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.
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18 February 2002
Article|
February 11 2002
Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold
Samuel Y. Cho,
Samuel Y. Cho
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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Richard L. Klemke
Richard L. Klemke
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
Search for other works by this author on:
Samuel Y. Cho
Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037
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: [email protected]
The online version of this article contains supplemental material.
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Abbreviations used in this paper ECM, extracellular matrix; ERK, extracellular-regulated kinase; FAK, focal adhesion kinase; LPA, lysophosphatidic acid; PH, pleckstrin homology; SD, substrate domain; SH, src-homology.
Received:
November 09 2001
Revision Received:
January 14 2002
Accepted:
January 14 2002
Online ISSN: 1540-8140
Print ISSN: 0021-9525
The Rockefeller University Press
2002
J Cell Biol (2002) 156 (4): 725–736.
Article history
Received:
November 09 2001
Revision Received:
January 14 2002
Accepted:
January 14 2002
Citation
Samuel Y. Cho, Richard L. Klemke; Purification of pseudopodia from polarized cells reveals redistribution and activation of Rac through assembly of a CAS/Crk scaffold . J Cell Biol 18 February 2002; 156 (4): 725–736. doi: https://doi.org/10.1083/jcb.200111032
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