The Ras and Rho GTPases genetically interact to co-ordinately regulate cell polarity during development in Penicillium marneffei

Ras and Rho GTPases have been examined in a wide variety of eukaryotes and play varied and often overlapping roles in cell polarization and development. Studies in Saccharomyces cerevisiae and mammalian cells have defined some of the central activities of these GTPases. However, these paradigms do not explain the role of these proteins in all eukaryotes. Unlike yeast, but like more complex eukaryotes, filamentous fungi have Rac-like proteins in addition to Ras and Cdc42. To investigate the unique functions of these proteins and determine how they interact to co-ordinately regulate morphogenesis during growth and development we undertook a genetic analysis of GTPase function by generating double mutants of the Rho GTPases cflA and cflB and the newly isolated Ras GTPase rasA from the dimorphic pathogenic fungus, Penicillium marneffei. P. marneffei growth at 25 degrees C is as multinucleate, septate, branched hyphae which are capable of undergoing asexual development (conidiation), while at 37 degrees C, uninucleate pathogenic yeast cells which divide by fission are produced. Here we show that RasA (Ras) acts upstream of CflA (Cdc42) to regulate germination of spores and polarized growth of both hyphal and yeast cells, while also exhibiting CflA-independent activities. CflA (Cdc42) and CflB (Rac) co-ordinately control hyphal cell polarization despite also having unique roles in regulating conidial germination and polarized growth of yeast cells (CflA) and polarized growth of conidiophore cell types and hyphal branching (CflB).     

Kinase suppressor of Ras couples Ras to the ERK cascade during T cell development

Ras signaling is critical for many developmental processes and requires the precise coordination of interactions among multiple downstream components. One mechanism by which this regulation is achieved is through the use of scaffolding molecules that coordinate the assembly of multimolecular complexes. Recently, the scaffolding molecule kinase suppressor of Ras (KSR) was isolated in genetic screens as a modifier of Ras signaling, although its contribution to regulating Ras-mediated activation of its different downstream effectors is not well understood. We have analyzed the role of KSR in linking Ras to the ERK cascade during positive selection. Our results demonstrate that KSR overexpression interferes with T cell development, an effect that requires the direct interaction between KSR and MEK. This functional effect correlates with the ability of KSR to uncouple Ras from the ERK cascade when overexpressed.     

Bud-site selection and cell polarity in budding yeast

Polarized growth involves a hierarchy of events such as selection of the growth site, polarization of the cytoskeleton to the selected growth site, and transport of secretory vesicles containing components required for growth. The budding yeast Saccharomyces cerevisiae is an excellent model system for the study of polarized cell growth. A large number of proteins have been found to be involved in these processes, although their mechanisms of action are not yet well-understood. Recent discoveries have helped elucidate many of the processes involved in cell polarity and bud-site selection in yeast and have modified the traditional view of cellular structures involved in these processes. This review focuses on recent advances on the roles of cortical tags, GTPases and the cytoskeleton in the generation and maintenance of cell polarity in yeast.     

Localization of the Rsr1/Bud1 GTPase involved in selection of a proper growth site in yeast

Yeast cells organize their actin cytoskeleton in a highly polarized manner during vegetative growth. The Ras-like GTPase Rsr1/Bud1 and its regulators are required for selection of a specific site for growth. Here we showed that Rsr1/Bud1 was broadly distributed on the plasma membrane and highly concentrated at the incipient bud site and polarized growth sites. We also showed that localization of Cdc24, a guanine nucleotide exchange factor for the Cdc42 GTPase, to the proper bud site was dependent on Rsr1/Bud1. Surprisingly, Rsr1/Bud1 also localized to intracellular membranes. A mutation in the lysine repeat in the hypervariable region of Rsr1/Bud1 specifically abolished its plasma membrane localization, whereas a mutation at the CAAX motif eliminated both plasma membrane and internal membrane association of Rsr1/Bud1. Thus the lysine repeat and the CAAX motif of Rsr1/Bud1 are important for its localization to the plasma membrane and to the polarized growth sites. This localization of Rsr1/Bud1 is essential for its function in proper bud site selection because both mutations resulted in random bud site selection.     

Rop GTPase: a master switch of cell polarity development in plants

Cell polarity is fundamentally important to plant growth and development, yet the mechanism governing its development is understood poorly. Several studies have revealed a role for Rop GTPases in pollen polar tip growth. Rop is also localized to the future site of root hair development and the tip of root hairs, and expression of constitutively active Rop mutants impacts on the morphogenesis of tip-growing root hairs as well as on non-tip-growing cells. These findings highlight the importance of Rop as a common switch in cell polarity control in plants.     

