Small GTPase Rac promotes hyphal formation and microconidiogenesis in Trichophyton rubrum

Polarity Proteins in Axon Specification and Synaptogenesis

Extracellular guidance cues have a key role in orchestrating cell behaviour. They can take many forms, including soluble and cell-bound ligands (proteins, lipids, peptides or small molecules) and insoluble matrix substrates, but to act as guidance cues, they must be presented to the cell in a spatially restricted manner. Cells that recognize such cues respond by activating intracellular signal transduction pathways in a spatially restricted manner and convert the extracellular information into intracellular polarity. Although extracellular cues influence a broad range of cell polarity decisions, such as mitotic spindle orientation during asymmetric cell division, or the establishment of apical-basal polarity in epithelia, this review will focus specifically on guidance cues that promote cell migration (chemotaxis), or localized cell shape changes (chemotropism).

Scrib Controls Cdc42 Localization and Activity to Promote Cell Polarization during Astrocyte Migration

Binding of integrins to the extracellular matrix results in actin cytoskeletal rearrangements, e.g. during cell spreading, by regulating the activity of Rho GTP-ases. We have shown previously that alphaPIX (Cool-2 or ARHGEF6), a Rac1/Cdc42-specific guanine nucleotide exchange factor (GEF), binds to beta-parvin/affixin and colocalizes with integrin-linked kinase in actively spreading cells, suggesting that alphaPIX is involved in integrin-induced signaling leading to activation of Rac1/Cdc42. Here we report calpain 4, the small subunit of the proteases mu-calpain and m-calpain, as a novel binding partner of alphaPIX. This association was identified by the CytoTrap system and confirmed by coimmunoprecipitation and glutathione S-transferase pull-down assays. The alphaPIX triple domain SH3-DH-PH was found to be required for calpain 4 binding. During integrin-dependent spreading of CHO-K1 cells, alphaPIX colocalized with mu- and m-calpain, integrin-linked kinase, and beta1 integrin in early integrin-containing clusters. Overexpression of alphaPIX wild type but not the GEF-deficient mutant (L386R/L387S) resulted in enhanced formation of characteristic cellular protrusions during cell spreading, suggesting that alphaPIX GEF activity is necessary for this specific actin cytoskeletal reorganization. The calpain inhibitors calpeptin and calpain inhibitor IV significantly inhibited integrin-dependent cell spreading. However, concomitant overexpression of alphaPIX wild type or the L386R/L387S mutant restored cell spreading. Together, these data suggest that alphaPIX is a component of early integrin clusters and plays a dual role in integrin-dependent cell spreading. Whereas alphaPIX GEF activity contributes to enhanced formation of cellular protrusions, the GEF-independent association with calpain 4 leads to induction of a yet unknown signaling cascade resulting in cell spreading.

The PAR-6 Polarity Protein Regulates Dendritic Spine Morphogenesis through p190 RhoGAP and the Rho GTPase

The Cdc42 Guanine Nucleotide Exchange Factor FGD1 Regulates Osteogenesis in Human Mesenchymal Stem Cells

Cdc42 Explores the Cell Periphery for Mate Selection in Fission Yeast

The phytopathogenic fungus Ustilago maydis exists in two stages, the yeast-like haploid form and the filamentous dikaryon. Both pathogenicity and dimorphism are genetically controlled by two mating-type loci, with only the filamentous stage being pathogenic on corn. We have identified two genes (kin1 and kin2) encoding motor proteins of the kinesin family. Kin1 is most similar to the human CENP-E gene product, while Kin2 is most closely related to the conventional kinesin Nkin of Neurospora crassa. Deletion mutants of kin1 had no discernible phenotype; delta kin2 mutants, however, were severely affected in hyphal extension and pathogenicity. The wild-type dikaryon showed rapid tip growth, with all the cytoplasm being moved to the tip compartment. Left behind are septate cell wall tubes devoid of cytoplasm. In delta kin2 mutants, dikaryotic cells were formed after cell fusion, but these hyphal structures remained short and filled with cytoplasm. A functional green fluorescent protein (GFP)-Kin2 fusion was generated and used to determine the localization of the motor protein by fluorescence microscopy. Inspection of the hyphal tips by electron microscopy revealed a characteristic accumulation of darkly stained vesicles which was absent in mutant cells. We suggest that the motor protein Kin2 is involved in organizing this specialized growth zone at the hyphal tip, probably by affecting the vectorial transport of vesicles.

