Ras1 Acts through Duplicated Cdc42 and Rac Proteins to Regulate Morphogenesis and Pathogenesis in the Human Fungal Pathogen Cryptococcus neoformans

Proliferation and morphogenesis in eukaryotic cells depend on the concerted activity of Rho-type GTPases, including Ras, Cdc42, and Rac. The sexually dimorphic fungus Cryptococcus neoformans, which encodes paralogous, non-essential copies of all three, provides a unique model in which to examine the interactions of these conserved proteins. Previously, we demonstrated that RAS1 mediates C. neoformans virulence by acting as a central regulator of both thermotolerance and mating. We report here that ras1Δ mutants accumulate defects in polarized growth, cytokinesis, and cell cycle progression. We demonstrate that the ras1Δ defects in thermotolerance and mating can be largely explained by the compromised activity of four downstream Rho-GTPases: the Cdc42 paralogs, Cdc42 and Cdc420; and the Rac paralogs, Rac1 and Rac2. Further, we demonstrate that the separate GTPase classes play distinct Ras-dependent roles in C. neoformans morphogenesis and pathogenesis. Cdc42 paralogs primarily control septin localization and cytokinesis, while Rac paralogs play a primary role in polarized cell growth. Together, these duplicate, related signaling proteins provide a robust system to allow microbial proliferation in the presence of host-derived cell stresses.     

Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans

Septins are conserved, cytoskeletal GTPases that contribute to cytokinesis, exocytosis, cell surface organization and vesicle fusion by mechanisms that are poorly understood. Roles of septins in morphogenesis and virulence of a human pathogen and basidiomycetous yeast Cryptococcus neoformans were investigated. In contrast to a well‐established paradigm in S. cerevisiae, Cdc3 and Cdc12 septin homologues are dispensable for growth in C. neoformans yeast cells at 24°C but are essential at 37°C. In a bilateral cross between septin mutants, cells fuse but the resulting hyphae exhibit morphological abnormalities, including lack of properly fused specialized clamp cells and failure to produce spores. Interestingly, post‐mating hyphae of the septin mutants have a defect in nuclear distribution. Thus, septins are essential for the development of spores, clamp cell fusion and also play a specific role in nuclear dynamics in hyphae. In the post‐mating hyphae the septins localize to discrete sites in clamp connections, to the septa and the bases of the initial emerging spores. Strains lacking CDC3 or CDC12 exhibit significantly reduced virulence in a Galleria mellonella model of infection. Thus, C. neoformans septins are vital to morphology of the hyphae and contribute to virulence.     

Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species.     

Borg proteins control septin organization and are negatively regulated by Cdc42

The Cdc42 GTPase binds to numerous effector proteins that control cell polarity, cytoskeletal remodelling and vesicle transport. In many cases the signalling pathways downstream of these effectors are not known. Here we show that the Cdc42 effectors Borg1 to Borg3 bind to septin GTPases. Endogenous septin Cdc10 and Borg3 proteins can be immunoprecipitated together by an anti-Borg3 antibody. The ectopic expression of Borgs disrupts normal septin organization. Cdc42 negatively regulates this effect and inhibits the binding of Borg3 to septins. Borgs are therefore the first known regulators of mammalian septin organization and provide an unexpected link between the septin and Cdc42 GTPases.     

Members of the CIP4 family of proteins participate in the regulation of platelet‐derived growth factor receptor‐β‐dependent actin reorganization and migration