Interaction of anesthetics with the Rho GTPase regulator Rho GDP dissociation inhibitor

The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599-601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIalpha), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIalpha was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short-range and W192 and W194 of the RhoGDIalpha are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols ( n < 6) also reversed tryptophan quenching, suggesting that RhoGDIalpha may also bind n-alkanols. Consistent with this, E193 was photolabeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photolabeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIalpha, whereas alcohols bind to a distal site that interacts allosterically with this pocket.     

Small GTPase Rho5 is a functional homologue of Rho1, which controls cell shape and septation in fission yeast

The small GTPase Rho1 plays an essential role in controlling the organization of the actin cytoskeleton and synthesis of the cell wall in the fission yeast Schizosaccharomyces pombe. Here we studied the role of Rho5 whose primary structure is very similar to that of Rho1. It was found that elevated expression of Rho5 was able to compensate for the lethality of cells lacking Rho1. Rho5 was localized to the ends of interphase cells and the mid-region of mitotic cells. Overexpression of Rho5 caused depolarization of F-actin patches and abnormal formation of the cell wall, as did Rho1. Although rho5(+) was not essential for maintaining the cell shape, rho1 rho5-double null cells showed more severe defects in cell viability than rho1-null cells. Thus, it is likely that Rho5 has an overlapping function with Rho1 in controlling cell growth and division in S. pombe.     

Gbetagamma recruits Rho1 to the site of polarized growth during mating in budding yeast

In mating mixtures of Saccharomyces cerevisiae, cells polarize their growth toward their conjugation partners along a pheromone gradient. This chemotropic phenomenon is mediated by structural proteins such as Far1 and Bem1 and by signaling proteins such as Cdc24, Cdc42, and Gbetagamma. The Gbetagamma subunit is thought to provide a positional cue that recruits the polarity establishment proteins, and thereby induces polarization of the actin cytoskeleton. We identified RHO1 in a screen for allele-specific high-copy suppressors of Gbetagamma overexpression, suggesting that Rho1 binds Gbetagamma in vivo. Inactivation of Rho1 GTPase activity augmented the rescue phenotype, suggesting that it is the activated form of Rho1 that binds Gbetagamma. We also found, in a pull-down assay, that Rho1 associates with GST-Ste4 and that Rho1 is localized to the neck and tip of mating projections. Moreover, a mutation in STE4 that disrupts Gbetagamma-Rho1 interaction reduces the projection tip localization of Rho1 and compromises the integrity of pheromone-treated cells deficient in Rho1 activity. In addition to its roles as a positive regulator of 1,3-beta-glucan synthase and of the cell integrity MAP kinase cascade, it was recently shown that Rho1 is necessary for the formation of mating projections. Together, these results suggest that Gbetagamma recruits Rho1 to the site of polarized growth during mating.     

Novel Rho GTPase involved in cytokinesis and cell wall integrity in the fission yeast Schizosaccharomyces pombe

The Rho family of GTPases is present in all eukaryotic cells from yeast to mammals; they are regulators in signaling pathways that control actin organization and morphogenetic processes. In yeast, Rho GTPases are implicated in cell polarity processes and cell wall biosynthesis. It is known that Rho1 and Rho2 are key proteins in the construction of the cell wall, an essential structure that in Schizosaccharomyces pombe is composed of beta-glucan, alpha-glucan, and mannoproteins. Rho1 regulates the synthesis of 1,3-beta-D-glucan by activation of the 1,3-beta-D-glucan synthase, and Rho2 regulates the synthesis of alpha-glucan by the 1,3-alpha-D-glucan synthase Mok1. Here we describe the characterization of another Rho GTPase in fission yeast, Rho4. rho4Delta cells are viable but display cell separation defects at high temperature. In agreement with this observation, Rho4 localizes to the septum. Overexpression of rho4(+) causes lysis and morphological defects. Several lines of evidence indicate that both rho4(+) deletion or rho4(+) overexpression result in a defective cell wall, suggesting an additional role for Rho4 in cell wall integrity. Rho4Delta cells also accumulate secretory vesicles around the septum and are defective in actin polarization. We propose that Rho4 could be involved in the regulation of the septum degradation during cytokinesis.     

Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFP-based FRET probes

GFP-based fluorescence resonance energy transfer (FRET) probes that visualize local activity-changes of Ras and Rho GTPases in living cells are now available for examining the spatio-temporal regulation of these proteins. This article describes principles and strategies to develop intramolecular FRET probes for Ras- and Rho-family GTPases. The procedure for characterizing candidate probes, and image acquisition and processing are also explained. An optimal FRET probe should have (i) a wide dynamic range (which means a high sensitivity), (ii) a high fluorescence intensity, (iii) target specificity, and (iv) a minimal perturbation to endogenous signaling cascades. Although an improvement of FRET probes should be executed in a trial-and-error manner, practical tips for optimization are provided here. In addition, we illustrate some applications of FRET probes for neuronal cells, which are composed of diverse subcellular compartments with different functions; thus, tools to decipher the dynamics of GTPase activity in each compartment have long been desired.     

Dystrophin-glycoprotein complex and Ras and Rho GTPase signaling are altered in muscle atrophy

The dystrophin-glycoprotein complex (DGC)is a sarcolemmal complex whose defects cause muscular dystrophies. Thenormal function of this complex is not clear. We have proposed thatthis is a signal transduction complex, signaling normal interactionswith matrix laminin, and that the response is normal growth andhomeostasis. If so, the complex and its signaling should be altered inother physiological states such as atrophy. The amount of some of the DGC proteins, including dystrophin, -dystroglycan, and-sarcoglycan, is reduced significantly in rat skeletal muscleatrophy induced by tenotomy. Furthermore, H-Ras, RhoA, and Cdc42decrease in expression levels and activities in muscle atrophy. Whenthe small GTPases were assayed after laminin or -dystroglycandepletion, H-Ras, Rac1, and Cdc42 activities were reduced, suggesting aphysical linkage between the DGC and the GTPases. Dominant-negativeCdc42, introduced with a retroviral vector, resulted in fibers thatappeared atrophic. These data support a putative role for the DGC intransduction of mechanical signals in muscle.

     

Rho GTPase signaling in the development of colorectal cancer

The involvement of Rho GTPases in major aspects of cancer development, such as cell proliferation, apoptosis, cell polarity, adhesion, migration, and invasion, have recently been attracting increasing attention. In this review, we have summarized the current findings in the literature, and we discuss the participation of the Rho GTPase members RhoA, Rac1, and Cdc42 in the development of colorectal cancer, the second most lethal neoplasia worldwide. First, we present an overview of the mechanisms of Rho GTPase regulation and the impact that regulator proteins exert on GTPase signaling. Second, we focus on the participation of Rho GTPases as modulators of colorectal cancer development. Third, we emphasize the involvement of activation and expression alterations of Rho GTPases in events associated with cancer progression, such as loss of cell-cell adhesion, proliferation, migration, and invasion. Finally, we highlight the potential use of novel anticancer drugs targeting specific components of the Rho GTPase signaling pathway with antineoplastic activity in this cancer type.     

Conformational selection in G-proteins: lessons from Ras and Rho

The induced fit model has traditionally been invoked to describe the activating conformational change of the monomeric G-proteins, such as Ras and Rho. With this scheme, the presence or absence of the γ-phosphate of GTP leads to an instantaneous switch in conformation. Here we describe atomistic molecular simulations that demonstrate that both Ras and Rho superfamily members harbor an intrinsic susceptibility to sample multiple conformational states in the absence of nucleotide ligand. By comparing the distribution of conformers in the presence and absence of nucleotide, we show that conformational selection is the dominant mechanism by which Ras and Rho undergo nucleotide-dependent conformational changes. Furthermore, the pattern of correlated motions revealed by these simulations predicts a preserved allosteric coupling of the nucleotide-binding site with the membrane interacting C-terminus in both Rho and Ras.     

Endocytosis and the development of cell polarity in yeast require a dynamic F-actin cytoskeleton

Studies using drugs that cause the disassembly of filamentous actin (F-actin) have demonstrated the importance of an intact actin cytoskeleton for polarised secretion by yeast cells [1,2]. To address the level of dynamic turnover needed for such processes, however, drugs or mutants that confer stabilising properties on F-actin are needed. Jasplakinolide is the only readily available drug that stabilises F-actin structures both in vivo and in vitro [3-6]. Yeast strains have been generated in which two of the ABC multidrug resistance transporter genes have been deleted, rendering normally jasplakinolide-resistant yeast cells sensitive to its effects. Treatment of these cells with jasplakinolide caused rapid and dramatic effects on the actin cytoskeleton, resulting in the accumulation of single large actin structures in cells. These structures, however, still contained components that are normally associated with cortical actin patches. A dynamic actin cytoskeleton was found to be critical for the generation of cell polarity and endocytosis.     

