Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton.
We have examined functions of the cytoplasmic domain of E-selectin, an inducible endothelial transmembrane protein, especially its ability to associate with the cytoskeleton during leukocyte adhesion. Confocal microscopy of interleukin-1 beta (IL-1 beta)-activated human umbilical vein endothelial cells (HUVEC) visualized clustering of E-selectin molecules in the vicinity of leukocyte-endothelial cell attachment sites. A detergent based extraction and Western blotting procedure demonstrated an association of E-selectin with the insoluble (cytoskeletal) fraction of endothelial monolayers that correlated with adhesion of leukocytes via an E-selectin-dependent mechanism. A mutant form of E-selectin lacking the cytoplasmic domain (tailless E-selectin) was expressed in COS-7 cell and supported leukocyte attachment (in a nonstatic adhesion assay) in a fashion similar to the native E-selectin molecule, but failed to become associated with the cytoskeletal fraction. To identify the cytoskeletal components that associate with the cytoplasmic domain of E-selectin, paramagnetic beads coated with the adhesion-blocking anti-E-selectin monoclonal antibody H18/7 were incubated with IL-1 beta-activated HUVEC, and then subjected to detergent extraction and magnetic separation. Certain actin-associated proteins, including alpha-actinin, vinculin, filamin, paxillin, as well as focal adhesion kinase (FAK), were copurified by this procedure, however talin was not. When a mechanical stress was applied to H18/7-coated ferromagnetic beads bound to the surface of IL-1 beta-activated HUVEC, using a magnetical twisting cytometer, the observed resistance to the applied stress was inhibited by cytochalasin D, thus demonstrating transmembrane cytoskeletal mechanical linkage. COS-7 cells transfected with the tailless E-selectin failed to show resistance to the twisting stress. Taken together, these data indicate that leukocyte adhesion to cytokine-activated HUVEC induces transmembrane cytoskeletal linkage of E-selectin through its cytoplasmic domain, a process which may have important implications for cell-cell signaling as well as mechanical anchoring during leukocyte-endothelial adhesive interactions.
Yoshida
M
M
Department of Pathology, Brigham and Women's Hospital, Boston, MA 02115, USA.
Westlin
W F
WF
Wang
N
N
Ingber
D E
DE
Rosenzweig
A
A
Resnick
N
N
Gimbrone
M A
MA
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D019040:E-Selectin / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D018922:HL-60 Cells; D006801:Humans; D018189:Immunomagnetic Separation; D007375:Interleukin-1 / Q000494:pharmacology; D007962:Leukocytes / Q000166:cytology*; D008969:Molecular Sequence Data; D014471:Umbilical Veins / Q000166:cytology
D000199:Actins; D003598:Cytoskeletal Proteins; D019040:E-Selectin; D007375:Interleukin-1