title: 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.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104, USA.
authors.forename: Gurunadh R
authors.initials: GR
authors.lastname: Chichili
authors.forename: William
authors.initials: W
authors.lastname: Rodgers
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002784:Cholesterol / Q000378:metabolism; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D047630:Depsipeptides / Q000494:pharmacology; D005372:Filipin / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000235:genetics / Q000378:metabolism; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000235:genetics / Q000378:metabolism; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D053778:Thiazolidines / Q000494:pharmacology
chemical_list: D000199:Actins; D000900:Anti-Bacterial Agents; D000970:Antineoplastic Agents; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D053778:Thiazolidines; C057531:jasplakinolide; D049452:Green Fluorescent Proteins; D005372:Filipin; D002784:Cholesterol; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; C037068:latrunculin B