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Published online 11 February 2002. doi:10.1083/jcb.200111032
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© The Rockefeller University Press, 0021-9525/2002/2/725 $5.00
The Journal of Cell Biology, Volume 156, Number 4, February 18, 2002 725-736

Article

Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold



Samuel Y. Cho and Richard L. Klemke

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037

Address correspondence to Richard L. Klemke, Dept. of Immunology, SP231, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: (858) 784-7750. Fax: (858) 784-7785. E-mail: klemke{at}scripps.edu

Initiation of cell migration requires morphological polarization with formation of a dominant leading pseudopodium and rear compartment. A molecular understanding of this process has been limited, due to the inability to biochemically separate the leading pseudopodium from the rear of the cell. Here we examine the spatio-temporal localization and activation of cytoskeletal-associated signals in purified pseudopodia directed to undergo growth or retraction. Pseudopodia growth requires assembly of a p130Crk-associated substrate (CAS)/c-CrkII (Crk) scaffold, which facilitates translocation and activation of Rac1. Interestingly, Rac1 activation then serves as a positive-feedback loop to maintain CAS/Crk coupling and pseudopodia extension. Conversely, disassembly of this molecular scaffold is critical for export and down regulation of Rac1 activity and induction of pseudopodia retraction. Surprisingly, the uncoupling of Crk from CAS during pseudopodium retraction is independent of changes in focal adhesion kinase activity and CAS tyrosine phosphorylation. These findings establish CAS/Crk as an essential scaffold for Rac1-mediated pseudopodia growth and retraction, and illustrate spatio-temporal segregation of cytoskeletal signals during cell polarization.

Key Words: cell migration; chemotaxis; signal transduction; pseudopodium; Rac GTPase


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