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. 2000 May 9;97(10):5225-30.
doi: 10.1073/pnas.97.10.5225.

Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells

C Y Chung  1 , S Lee, C Briscoe, C Ellsworth, R A Firtel
Affiliations

Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells

C Y Chung et al. Proc Natl Acad Sci U S A. .

Abstract

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B(61L) or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B(61L) and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B(17N) do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.

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Figures

Figure 1
Figure 1
DdRac1B activity controls the assembly and organization of the actin cytoskeleton of aggregation-stage cells and induction of gene expression. F-actin organization in cells lacking DdRacGAP1 or expressing constitutively active or dominant-negative mutants of DdRac1B was examined by FITC- or tetramethylrhodamine B isothiocyanate-labeled phalloidin staining. (A) Cells starved for 5 h. Wild-type cells have F-actin-rich lamellipodia at the leading edge and mutants have aberrant F-actin organization. (Bar = 5 μm.) (B) Cells pulsed for 5 h with 30 nM cAMP every 6 min (see legend to Fig. 3) and then either directly fixed or placed in a cAMP gradient established from a micropipette (see Fig. 3). Arrows show the direction of the micropipette containing cAMP and cell movement. (C) Expression of cAR1 protein and GBF transcripts in wild-type (KAx-3) cells and cells expressing DdRac1B17N. For cAR1 expression, cells were pulsed for 5 h as described in Materials and Methods, the same conditions used for the chemotaxis assays. Cell lysates were run on a 10% SDS/PAGE gel and proteins were transferred to a membrane and blotted with anti-cAR1 polyclonal antibody, a gift of the Devreotes laboratory (Johns Hopkins University School of Medicine, Baltimore, MD). To examine GBF expression, total RNA was prepared from wild-type cells and cells expressing DdRac1B17N at different stages of development on non-nutrient agar plates. RNA (6 μg) was loaded in each lane and probed with the GBF probe. t = 0 h, vegetative cells.
Figure 2
Figure 2
F-actin polymerization responses on cAMP stimulation of Dictyostelium mutants. The F-actin content was determined by tetramethylrhodamine B isothiocyanate-phalloidin staining of cells fixed at various times after stimulation with 50 μM cAMP (see Materials and Methods). The amount of F-actin was normalized relative to the F-actin level of unstimulated wild-type cells (t = 0). Each data point represents the average of two or three independent measurements. Error bars are shown only at 0 and 10 s to avoid complexity.
Figure 3
Figure 3
Chemotactic movement of wild-type and mutant cells to a micropipette containing cAMP. Cells were pulsed with 30 nM cAMP at 6-min intervals for 5 h and cells were washed and resuspended in Na/KPO4 buffer containing 200 μM CaCl2 and MgCl2. A small volume of cells were plated on glass-bottomed microwell plates (MarTek, Ashland, MA) and allowed to adhere to the surface for approximately 20 min. A micropipette filled with 100 μm cAMP was positioned and images of chemotaxing cells were captured every 6 s. The movement of cells and changes in cell shape were analyzed with the dias program, a newly developed image analysis system. Superimposed images representing cell shape at 1-min intervals are shown. Movement of cells during chemotaxis was traced and is presented in boxes. Wild-type cells are very polarized, their migration is rapid and directed toward the tip of the micropipette, and the vast majority of pseudopodia are extended only in the direction of the micropipette. The majority of Ddracgap1 null cells move to the cAMP source, but they make many turns and lateral pseudopodia. Cells expressing DdRac1B61L show chemotactic defects similar to those of Ddracgap1 null cells. Cell migration is severely impaired in cells expressing DdRac1B17N. The star indicates the position of the cAMP source.
Figure 4
Figure 4
Images at a 1-min interval of migrating wild-type and Ddracgap1 null cells. Regions of the cell in the earlier picture that are not present in the later picture are shown in red. Regions of the cell in the later picture that are not present in the earlier picture are shown in green.
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