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Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin. [ABSTRACT FROM AUTHOR]

The WD-repeat protein factor associated with nSMase activity (FAN) is a member of the family of TNF receptor adaptor proteins that are coupled to specific signaling cascades. However, the precise functional involvement of FAN in specific cellular TNF responses remain unclear. Here, we report the involvement of FAN in TNF-induced actin reorganization and filopodia formation mediated by activation of Cdc42. The pleckstrin-homology (PH) domain of FAN specifically binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P), which targets FAN to the plasma membrane. Site-specific mutagenesis revealed that the ability of FAN to mediate filopodia formation was blunted either by the destruction of the PtdIns(4,5)P binding motif, or by the disruption of intramolecular interactions between the PH domain and the adjacent beige and Chediak-Higashi (BEACH) domain. Furthermore, FAN was shown to interact with the actin cytoskeleton in TNF-stimulated cells via direct filamentous actin (F-actin) binding. The results of this study suggest that PH-mediated plasma membrane targeting of FAN is critically involved in TNF-induced Cdc42 activation and cytoskeleton reorganization. [ABSTRACT FROM AUTHOR]

Background: Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor. Results: We report on GxcDD (Guanine exchange factor for Rac GTPases), a Dictyostelium RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling. Conclusion: The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in Dictyostelium, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further. [ABSTRACT FROM AUTHOR]

Dictyostelium RacH localizes predominantly to membranes of the nuclear envelope, endoplasmic recticulum and Golgi apparatus. To investigate the role of this protein, we generated knockout and overexpressor strains. RacH-deficient cells displayed 50% reduced fluid-phase uptake and a moderate exocytosis defect, but phagocytosis was unaffected. Detailed examination of the endocytic pathway revealed defective acidification of early endosomes and reduced secretion of acid phosphatase in the presence of sucrose. The distribution of the postlysosomal marker vacuolin was altered, with a high proportion of cells showing a diffuse vesicular pattern in contrast to the wild-type strain, where few intensely stained vacuoles predominate. Cytokinesis, cell motility, chemotaxis and development appeared largely unaffected. In a cell-free system, RacH stimulates actin polymerization, suggesting that this protein is involved in actin-based trafficking of vesicular compartments. We also investigated the determinants of subcellular localization of RacH by expression of green-fluorescent-protein -tagged chimeras in which the C-terminus of RacH and the plasma-membrane-targeted RacG were exchanged, the insert region was deleted or the net positive charge of the hypervariable region was increased. We show that several regions of the molecule, not only the hypervariable region, determine targeting of RacH. Overexpression of mistargeted RacH mutants did not recapitulate the phenotypes of a strain overexpressing nonmutated RacH, indicating that the function of this protein is in great part related to its subcellular localization. [ABSTRACT FROM AUTHOR]

Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H+-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium. [ABSTRACT FROM AUTHOR]

Chemotaxis requires localized F-actin polymerization at the site of the plasma membrane closest to the chemoattractant source, a process controlled by Rac/Cdc42 GTPases. We identify Dictyostelium RacB as an essential mediator of this process. RacB is activated upon chemoattractant stimulation, exhibiting biphasic kinetics paralleling F-actin polymerization. racB null cells have strong chemotaxis and morphogenesis defects and a severely reduced chemoattractant-mediated F-actin polymerization and PAKc activation. RacB activation is partly controlled by the PI3K pathway. pi3k½ null cells and wild-type cells treated with LY294002 exhibit a significantly reduced second peak of RacB activation, which is linked to pseudopod extension, whereas a PTEN hypomorph exhibits elevated RacB activation. We identify a RacGEF, RacGEF1, which has specificity for RacB in vitro. racgef1 null cells exhibit reduced RacB activation and cells expressing mutant RacGEF1 proteins display chemotaxis and morphogenesis defects. RacGEF1 localizes to sites of F-actin polymerization. Inhibition of this localization reduces RacB activation, suggesting a feedback loop from RacB via F-actin polymerization to RacGEF1. Our findings provide a critical linkage between chemoattractant stimulation, F-actin polymerization, and chemotaxis in Dictyostelium. [ABSTRACT FROM AUTHOR]

Wound repair of cell membranes is essential for cell survival. Myosin II contributes to wound pore closure by interacting with actin filaments in larger cells; however, its role in smaller cells is unclear. In this study, we observed wound repair in dividing cells for the first time. The cell membrane in the cleavage furrow, where myosin II localized, was wounded by laserporation. Upon wounding, actin transiently accumulated, and myosin II transiently disappeared from the wound site. Ca2+ influx from the external medium triggered both actin and myosin II dynamics. Inhibition of calmodulin reduced both actin and myosin II dynamics. The wound closure time in myosin II-null cells was the same as that in wild-type cells, suggesting that myosin II is not essential for wound repair. We also found that disassembly of myosin II filaments by phosphorylation did not contribute to their disappearance, indicating a novel mechanism for myosin II delocalization from the cortex. Furthermore, we observed that several furrow-localizing proteins such as GAPA, PakA, myosin heavy chain kinase C, PTEN, and dynamin disappeared upon wounding. Herein, we discuss the possible mechanisms of myosin dynamics during wound repair. [ABSTRACT FROM AUTHOR]

