XB-ART-6203Methods Mol Med. November 1, 2002; 13 (11): 4045-59.
Motility determinants in WASP family proteins.
In response to upstream signals, proteins in the Wiskott-Aldrich Syndrome protein (WASP) family regulate actin nucleation via the Arp2/3 complex. Despite intensive study of the function of WASP family proteins in nucleation, it is not yet understood how their distinct structural organization contributes to actin-based motility. Herein, we analyzed the activities of WASP and Scar1 truncation derivatives by using a bead-based motility assay. The minimal region of WASP sufficient to direct movement was the C-terminal WCA fragment, whereas the corresponding region of Scar1 was insufficient. In addition, the proline-rich regions of WASP and Scar1 and the Ena/VASP homology 1 (EVH1) domain of WASP independently enhanced motility rates. The contributions of these regions to motility could not be accounted for by their direct effects on actin nucleation with the Arp2/3 complex, suggesting that they stimulate motility by recruiting additional factors. We have identified profilin as one such factor. WASP- and Scar1-coated bead motility rates were significantly reduced by depletion of profilin and VASP and could be more efficiently rescued by a combination of VASP and wild-type profilin than by VASP and a mutant profilin that cannot bind proline-rich sequences. Moreover, motility of WASP WCA beads was not affected by the depletion or addback of VASP and profilin. Our results suggest that recruitment of factors, including profilin, by the proline-rich regions of WASP and Scar1 and the EVH1 domain of WASP stimulates cellular actin-based motility.
PubMed ID: 12429845
PMC ID: PMC133613
Article link: Methods Mol Med.
Grant support: GM-59609 NIGMS NIH HHS , R01 GM059609-04 NIGMS NIH HHS , R01 GM059609 NIGMS NIH HHS , R01 GM059609 NIGMS NIH HHS , R01 GM059609-04 NIGMS NIH HHS , GM-59609 NIGMS NIH HHS
Genes referenced: actl6a aicda enah pfn1 vasp was wasf1