Visualization and Molecular Analysis of Actin Assembly in Living Cells
Dorothy A. Schafer, Matthew D. Welch*, Laura M. Machesky**, Paul C. Bridgman***, Shelley M. Meyer and John A. Cooper
Department of Cell Biology and Physiology, ***Department of Neurobiology and Anatomy, Washington University School of Medicine, St. Louis, MO, *Department of Molecular and Cellular Biology, University of California, Berkeley, CA, and **School of Biochemistry, University of Birmingham, Edgbaston, Birmington B15 2TT, UK.
J. Cell Biol. 143: 1919-1930 (1998). Online Journal.
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 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.
Movies Related to Figures
Figure 1. The distributions of GFP-CP (panels A, C, D and F) and GFP-Arp3 (panel B) in living fibroblasts change dramatically over time. The distribution of GFP (panel E) is diffuse, as expected.
A. GFP-CP (4 MB) |
B. GFP-Arp3 (7 MB) |
C. GFP-CP (5 MB) |
D. GFP-CP (5 MB) |
E. GFP (8 MB) |
F. GFP-CP (3 MB) |
Figure 3. Spots were reduced in number and spot motility was suppressed in migrating cells. Confluent monolayers of PtK1 fibroblasts expressing GFP-Arp3 (panel A) or GFP-CP (panel B) were "wounded" to obtain a clear are on the coverslip.
Figure 5. Actin polymerization is required for formation and movement of spots and for movements at the cell periphery. Fibroblasts expressing GFP-CP were filmed within 2 min after treatment with Cytochalasin or Latrunculin.
Figure 8. Small GTPases regulate spot formation and motility at the cell periphery. GFP-Arp3 fibroblasts were microinjected with plasmids for expression of constitutively active (V12) or dominant negative (N17) forms of rac1 and cdc42. Other cells were microinjected with an active form of RhoA (V14RhoA) or C3 toxin.
A. V12rac1 (6 MB) |
B. N17rac1 (5 MB) |
C. V12cdc42 (4 MB) |
D. N17cdc42 (5 MB) |
E. V14RhoA (7 MB) |
F. C3 Toxin (5 MB) |
Figure 9. Expression of PI 5-kinase promotes the movement of spots.
Figure 10. Expression of LIMK-1, which inactivates cofilin, results in decreased motility of spots and of the cell periphery. Nuclei were injected with DNA for expression of LIMK-1 (A) or a mutant form of LIMK-1 (B) lacking a portion of the catalytic kinase domain.
