Visualization and molecular analysis of actin assembly in living cells

Schafer, D. A., Welch, M. D., Machesky, L. M. , Bridgman, P. C., Meyer, S. M. and Cooper, J. A. (1998) Visualization and molecular analysis of actin assembly in living cells. Journal of Cell Biology, 143(7), pp. 1919-1930. (doi: 10.1083/jcb.143.7.1919) (PMID:9864364) (PMCID:PMC2175235)

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Abstract

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Machesky, Professor Laura
Authors: Schafer, D. A., Welch, M. D., Machesky, L. M., Bridgman, P. C., Meyer, S. M., and Cooper, J. A.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
Journal Name:Journal of Cell Biology
Publisher:Rockefeller University Press
ISSN:0021-9525
ISSN (Online):1540-8140

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