title: Cytoskeletal modulation of lipid interactions regulates Lck kinase activity.
abstract: The actin cytoskeleton promotes clustering of proteins associated with cholesterol-dependent rafts, but its effect on lipid interactions that form and maintain rafts is not understood. We addressed this question by determining the effect of disrupting the cytoskeleton on co-clustering of dihexadecyl-(C(16))-anchored DiO and DiI, which co-enrich in ordered lipid environments such as rafts. Co-clustering was assayed by fluorescence resonance energy transfer (FRET) in labeled T cells, where rafts function in the phosphoregulation of the Src family kinase Lck. Our results show that probe co-clustering was sensitive to depolymerization of actin filaments with latrunculin B (Lat B), inhibition of myosin II with blebbistatin, and treatment with neomycin to sequester phosphatidylinositol 4,5-bisphosphate. Cytoskeletal effects on lipid interactions were not restricted to order-preferring label because co-clustering of C(16)-anchored DiO with didodecyl (C(12))-anchored DiI, which favors disordered lipids, was also reduced by Lat B and blebbistatin. Furthermore, conditions that disrupted probe co-clustering resulted in activation of Lck. These data show that the cytoskeleton globally modulates lipid interactions in the plasma membrane, and this property maintains rafts that function in Lck regulation.
authors.affiliation: Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
authors.forename: Gurunadh R
authors.initials: GR
authors.lastname: Chichili
authors.forename: Robert C
authors.initials: RC
authors.lastname: Cail
authors.forename: William
authors.initials: W
authors.lastname: Rodgers
mesh_terms: D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D008055:Lipids; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000378:metabolism*; D009355:Neomycin / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D053778:Thiazolidines / Q000494:pharmacology
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D006576:Heterocyclic Compounds, 4 or More Rings; D008055:Lipids; D019269:Phosphatidylinositol 4,5-Diphosphate; D053778:Thiazolidines; C472645:blebbistatin; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D009355:Neomycin; C037068:latrunculin B; D002118:Calcium