DictyosteliumDock180-related RacGEFs Regulate the Actin Cytoskeleton during Cell Motility

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. INTRODUCTIONIn eukaryotes, cell migration is essential for many biological processes such as embryonic development and tissue renewal and in humans it is also linked to numerous pathologies including cancer. Cells are set in motion through reorganization of the actin cytoskeleton that creates cytoplasmic protrusions and promotes forward movement. Members of the Rac subfamily of Rho small GTPases are key regulators of cytoskeletal dynamics in virtually all eukaryotes. Activated Rac proteins relay directional signals from the leading edge of migrating cells to downstream effectors such as SCAR/WAVE and WASP proteins that mediate F-actin polymerization and power cell motility (Jaffe and Hall, 2005;Ridley, 2006;Charest and Firtel, 2007;Kolsch et al., 2008;Ladwein and Rottner, 2008). Despite their importance, the upstream signaling mechanisms that mediate Rac activation during cell migration are still unclear.The activity of the small GTPases is regulated through a GDP/GTP exchange mechanism that is catalyzed by the guanine nucleotide exchange factors (GEF), GTPase-activating proteins (GAPs), and guanine nucleotide-dissociation inhibitors (GDIs). GEFs activate GTPases by catalyzing the dissociation of GDP from the protein, thereby allowing the binding of GTP and their switch to an active state. Members of the classical Dbl homology-pleckstrin homology (DH-PH) domain-containing family were thought to be the universal activators of Rho GTPases until the CZH (CDM-zizimin homology) family of unconventional Rho-GEFs was discovered (Côté and Vuori, 2002;Meller et al., 2005;Côté and Vuori, 2007). In the CZH proteins, a CZH2 domain, rather than a DH domain, interacts wi...

ZizB, a novel RacGEF regulates development, cell motility and cytokinesis in Dictyostelium.

“…The lack of developmental changes following ZizA loss does not preclude an important role for the protein in development because the cellular roles for the protein might be, at least partially, compensated for by the other zizimin proteins. This functional redundancy has been shown in other small GTPase-related signalling families of D. discoideum Doc proteins (Para et al, 2009), RacB and Rac GEF1 (Park et al, 2004) and Trix Rac GEF (Strehle et al, 2006). However, ablation of ZizB did perturb development, suggesting a crucial role that cannot be replaced by related proteins, or that the relatively low expression levels of ZizA, ZizC and ZizD cannot effectively complement loss of ZizB.…”

“…The D. discoideum Dock180-related proteins, DocA and DocD regulate the actin cytoskeleton and cell motility (Para et al, 2009). In our study, we explore the role of the highest expressing D. discoideum zizimins, ZizA and ZizB and show both novel and conserved functions of these members of the ancient zizimin protein family.…”

“…These small G proteins are involved in the regulation of many processes within the cell, such as cytoskeletal organisation, cytokinesis, cell morphogenesis, cell migration and development (Dumontier et al, 2000;Jaffe and Hall, 2005;Kolsch et al, 2008;Mondal et al, 2007;Para et al, 2009). The activity of these small GTPases is regulated by the exchange of GDP and GTP, catalysed by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine nucleotide dissociation inhibitors (GDIs) (Jaffe and Hall, 2005).…”

“…The Dock proteins are composed of two main domains, the Dock-homology region 1 (DHR1) and the Dock-homology region 2 (DHR2) domains. The DHR1 domain has been shown to bind phospholipids (Côté et al, 2005;Kobayashi et al, 2001;Para et al, 2009). The DHR2 domain interacts with the target GTPase and is responsible for the GEF activity (Brugnera et al, 2002;Côté and Vuori, 2002;Côté and Vuori, 2006).…”

“…5A,C,D). The D. discoideum Dock180-related Dock A and Dock D proteins have been previously shown to rapidly translocate to the cell cortex upon global stimulation with cAMP (Para et al, 2009). We investigated whether the D. discoideum zizimin proteins are also involved in the chemotactic signalling response.…”