title: Cytoskeletal regulation of CD44 membrane organization and interactions with E-selectin.
abstract: Interactions of CD44 on neutrophils with E-selectin on activated endothelial cells mediate rolling under flow, a prerequisite for neutrophil arrest and migration into perivascular tissues. How CD44 functions as a rolling ligand despite its weak affinity for E-selectin is unknown. We examined the nanometer scale organization of CD44 on intact cells. CD44 on leukocytes and transfected K562 cells was cross-linked within a 1.14-nm spacer. Depolymerizing actin with latrunculin B reduced cross-linking. Fluorescence resonance energy transfer (FRET) revealed tight co-clustering between CD44 fused to yellow fluorescent protein (YFP) and CD44 fused to cyan fluorescent protein on K562 cells. Latrunculin B reduced FRET-reported co-clustering. Number and brightness analysis confirmed actin-dependent CD44-YFP clusters on living cells. CD44 lacking binding sites for ankyrin and for ezrin/radixin/moesin (ERM) proteins on its cytoplasmic domain (ΔANKΔERM) did not cluster. Unexpectedly, CD44 lacking only the ankyrin-binding site (ΔANK) formed larger but looser clusters. Fluorescence recovery after photobleaching demonstrated increased CD44 mobility by latrunculin B treatment or by deleting the cytoplasmic domain. ΔANKΔERM mobility increased only modestly, suggesting that the cytoplasmic domain engages the cytoskeleton by an additional mechanism. Ex vivo differentiated CD44-deficient neutrophils expressing exogenous CD44 rolled on E-selectin and activated Src kinases after binding anti-CD44 antibody. In contrast, differentiated neutrophils expressing ΔANK had impaired rolling and kinase activation. These data demonstrate that spectrin and actin networks regulate CD44 clustering and suggest that ankyrin enhances CD44-mediated neutrophil rolling and signaling.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: From the Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation and.
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Yago
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Biomedical Engineering, University of Texas, Arlington, Texas 76010.
authors.forename: Salim
authors.initials: S
authors.lastname: Abdisalaam
authors.affiliation: Department of Biomedical Engineering, University of Texas, Arlington, Texas 76010.
authors.forename: George
authors.initials: G
authors.lastname: Alexandrakis
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and.
authors.forename: William
authors.initials: W
authors.lastname: Rodgers
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104 and From the Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation and rodger-mcever@omrf.org.
authors.forename: Rodger P
authors.initials: RP
authors.lastname: McEver
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D017487:Ankyrins / Q000235:genetics / Q000378:metabolism; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism; D019040:E-Selectin / Q000235:genetics / Q000378:metabolism*; D005260:Female; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D036904:Leukocyte Rolling / Q000187:drug effects; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D053778:Thiazolidines / Q000494:pharmacology; D000099149:Ezrin
chemical_list: D017487:Ankyrins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003598:Cytoskeletal Proteins; D019040:E-Selectin; D018960:Hyaluronan Receptors; D008164:Luminescent Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D053778:Thiazolidines; D000099149:Ezrin; C476399:Cd44 protein, mouse; C071058:moesin; C071776:radixin; C037068:latrunculin B
keywords: Cell Adhesion; Cytoskeleton; Fluorescence Recovery after Photobleaching (FRAP); Fluorescence Resonance Energy Transfer (FRET); Neutrophil; Signal Transduction