Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton in Saccharomyces cerevisiae

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.     

Clostridial ADP-ribosylating toxins: effects on ATP and GTP-binding proteins

The actin cytoskeleton appears to be as the cellular target of various clostridial ADP-ribosyltransferases which have been described during recent years.Clostridium botulinum C2 toxin,Clostridium perfringens iota toxin andClostridium spiroforme toxin ADP-ribosylate actin monomers and inhibit actin polymerization.Clostridium botulium exoenzyme C3 andClostridium limosum exoenzyme ADP-ribosylate the low-molecular-mass GTP-binding proteins of the Rho family, which participate in the regulation of the actin cytoskeleton. ADP-ribosylation inactivates the regulatory Rho proteins and disturbs the organization of the actin cytoskeleton.     

Cytoskeleton and morphogenesis in brown algae

BACKGROUND: Morphogenesis on a cellular level includes processes in which cytoskeleton and cell wall expansion are strongly involved. In brown algal zygotes, microtubules (MTs) and actin filaments (AFs) participate in polarity axis fixation, cell division and tip growth. Brown algal vegetative cells lack a cortical MT cytoskeleton, and are characterized by centriole-bearing centrosomes, which function as microtubule organizing centres. SCOPE: Extensive electron microscope and immunofluorescence studies of MT organization in different types of brown algal cells have shown that MTs constitute a major cytoskeletal component, indispensable for cell morphogenesis. Apart from participating in mitosis and cytokinesis, they are also involved in the expression and maintenance of polarity of particular cell types. Disruption of MTs after Nocodazole treatment inhibits cell growth, causing bulging and/or bending of apical cells, thickening of the tip cell wall, and affecting the nuclear positioning. Staining of F-actin using Rhodamine-Phalloidin, revealed a rich network consisting of perinuclear, endoplasmic and cortical AFs. AFs participate in mitosis by the organization of an F-actin spindle and in cytokinesis by an F-actin disc. They are also involved in the maintenance of polarity of apical cells, as well as in lateral branch initiation. The cortical system of AFs was found related to the orientation of cellulose microfibrils (MFs), and therefore to cell wall morphogenesis. This is expressed by the coincidence in the orientation between cortical AFs and the depositing MFs. Treatment with cytochalasin B inhibits mitosis and cytokinesis, as well as tip growth of apical cells, and causes abnormal deposition of MFs. CONCLUSIONS: Both the cytoskeletal elements studied so far, i.e. MTs and AFs are implicated in brown algal cell morphogenesis, expressed in their relationship with cell wall morphogenesis, polarization, spindle organization and cytokinetic mechanism. The novelty is the role of AFs and their possible co-operation with MTs.     

The Rho-mDia1 pathway regulates cell polarity and focal adhesion turnover in migrating cells through mobilizing Apc and c-Src

Directed cell migration requires cell polarization and adhesion turnover, in which the actin cytoskeleton and microtubules work critically. The Rho GTPases induce specific types of actin cytoskeleton and regulate microtubule dynamics. In migrating cells, Cdc42 regulates cell polarity and Rac works in membrane protrusion. However, the role of Rho in migration is little known. Rho acts on two major effectors, ROCK and mDia1, among which mDia1 produces straight actin filaments and aligns microtubules. Here we depleted mDia1 by RNA interference and found that mDia1 depletion impaired directed migration of rat C6 glioma cells by inhibiting both cell polarization and adhesion turnover. Apc and active Cdc42, which work together for cell polarization, localized in the front of migrating cells, while active c-Src, which regulates adhesion turnover, localized in focal adhesions. mDia1 depletion impaired localization of these molecules at their respective sites. Conversely, expression of active mDia1 facilitated microtubule-dependent accumulation of Apc and active Cdc42 in the polar ends of the cells and actin-dependent recruitment of c-Src in adhesions. Thus, the Rho-mDia1 pathway regulates polarization and adhesion turnover by aligning microtubules and actin filaments and delivering Apc/Cdc42 and c-Src to their respective sites of action.     