The Rho-related family of GTPases are pivotal regulators of morphogenetic processes in diverse eukaryotic organisms. In the filamentous fungi two related members of this family, Cdc42 and Rac1, perform particularly important roles in the establishment and maintenance of hyphal polarity. The activity of these GTPases is tightly controlled by two sets of regulators: guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Despite the importance of Cdc42 and Rac1 in polarized hyphal growth, the morphogenetic functions of their cognate GEFs and GAPs have not been widely characterized in filamentous fungi outside the Saccharomycotina. Here we present a functional analysis of the Aspergillus nidulans homologs of the yeast GEF Cdc24 and the yeast GAP Rga1. We show that Cdc24 is required for the establishment of hyphal polarity and localizes to hyphal tips. We also show that Rga1 is necessary for the suppression of branching in developing conidiophores. During asexual development Rga1 appears to act primarily via Cdc42 and in doing so serves as a critical determinant of conidiophore architecture. Our results provide new insight into the roles of Cdc42 during development in A. nidulans.

A Rich1/Amot Complex Regulates the Cdc42 GTPase and Apical-Polarity Proteins in Epithelial Cells

Epithelial cell sheets line the organ and body surfaces and the specialized barrier functions of these epithelia regulate the exchange of substances with the outside environment and between different body compartments. Epithelia play a role in a wide range of physiological processes such as digestion, excretion, and leukocyte trafficking. In addition, during development, some epithelia form transient primitive structures, including the neural tube and somites, which are essential for the development of more complex organs. The establishment and maintenance of epithelial cell polarity is critical for the development and functioning of multicellular organisms (Nelson 2000). A multi-step model for the establishment of cell polarity has been proposed by Drubin and Nelson (1996). Cell polarity is initiated by a spatial cue, such as generated by cell–cell contact sites. This cue is interpreted and marked by the formation of signaling complexes that relay the spatial information to the actin cytoskeleton. Localized actin assembly then leads to the formation of a targeting patch, which functions to reinforce the initial cue. Subsequently, this cue can further be propagated via a reorganization of the microtubule cytoskeleton, which in turn causes a redistribution of the membrane trafficking apparatus. In addition to actin cytoskeletal dynamics and vesicle trafficking, epithelial morphogenesis also depends on cell–substrate and cell–cell adhesion. Members of the Rho family of GTPases play essential roles in each of these processes (for reviews, see Hall 1998; Kaibuchi et al. 1999a,b; Braga 2000; Ellis and Mellor 2000; Schwartz and Shattil 2000; Ridley 2001a,b) and therefore it is not surprising to see that Rho GTPases have emerged as critical players at multiple stages of epithelial morphogenesis. In this review we will discuss the involvement of Rho family members in the development and maintenance of epithelial morphology and highlight recent advances in our understanding of the roles of these GTPases in the establishment of epithelial polarity. We will also discuss the participation of these GTPases in epithelial remodeling during wound-healing and epithelial-mesenchymal transitions. As other members of the Ras superfamily, Rho GTPases cycle between a GDP-bound (inactive) state and a GTP-bound (active) state. In the active state, these GTPases relay signals from growth factors, cytokines, and adhesion molecules to regulate a wide range of biological processes, including actin cytoskeleton organization, transcriptional regulation, and vesicle trafficking (Van Aelst and D’Souza-Schorey 1997; Hall 1998). The nucleotide state of Rho family proteins is controlled by three classes of regulatory proteins: guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and guanine nucleotide dissociation inhibitors (GDIs) (Boguski and McCormick 1993). GEFs catalyze the exchange of GDP for GTP by facilitating the release of GDP and transient stabilization of the nucleotide-free protein. GAPs promote the intrinsic GTP hydrolyzing activity of Rho proteins, thereby enhancing their conversion to the GDP-bound form. GDIs preferentially bind to GDP-bound GTPases and prevent spontaneous and GEF-catalyzed release of nucleotide, thereby maintaining the GTPases in the inactive state. Although activation of Rho GTPases in response to extracellular signals in principle could occur either via the activation of GEFs or inhibition of GAPs and GDIs, studies on oncogenic forms of GEFs suggest that nucleotide exchange is the rate-limiting step in GTPase activation. The localized activation of GEFs is likely to be of critical importance in polarity establishment and morphogenesis. Localized control of GEFs and GTPases has been extensively characterized in the budding yeast Saccharomyces cerevisiae. Polarized growth is important at several stages of the budding yeast life cycle, including bud formation during vegetative growth and shmoo formation during mating. Recent genetic and biochemical analyses of the roles of the GTPase Cdc42 and its GEF Cdc24 in these processes has led to a model in which GEF activity is regulated in four distinct steps: GEF recruitment to the plasma membrane and subsequent activation, stabilization by adaptor proteins, and termination of signaling by GEF inactivation (Gulli and Peter 2001). Less is known about the regulation of GEFs in 3Corresponding author. E-MAIL vanaelst@cshl.org; FAX (516) 367-8815. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.978802.