Remodeling of the actin cytoskeleton is one of the critical events that allows platelets to undergo morphological and functional changes in response to receptor-mediated signaling cascades. Coronins are a family of evolutionarily conserved proteins implicated in the regulation of the actin cytoskeleton, represented by the abundant coronins 1, 2, and 3 and the less abundant coronin 7 in platelets, but their functions in these cells are poorly understood. A recent report revealed impaired agonist-induced actin polymerization and cofilin phosphoregulation and altered thrombus formation in vivo as salient phenotypes in the absence of an overt hemostasis defect in vivo in a knockout mouse model of coronin 1. Here we show that the absence of coronin 1 is associated with impaired translocation of integrin β2 to the platelet surface upon stimulation with thrombin while morphological and functional alterations, including defects in Arp2/3 complex localization and cAMP-dependent signaling, are absent. Our results suggest a large extent of functional overlap among coronins 1, 2, and 3 in platelets, while aspects like integrin β2 translocation are specifically or predominantly dependent on coronin 1. [ABSTRACT FROM AUTHOR]

WASP family proteins are key players for connecting multiple signaling pathways to F-actin polymerization. To dissect the highly integrated signaling pathways controlling WASP activity, we identified a Rac protein that binds to the GTPase binding domain of WASP. Using two-hybrid and FRET-based functional assays, we identified RacC as a major regulator of WASP. RacC stimulates F-actin assembly in cell-free systems in a WASP-dependent manner. A FRET-based microscopy approach showed local activation of RacC at the leading edge of chemotaxing cells. Cells overexpressing RacC exhibit a significant increase in the level of F-actin polymerization upon cAMP stimulation, which can be blocked by a phosphatidylinositol (PI) 3-kinase inhibitor. Membrane translocation of PI 3-kinase and PI 3,4,5-trisphosphate reporter is absent in racC null cells. Cells overexpressing dominant negative RacC mutants and racC null cells move at a significantly slower speed and show a poor directionality during chemotaxis. Our results suggest that RacC plays an important role in PI 3-kinase activation and WASP activation for dynamic regulation of F-actin assembly during Dictyostelium chemotaxis. [ABSTRACT FROM AUTHOR]

With their essential role in inner ear function, stereocilia of sensory hair cells demonstrate the importance of cellular actin protrusions. Actin packing in stereocilia is mediated by cross-linkers of the plastin, fascin, and espin families. Although mice lacking espin (ESPN) have no vestibular or auditory function, we found that mice that either lacked plastin 1 (PLS1) or had nonfunctional fascin 2 (FSCN2) had reduced inner ear function, with double-mutant mice most strongly affected. Targeted mass spectrometry indicated that PLS1 was the most abundant cross-linker in vestibular stereocilia and the second most abundant protein overall; ESPN only accounted for ~15% of the total cross-linkers in bundles. Mouse utricle stereocilia lacking PLS1 were shorter and thinner than wild-type stereocilia. Surprisingly, although wild-type stereocilia had random liquid packing of their actin filaments, stereocilia lacking PLS1 had orderly hexagonal packing. Although all three cross-linkers are required for stereocilia structure and function, PLS1 biases actin toward liquid packing, which allows stereocilia to grow to a greater diameter. [ABSTRACT FROM AUTHOR]

Ras guanine nucleotide exchange factor (GEF) Q, a nucleotide exchange factor from Dictyostelium discoideum, is a 143-kD protein containing RasGEF domains and a DEP domain. We show that RasGEF Q can bind to F-actin, has the potential to form complexes with myosin heavy chain kinase (MHCK) A that contain active RasB, and is the predominant exchange factor for RasB. Overexpression of the RasGEF Q GEF domain activates RasB, causes enhanced recruitment of MHCK A to the cortex, and leads to cytokinesis defects in suspension, phenocopying cells expressing constitutively active RasB, and myosin-null mutants. RasGEF Q[sup -] mutants have defects in cell sorting and slug migration during later stages of development, in addition to cell polarity defects. Furthermore, RasGEF Q[sup -] mutants have increased levels of unphosphorylated myosin II, resulting in myosin II overassembly. Collectively, our results suggest that starvation signals through RasGEF Q to activate RasB, which then regulates processes requiring myosin II. [ABSTRACT FROM AUTHOR]