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. 2009 Jan;20(2):699-707.
doi: 10.1091/mbc.e08-09-0899. Epub 2008 Nov 26.

Dictyostelium Dock180-related RacGEFs regulate the actin cytoskeleton during cell motility

Alessia Para  1 , Miriam Krischke, Sylvain Merlot, Zhouxin Shen, Michael Oberholzer, Susan Lee, Steven Briggs, Richard A Firtel
Affiliations

Dictyostelium Dock180-related RacGEFs regulate the actin cytoskeleton during cell motility

Alessia Para et al. Mol Biol Cell. 2009 Jan.

Abstract

Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P(3) production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved.

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Figures

Figure 1.
Figure 1.
(A) Schematic representation of the Dictyostelium Dock180 protein family. The domain organization of the Homo sapiens Dock180 protein and the Dictyostelium Dock180 protein family is shown. (B) Computer-assisted analysis of chemotaxis (DIAS) of aggregation-competent wild-type (strain Ax2), dckA−, dckD−, and dckA−/D− cells in a cAMP gradient. Speed refers to the speed of the cell's centroid movement along the total path. Roundness is an indication of the polarity of the cells. Directionality is the distance from the start to finish divided by the total distance moved.
Figure 2.
Figure 2.
DockA and DockD roles in random cell movement. (A) Vegetative grown cells were plated in Na/K phosphate buffer and starved on plates for 1 h. Cell movement was monitored by DIC imaging for 30 min at 24-s intervals. Speed and roundness were analyzed with the DIAS program (Soll and Voss, 1998). (a) wild-type, (b) dckA−,(c) dckD−, and dckAA−/D−. (B) Overexpression of either DockA or DockD rescues the motility phenotype of dckA−/D−. (a) DockA in dckA−/D− and DockD in dckA−/D−. (C) Overexpression of GFP-DockD in wild-type cells increases cell motility.
Figure 3.
Figure 3.
Increased levels of DockD produce multiple cell protrusions and trigger F-actin polymerization in vegetative cells and chemotaxing cells. (A) Cell shape of vegetative cells: (a) wild-type; (b) GFP-DockD, (c) detail of a; and (d) detail of b. (B) Phalloidin staining of vegetative (a) wild-type, (b) dckA−/D−, and (c) wild-type cells overexpressing GFP-DockD. (C) (a) GFP localization, (b) phalloidin staining, and (c) overlay of the same cells as in B, panel c. Scale bar, 10 μm.
Figure 4.
Figure 4.
DockD localizes at the leading edge of migrating cells and colocalizes with sites of F-actin polymerization. (A) Time-lapse images of GFP-DockD in aggregation-competent wild-type cells moving toward a micropipette filled with cAMP. An asterisk indicates the position of the micropipette. Scale bar, 10 μm. (B) ABP-GFP in wild-type cells (a) and wild-type/V5-DockD cells (b). Open arrowhead points to the lamellipod.
Figure 5.
Figure 5.
DockD translocation to the membrane is not impaired by inhibition of F-actin polymerization. Wild-type cells overexpressing GFP-DockD without (A) and with LatB (B). Aggregation-competent cells were treated with LatB before being subjected to uniform stimulation with cAMP. Time points are shown in seconds. Scale bar, 10 μm.
Figure 6.
Figure 6.
DockD translocation to the membrane is regulated by PtdIns(3,4,5)P3. (A) Translocation kinetics. Fluorescent images at the times after stimulation are shown. GFP-DockD in wild-type cells (B), pi3k1−/2− cells (C), pi3k1−/2−/3− cells (D), wild-type cells treated for 1 h with 30 μM LY294002 (E), and pten− cells (F) after uniform stimulation with cAMP. Scale bar, 10 μm. See Materials and Methods for description of data analysis.
Figure 7.
Figure 7.
DdELMO1 is associated with the DockD complex. DockD-associated proteins were coimmunoprecipitated from V5-DockD aggregation-competent cells at 0, 10, and 60 s after stimulation with cAMP, and wild-type Ax2 cells were used as a control. The purified proteins were separated on an acrylamide gel and detected by silver nitrate staining. The upper portion of the gel contains the DockD band (upper arrow) and a band at 109 kDa (lower arrow) that corresponds to DdELMO1.
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