr505 and a in kinase W. Rose J.K. J. Cell Biol. PubMed Scopus Google Scholar, J. Magee Ley S.C. J. Immunol. 2000; PubMed Scopus Google Scholar). discrete regulation of raft Lck to from the membrane because CD45 is from and it Lck by of raft-associated Lck also be by phosphorylation of Tyr505 by which with rafts through binding to protein with glycolipid-enriched membranes D. M. A. Mol. Cell Biol. 2003; PubMed Scopus Google Scholar, T. D. A. E. P. J. A. I. J. K. Y. J. Exp. 2000; PubMed Scopus Google Scholar). the from we an and cholesterol-dependent regulation of Lck by promoting of Lck from the nonraft this we the phosphorylation of Tyr505 of Lck in cells treated with Lat of which of Lck with the detergent-resistant raft fraction with the filipin and Lat B decreased the of the Lck to that of the Furthermore, the in Tyr505 phosphorylation occurred in the with filipin and Lat This to where the raft-associated probes a in the membrane and where we the to CD45 to be the the changes in Tyr505 phosphorylation in are we the Lck in cells, which are a of In to the with the cells, the Lat phosphorylation of Lck Tyr505 in cells These data in and are with a and actin-dependent regulation of Lck by from This by the and cholesterol-dependent co-clustering of raft such as that by of the Cell membranes are represented in by a of protein and lipid molecules through the a that a of membrane-associated this lateral heterogeneity is established and maintained an show such is through an actin-dependent co-clustering of membrane raft-associated experiments show that the actin cytoskeleton by treating cells with Lat B disrupts co-clustering of raft-associated donor and acceptor fluorescent in some more than by cholesterol with Furthermore, promoting actin polymerization using jasplakinolide co-clustering of the raft-associated probes that of as a for the model in the specific co-clustering of the and probes with and actin-dependent raft Accordingly, we that the in this co-clustering by Lat B and from of the domains by We also that of the and with the domains proteins from in the nonraft In this the Lat and in co-clustering of the and with from a of this by model that the of the donor and acceptor in cells with Lat B and filipin a more of the domains in with jasplakinolide increased co-clustering of the FPs in a cholesterol-dependent Accordingly, we this as of a of the raft through of the actin cytoskeleton by the In the in co-clustering between raft and nonraft probes and by jasplakinolide was and occurred by to the cholesterol-dependent studies demonstrated that an of with membrane enriched with raft-associated proteins and W. 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PubMed Scopus Google Scholar). a protein clustering and for the in co-clustering of and in the However, it is to how such a for the co-clustering that was specific to the raft-associated as as the cholesterol-dependent nature of this Accordingly, we the model where the co-clustering through an actin-dependent in the membrane that in the raft-associated An was that protein co-clustering using Lat B no the of with the detergent-resistant raft fraction and a Lck These the that in membrane membrane rafts are heterogeneous in nature, it is to the size and of the domains represented by the raft fraction. In with Lat the in protein co-clustering detergent a of raft into nanoclusters because the are to significant protein co-clustering as protein molecules (14Sharma P. Varma R. Sarasij R.C. Ira Gousset K. Krishnamoorthy G. Rao M. Mayor S. 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This with with cells and other of T cells because we no more than of the protein in the raft fraction. of proteins and with detergent-resistant membranes are and it is to that cell have separate properties in this using are with of the from membrane rafts containing the actin cytoskeleton is an in to Furthermore, the nature of the actin cytoskeleton to and protein in the membrane to enhance signaling with this we observed an actin-dependent regulation of the Src family kinase by an of phosphorylation of Tyr505 by treating cells with Lat Furthermore, the of Lat B the Tyr505 phosphorylation required CD45 because it was observed in In an study, it was that of raft-associated Lck is to from CD45 W. Rose J.K. J. Cell Biol. PubMed Scopus Google Scholar), the from the raft fraction. this by that the actin cytoskeleton is toward this polymerization and of the cytoskeleton are to many of the that the of the such as cell and cell These often proteins that associate with membrane and this for the of rafts and actin in the membrane such as in of W. Zavzavadjian J. Exp. Cell Res. 2001; 267: 173-183Crossref PubMed Scopus (59) 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). data show that a between the actin and the membrane where are to co-clustering between proteins that in the rafts. studies define the by which this between membrane rafts and the actin cytoskeleton We K. Rodgers for the the fitting and of the for the of the with