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Vol. 13, Issue 12, 4231-4242, December 2002
and
*Department of Structural Analysis, National Cardiovascular Center
Research Institute, Suita, Osaka 565-8565, Japan;
Endothelial cell migration is an essential step in vasculogenesis
and angiogenesis, in which receptor tyrosine kinases play a pivotal
role. We investigated the mechanism by which ephrin-B1 promotes
membrane ruffling in human aortic endothelial cells, because
membrane ruffling heralds cell body migration. We especially focused on
the role of Crk adaptor protein in EphB-mediated signaling. Using
DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed
that Crk was recruited to the nascent focal complex after ephrin-B1
stimulation. Furthermore, we found that p130Cas, but not
paxillin, recruited Crk to the nascent focal complex. The necessity of
Crk in ephrin-B1-induced membrane ruffling was shown both by the
overexpression of dominant negative Crk mutants and by the depletion of
Crk by using RNA interference. Then, we examined the role of two major
downstream molecules of Crk, Rac1 and Rap1. The dominant negative
mutant of Rac1 completely inhibited ephrin-B1-induced membrane
ruffling and focal complex assembly. In contrast, rap1GAPII, a negative
regulator of Rap1, did not inhibit ephrin-B1-induced membrane
ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not
induce membrane spreading, probably due to instability of the focal
complex. These results indicated that Crk plays a critical role in
Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex
stabilization contributing to ephrin-B1-induced human aortic
endothelial cells migration.
Department of Cardiovascular Medicine, Hokkaido
University School of Medicine, Sapporo 060-6833, Japan; and
Department of Tumor Virology, Research Institute for
Microbial Diseases, Osaka University, Osaka 565-0871, Japan
Online
version of this article contains video material for some figures.
Online version available at www.molbiolcell.org.
§
Corresponding author. E-mail address:
nmochizu{at}ri.ncvc.go.jp.
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