Background information. The F‐BAR {Fes/CIP4 [Cdc42 (cell division cycle 42)‐interacting protein 4] homology and BAR (Bin/amphiphysin/Rvs)} proteins have emerged as important co‐ordinators of signalling pathways that regulate actin assembly and membrane dynamics. The presence of the F‐BAR domain is the hallmark of this family of proteins and the CIP4 (Cdc42‐interacting protein 4) was one of the first identified vertebrate F‐BAR proteins. There are three human CIP4 paralogues, namely CIP4, FBP17 (formin‐binding protein 17) and Toca‐1 (transducer of Cdc42‐dependent actin assembly 1). The CIP4‐like proteins have been implicated in Cdc42‐dependent actin reorganization and in regulation of membrane deformation events visible as tubulation of lipid bilayers. Results. We performed side‐by‐side analyses of the three CIP4 paralogues. We found that the three CIP4‐like proteins vary in their effectiveness to catalyse membrane tubulation and actin reorganization. Moreover, we show that the CIP4‐dependent membrane tubulation is enhanced in the presence of activated Cdc42. Some F‐BAR members have been shown to have a role in the endocytosis of the EGF (epidermal growth factor) receptor and this prompted us to study the involvement of the CIP4‐like proteins in signalling of the PDGFRβ [PDGF (platelet‐derived growth factor) β‐receptor]. We found that knock‐down of CIP4‐like proteins resulted in a prolonged formation of PDGF‐induced dorsal ruffles, as well as an increased PDGF‐dependent cell migration. This was most likely a consequence of a sustained PDGFRβ activation caused by delayed internalization of the receptor in the cells treated with siRNA (small interfering RNA) specific for the CIP4‐like proteins. Conclusions. Our findings show that CIP4‐like proteins induced membrane tubulation downstream of Cdc42 and that they have important roles in PDGF‐dependent actin reorganization and cell migration by regulating internalization and activity of the PDGFRβ. Moreover, the results suggest an important role for the CIP4‐like proteins in the regulation of the activity of the PDGFRβ.     

Cdc24, the GDP-GTP exchange factor for Cdc42, is required for invasive hyphal growth of Candida albicans

Candida albicans, the most common human fungal pathogen, is particularly problematic for immunocompromised individuals. The reversible transition of this fungal pathogen to a filamentous form that invades host tissue is important for its virulence. Although different signaling pathways such as a mitogen-activated protein kinase and a protein kinase A cascade are critical for this morphological transition, the function of polarity establishment proteins in this process has not been determined. We examined the role of four different polarity establishment proteins in C. albicans invasive growth and virulence by using strains in which one copy of each gene was deleted and the other copy expressed behind the regulatable promoter MET3. Strikingly, mutants with ectopic expression of either the Rho G-protein Cdc42 or its exchange factor Cdc24 are unable to form invasive hyphal filaments and germ tubes in response to serum or elevated temperature and yet grow normally as a budding yeast. Furthermore, these mutants are avirulent in a mouse model for systemic infection. This function of the Cdc42 GTPase module is not simply a general feature of polarity establishment proteins. Mutants with ectopic expression of the SH3 domain containing protein Bem1 or the Ras-like G-protein Bud1 can grow in an invasive fashion and are virulent in mice, albeit with reduced efficiency. These results indicate that a specific regulation of Cdc24/Cdc42 activity is required for invasive hyphal growth and suggest that these proteins are required for pathogenicity of C. albicans.     

Immunodetection of Rho-like plant proteins with Rac1 and Cdc42Hs antibodies

A few small GTP-binding proteins of the Ras superfamily have been identified in plants, including members of the Rho family: the proteins belonging to this group are known in mammalian and yeast cells to be involved in the control of polarity, cell morphogenesis and movement by regulating cytoskeleton organization. An investigation into where some Rho-like proteins are located in plant cells was made. The antibodies used, anti-Cdc42Hs and anti-Rac 1, were raised against conserved characteristic sequences of Cdc42Hs and Rac1 mammalian proteins respectively. In fixed cells, Cdc42Hs antibody recognized epitopes generally co-localized with microtubules which may be implicated in the establishment of cell polarity, whereas the proteins recognized by Rac1 antibody seemed to be associated with organelle membranes. The same anti-bodies were used in Western blots of proteins from tobacco BY-2 and lucerne A2 suspension cells: Cdc42Hs antibody recognized three bands whereas Rac1 antibody revealed only one band of 18 kDa Mr. A [³⁵S]GTP overlay revealed four bands of the same Mr as those recognized in Western blots by Cdc42Hs and Rac1 antibodies.Key words: Rho G-proteins, Cdc42, Rac1, Immunofluorescence, plant cells.      