The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity

Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane. This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus. We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes. Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane. We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70. These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface.     

Differential expression of Rho1GTPase and Rho3GTPase during isotropic and polarized growth of Mucor circinelloides

Evidence has been obtained that indicates the presence of small 22 kDa GTP-binding Rho proteins through ADP-ribosylation by Clostridium botulinum C3 exotoxin in Mucor circinelloides. Rho protein was detected at all stages of growth studied. During polarized growth, both under aerobic conditions and during the yeast-mycelia transition, the radiolabeling of the [32P]ADP-ribosylated protein increased when tube formation occurred and decreased as the hyphae branched. However, when Mucor grew isotropically, the Rho protein band was thick and its intensity did not vary significantly even after bud formation and separation of daughter cells. Crude extracts of yeast and mycelial cells exhibited a broad 22 kDa band of the [32P]ADP-ribosylated Rho protein that was resolved into a protein with a pI of 6.0, after two-dimensional electrophoresis, corresponding to the Rho1p homolog. Furthermore, [32P]ADP-ribosylated Rho protein from soluble and particulate extracts of multipolarized mycelial cells obtained from the yeast-mycelia transition was separated into two proteins with pI of 6.0 and 6.4, respectively, after two-dimensional electrophoresis. These correspond to the Rho1p and Rho3p homologs, respectively. Therefore, our results show that an increase in Rho accumulation is associated with polarized growth.     

Interaction between bud-site selection and polarity-establishment machineries in budding yeast

Saccharomyces cerevisiae yeast cells polarize in order to form a single bud in each cell cycle. Distinct patterns of bud-site selection are observed in haploid and diploid cells. Genetic approaches have identified the molecular machinery responsible for positioning the bud site: during bud formation, specific locations are marked with immobile landmark proteins. In the next cell cycle, landmarks act through the Ras-family GTPase Rsr1 to promote local activation of the conserved Rho-family GTPase, Cdc42. Additional Cdc42 accumulates by positive feedback, creating a concentrated patch of GTP-Cdc42, which polarizes the cytoskeleton to promote bud emergence. Using time-lapse imaging and mathematical modelling, we examined the process of bud-site establishment. Imaging reveals unexpected effects of the bud-site-selection system on the dynamics of polarity establishment, raising new questions about how that system may operate. We found that polarity factors sometimes accumulate at more than one site among the landmark-specified locations, and we suggest that competition between clusters of polarity factors determines the final location of the Cdc42 cluster. Modelling indicated that temporally constant landmark-localized Rsr1 would weaken or block competition, yielding more than one polarity site. Instead, we suggest that polarity factors recruit Rsr1, effectively sequestering it from other locations and thereby terminating landmark activity.     

Analysis of binding site hot spots on the surface of Ras GTPase

We have recently discovered an allosteric switch in Ras, bringing an additional level of complexity to this GTPase whose mutants are involved in nearly 30% of cancers. Upon activation of the allosteric switch, there is a shift in helix 3/loop 7 associated with a disorder to order transition in the active site. Here, we use a combination of multiple solvent crystal structures and computational solvent mapping (FTMap) to determine binding site hot spots in the “off” and “on” allosteric states of the GTP-bound form of H-Ras. Thirteen sites are revealed, expanding possible target sites for ligand binding well beyond the active site. Comparison of FTMaps for the H and K isoforms reveals essentially identical hot spots. Furthermore, using NMR measurements of spin relaxation, we determined that K-Ras exhibits global conformational dynamics very similar to those we previously reported for H-Ras. We thus hypothesize that the global conformational rearrangement serves as a mechanism for allosteric coupling between the effector interface and remote hot spots in all Ras isoforms. At least with respect to the binding sites involving the G domain, H-Ras is an excellent model for K-Ras and probably N-Ras as well. Ras has so far been elusive as a target for drug design. The present work identifies various unexplored hot spots throughout the entire surface of Ras, extending the focus from the disordered active site to well-ordered locations that should be easier to target.     

GTPase signaling: bridging the GAP between ARF and Rho

Membrane traffic and actin cytoskeleton dynamics are intimately linked, and GTPases of the Rho and ARF families may work together to regulate both. Recent studies have identified a family of GTPase activating proteins (GAPs) that contain both ARF-GAP and Rho-GAP domains, providing the first direct link between these two signaling pathways.