Leukocyte adhesion to vascular endothelium induces E-selectin linkage to the actin cytoskeleton.

作者：Masayuki Yoshida, William Westlin, Ningning Wang, Donald E. Ingber, Anthony Rosenzweig, Neil M. Resnick, Michael A. Gimbrone · 发表于：The Journal of Cell Biology · 年份：1996 · DOI：10.1083/jcb.133.2.445 · 被引用次数：211 · 研究领域：Cell Adhesion Molecules Research、Angiogenesis and VEGF in Cancer、Cellular Mechanics and Interactions

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 appl...