Role of host GTPases in infection by Listeria monocytogenes

The bacterial pathogen Listeria monocytogenes induces internalization into mammalian cells and uses actin‐based motility to spread within tissues. Listeria accomplishes this intracellular life cycle by exploiting or antagonizing several host GTPases. Internalization into human cells is mediated by the bacterial surface proteins InlA or InlB. These two modes of uptake each require a host actin polymerization pathway comprised of the GTPase Rac1, nucleation promotion factors, and the Arp2/3 complex. In addition to Rac1, InlB‐mediated internalization involves inhibition of the GTPase Arf6 and participation of Dynamin and septin family GTPases. After uptake, Listeria is encased in host phagosomes. The bacterial protein GAPDH inactivates the human GTPase Rab5, thereby delaying phagosomal acquisition of antimicrobial properties. After bacterial‐induced destruction of the phagosome, cytosolic Listeria uses the surface protein ActA to stimulate actin‐based motility. The GTPase Dynamin 2 reduces the density of microtubules that would otherwise limit bacterial movement. Cell‐to‐cell spread results when motile Listeria remodel the host plasma membrane into protrusions that are engulfed by neighbouring cells. The human GTPase Cdc42, its activator Tuba, and its effector N‐WASP form a complex with the potential to restrict Listeria protrusions. Bacteria overcome this restriction through two microbial factors that inhibit Cdc42‐GTP or Tuba/N‐WASP interaction.     

Cryptococcus neoformans phospholipase B1 activates host cell Rac1 for traversal across the blood-brain barrier

Cryptococcus neoformans penetration into the central nervous system (CNS) requires traversal of the blood–brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP‐binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood–brain barrier involves host Rac1. While the levels of activated Rac1 (GTP‐Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down‐modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, andSTAT3 was observed to associate with GTP‐Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood–brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host–microbial interaction contributing to traversal of the blood–brain barrier may prevent C. neoformans penetration into the brain.     

A protein interaction map for cell polarity development

Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.     

Regulating polarity by directing traffic: Cdc42 prevents adherens junctions from Crumblin' aPart

The GTPase Cdc42 was among the original genes identified with roles in cell polarity, and interest in its cellular roles from yeast to humans remains high. Cdc42 is a well-known regulator of the actin cytoskeleton, but also plays important roles in vesicular trafficking. In this issue, Harris and Tepass (Harris, K.P, and U. Tepass. 2008. J. Cell. Biol. 183:1129–1143) provide new insights into how Cdc42 and Par proteins work together to modulate cell adhesion and polarity during embryonic morphogenesis by regulating the traffic of key cell junction proteins.     

Listeria monocytogenes antagonizes the human GTPase Cdc42 to promote bacterial spread

The bacterial pathogen Listeria monocytogenes uses actin‐based motility to spread from infected human cells to surrounding healthy cells. Cell–cell spread involves the formation of thin extensions of the host plasma membrane (‘protrusions’) containing motile bacteria. In cultured enterocytes, the Listeria protein InlC promotes protrusion formation by binding and antagonizing the human scaffolding protein Tuba. Tuba is a known activator of the GTPase Cdc42. In this work, we demonstrate an important role for Cdc42 in controlling Listeria spread. Infection of the enterocyte cell line Caco‐2 BBE1 induced a decrease in the level of Cdc42‐GTP, indicating that Listeria downregulates this GTPase. Genetic data involving RNA interference indicated that bacterial impairment of Cdc42 may involve inhibition of Tuba. Experiments with dominant negative and constitutively activated alleles of Cdc42 demonstrated that the ability to inactivate Cdc42 is required for efficient protrusion formation by Listeria. Taken together, these findings indicate a novel mechanism of bacterial spread involving pathogen‐induced downregulation of host Cdc42.     

A novel septin-associated protein, Syp1p, is required for normal cell cycle-dependent septin cytoskeleton dynamics in yeast

Septins are a family of GTP-binding proteins whose heterooligomeric complex is the basic structural element of the septin filaments found in many eukaryotic organisms. In budding yeast, septins are mainly confined at the mother–daughter junction and are required for cell morphogenesis and division. Septins undergo assembly and disassembly in accordance with the progression of the cell cycle. In this report, we identified the yeast protein Syp1p as a new regulator of septin dynamics. Syp1p colocalizes with septins throughout most of the cell cycle. Syp1p interacts with the septin subunit Cdc10p and can be precipitated by Cdc10p and Cdc12p. In the syp1Δ mutant, both formation of a complete septin ring at the incipient bud site and disassembly of the septin ring in later stages of cell division are significantly delayed. In addition, overexpression of Syp1p causes marked acceleration of septin disassembly. The fluorescence recovery after photobleaching (FRAP) assay further showed that Syp1p promotes septin turnover in different cell cycle stages. These results suggest that Syp1p is involved in the regulation of cell cycle-dependent dynamics of the septin cytoskeleton in yeast.     