P-Rex1 is a guanine-nucleotide exchange factor (GEF) for the small GTPase Rac. We have investigated here the mechanisms of stimulation of P-Rex1 Rac-GEF activity by the lipid second messenger phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) and the Gbetagamma subunits of heterotrimeric G proteins. We show that a P-Rex1 mutant lacking the PH domain (DeltaPH) cannot be stimulated by PtdIns(3,4,5)P3, which implies that the PH domain confers PtdIns(3,4,5)P3 regulation of P-Rex1 Rac-GEF activity. Consistent with this, we found that PtdIns(3,4,5)P3 binds to the PH domain of P-Rex1 and that the DH/PH domain tandem is sufficient for PtdIns(3,4,5)P3-stimulated P-Rex1 activity. The Rac-GEF activities of the DeltaPH mutant and the DH/PH domain tandem can both be stimulated by Gbetagamma subunits, which infers that Gbetagamma subunits regulate P-Rex1 activity by binding to the catalytic DH domain. Deletion of the DEP, PDZ, or inositol polyphosphate 4-phosphatase homology domains has no major consequences on the abilities of either PtdIns(3,4,5)P3 or Gbetagamma subunits to stimulate P-Rex1 Rac-GEF activity. However, the presence of any of these domains impacts on the levels of basal and/or stimulated P-Rex1 Rac-GEF activity, suggesting that there are important functional interactions between the DH/PH domain tandem and the DEP, PDZ, and inositol polyphosphate 4-phosphatase homology domains of P-Rex1.

PLEKHG2/FLJ00018 is a Gβγ-dependent guanine nucleotide exchange factor for the small GTPases Rac and Cdc42 and has been shown to mediate the signaling pathways leading to actin cytoskeleton reorganization. Here we showed that the zinc finger domain-containing protein four-and-a-half LIM domains 1 (FHL1) acts as a novel interaction partner of PLEKHG2 by the yeast two-hybrid system. Among the isoforms of FHL1 (i.e. FHL1A, FHL1B, and FHL1C), FHL1A and FHL1B interacted with PLEKHG2. We found that there was an FHL1-binding region at amino acids 58-150 of PLEKHG2. The overexpression of FHL1A but not FHL1B enhanced the PLEKHG2-induced serum response element-dependent gene transcription. The co-expression of FHL1A and Gβγ synergistically enhanced the PLEKHG2-induced serum response element-dependent gene transcription. Increased transcription activity was decreased by FHL1A knock-out with the CRISPR/Cas9 system. Compared with PLEKHG2-expressing cells, the number and length of finger-like protrusions were increased in PLEKHG2-, Gβγ-, and FHL1A-expressing cells. Our results provide evidence that FHL1A interacts with PLEKHG2 and regulates cell morphological change through the activity of PLEKHG2.

On the model of the systemic inflammatory response (SIRS), induced by lipopolysaccharide (LPS), the morphological and functional changes in the thymus and spleen and the subpopulation composition of peripheral blood lymphocytes of rats differing in resistance to hypoxia were studied. It was demonstrated that the level of endotoxin in blood serum after 3 hours of LPS administration in susceptible-to-hypoxia rats was 64 times higher than in the control group, while in tolerant-to-hypoxia animals it was only 8 times higher in 6 hours. After 24 hours of LPS injection, only in susceptible-to-hypoxia rats did the level of C-reactive protein in blood serum increase. There is a difference in the dynamics of morphological changes of lymphoid organs after LPS injection in tolerant- and susceptible-to-hypoxia animals. After 3 hours of LPS administration, the tolerant-to-hypoxia rats showed no changes in the thymus, spleen, and subpopulation composition of lymphocytes in peripheral blood. After 6 hours there was only a decrease in B-lymphocytes and increase in cytotoxic T-lymphocytes and NK cells. After 1 day of LPS injection, the tolerant-to-hypoxia rats had devastation in PALS of the spleen. After 3 hours of LPS injection the susceptible-to-hypoxia animals had reactive changes in the lymphoid organs: decrease of the thymus cortex, narrowing of the marginal zones of spleen lymphoid follicles, widening of their germinal centers, and a decrease in the absolute number of cytotoxic T-lymphocytes, NK cells, and B-lymphocytes. After 24 hours of LPS injection the tolerant-to-hypoxia animals had a greater absolute number of T-lymphocytes and NK cells in comparison with the susceptible rats. Thus, in animals with different resistance to hypoxia the LPS-induced SIRS is characterized by different dynamics of morphological and functional changes of the thymus and spleen. The obtained data will serve as a basis for the development of new individual approaches to the prevention and treatment of infectious and inflammatory diseases.

Phenotypic plasticity is common in development. For Candida albicans, the most common cause of invasive fungal infections in humans, morphological plasticity is its defining feature and is critical for its pathogenesis. Unlike other fungal pathogens that exist primarily in either yeast or hyphal forms, C. albicans is able to switch reversibly between yeast and hyphal growth forms in response to environmental cues. Although many regulators have been found involved in hyphal development, the mechanisms of regulating hyphal development and plasticity of dimorphism remain unclear. Here we show that hyphal development involves two sequential regulations of the promoter chromatin of hypha-specific genes. Initiation requires a rapid but temporary disappearance of the Nrg1 transcriptional repressor of hyphal morphogenesis via activation of the cAMP-PKA pathway. Maintenance requires promoter recruitment of Hda1 histone deacetylase under reduced Tor1 (target of rapamycin) signaling. Hda1 deacetylates a subunit of the NuA4 histone acetyltransferase module, leading to eviction of the NuA4 acetyltransferase module and blockage of Nrg1 access to promoters of hypha-specific genes. Promoter recruitment of Hda1 for hyphal maintenance happens only during the period when Nrg1 is gone. The sequential regulation of hyphal development by the activation of the cAMP-PKA pathway and reduced Tor1 signaling provides a molecular mechanism for plasticity of dimorphism and how C. albicans adapts to the varied host environments in pathogenesis. Such temporally linked regulation of promoter chromatin by different signaling pathways provides a unique mechanism for integrating multiple signals during development and cell fate specification.