The germinal centre kinase Don3 triggers the dynamic rearrangement of higher‐order septin structures during cytokinesis in Ustilago maydis

The dimorphic phytopathogenic fungus Ustilago maydis grows in its haploid phase by budding. Cytokinesis and separation of daughter cells are accomplished by the consecutive formation of two distinct septa. Here, we show that both septation events involve the dynamic rearrangement of septin assemblies from hourglass‐shaped collars into ring‐like structures. Using a chemical genetic approach we demonstrate that the germinal centre kinase Don3 triggers this septin reorganization during secondary septum formation. Although chemical inhibition of an analogue‐sensitive version of Don3 prevented septation, a stable septin collar was assembled at the presumptive septation site. Interestingly, the essential light chain of type II myosin, Cdc4, was already associated with this septin collar. Release of Don3 kinase inhibition triggered immediate dispersal of septin filaments and concomitant incorporation of Cdc4 into a contractile actomyosin ring, which also contained the F‐BAR domain protein Cdc15. Inhibition of actin polymerization or deletion of the cdc15 gene, did not affect assembly of the initial collar consisting of septin and myosin light chain. However, reassembly of septin filaments into a ring‐like structure was prevented in the absence of either F‐actin or Cdc15, indicating that septin ring formation in U. maydis depends on a functional contractile actomyosin ring.     

Cdc42 and Vesicle Trafficking in Polarized Cells

Cdc42, a highly conserved small GTPase of the Rho family, acts as a molecular switch to modulate a wide range of signaling pathways. Vesicle trafficking and cell polarity are two processes Cdc42 is known to regulate. Although the trafficking and polarity machineries are each well understood, how they interact to cross‐regulate each other in cell polarization is still a mystery. Cdc42 is an interesting candidate that may integrate these two networks within the cell. Here we review findings on the interplay between Cdc42 and trafficking in yeast, Caenorhabditis elegans, Drosophila and mammalian cell culture systems, and discuss recent advances in our understanding of the function of Cdc42 and two of its effectors, the WASp–Arp2/3 and Par complexes, in regulating polarized traffic. Work in yeast suggests that the polarized distribution of Cdc42, which acts here as a key polarity determinant, requires input from multiple processes including endocytosis and recycling. In metazoan cells, Cdc42 can regulate several steps in the biosynthetic as well as endocytotic and recycling pathways. The recent discovery that the Par polarity complex co‐operates with Cdc42 in the regulation of endocytosis and recycling opens exciting possibilities for the integration of polarity protein function and endocytotic machinery.     

Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae

In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.     

Cdc42 and Par proteins stabilize dynamic adherens junctions in the Drosophila neuroectoderm through regulation of apical endocytosis

Cell rearrangements require dynamic changes in cell–cell contacts to maintain tissue integrity. We investigated the function of Cdc42 in maintaining adherens junctions (AJs) and apical polarity in the Drosophila melanogaster neuroectodermal epithelium. About one third of cells exit the epithelium through ingression and become neuroblasts. Cdc42-compromised embryos lost AJs in the neuroectoderm during neuroblast ingression. In contrast, when neuroblast formation was suppressed, AJs were maintained despite the loss of Cdc42 function. Loss of Cdc42 function caused an increase in the endocytotic uptake of apical proteins, including apical polarity factors such as Crumbs, which are required for AJ stability. In addition, Cdc42 has a second function in regulating endocytotic trafficking, as it is required for the progression of apical cargo from the early to the late endosome. The Par complex acts as an effector for Cdc42 in controlling the endocytosis of apical proteins. This study reveals functional interactions between apical polarity proteins and endocytosis that are critical for stabilizing dynamic basolateral AJs.