TSC1 Controls Distribution of Actin Fibers through Its Effect on Function of Rho Family of Small GTPases and Regulates Cell Migration and Polarity

The tumor-suppressor genes TSC1 and TSC2 are mutated in tuberous sclerosis, an autosomal dominant multisystem disorder. The gene products of TSC1 and TSC2 form a protein complex that inhibits the signaling of the mammalian target of rapamycin complex1 (mTORC1) pathway. mTORC1 is a crucial molecule in the regulation of cell growth, proliferation and survival. When the TSC1/TSC2 complex is not functional, uncontrolled mTORC1 activity accelerates the cell cycle and triggers tumorigenesis. Recent studies have suggested that TSC1 and TSC2 also regulate the activities of Rac1 and Rho, members of the Rho family of small GTPases, and thereby influence the ensuing actin cytoskeletal organization at focal adhesions. However, how TSC1 contributes to the establishment of cell polarity is not well understood. Here, the relationship between TSC1 and the formation of the actin cytoskeleton was analyzed in stable TSC1-expressing cell lines originally established from a Tsc1-deficient mouse renal tumor cell line. Our analyses showed that cell proliferation and migration were suppressed when TSC1 was expressed. Rac1 activity in these cells was also decreased as was formation of lamellipodia and filopodia. Furthermore, the number of basal actin stress fibers was reduced; by contrast, apical actin fibers, originating at the level of the tight junction formed a network in TSC1-expressing cells. Treatment with Rho-kinase (ROCK) inhibitor diminished the number of apical actin fibers, but rapamycin had no effect. Thus, the actin fibers were regulated by the Rho-ROCK pathway independently of mTOR. In addition, apical actin fibers appeared in TSC1-deficient cells after inhibition of Rac1 activity. These results suggest that TSC1 regulates cell polarity-associated formation of actin fibers through the spatial regulation of Rho family of small GTPases.     

Fission yeast Aip3p (spAip3p) is required for an alternative actin-directed polarity program

Aip3p is an actin-interacting protein that regulates cell polarity in budding yeast. The Schizosaccharomyces pombe-sequencing project recently led to the identification of a homologue of Aip3p that we have named spAip3p. Our results confirm that spAip3p is a true functional homologue of Aip3p. When expressed in budding yeast, spAip3p localizes similarly to Aip3p during the cell cycle and complements the cell polarity defects of an aip3Delta strain. Two-hybrid analysis shows that spAip3p interacts with actin similarly to Aip3p. In fission yeast, spAip3p localizes to both cell ends during interphase and later organizes into two rings at the site of cytokinesis. spAip3p localization to cell ends is dependent on microtubule cytoskeleton, its localization to the cell middle is dependent on actin cytoskeleton, and both patterns of localization require an operative secretory pathway. Overexpression of spAip3p disrupts the actin cytoskeleton and cell polarity, leading to morphologically aberrant cells. Fission yeast, which normally rely on the microtubule cytoskeleton to establish their polarity axis, can use the actin cytoskeleton in the absence of microtubule function to establish a new polarity axis, leading to the formation of branched cells. spAip3p localizes to, and is required for, branch formation, confirming its role in actin-directed polarized cell growth in both Schizosaccharomyces pombe and Saccharomyces cerevisiae.     

Smurf1: a link between cell polarity and ubiquitination

Members of the Rho family of small guanosine triphosphatases are well known for their important functions in the dynamic regulation of actin cytoskeleton. We recently found that a HECT domain E3 ubiquitin ligase, called Smurf1, regulates cell polarity and protrusion formation by targeting RhoA for degradation at cellular protrusions. Smurf1 regulates these functions as a partner of protein kinase Cxi, a component of the polarity complex. Furthermore, using siRNA-mediated knockdown, we demonstrated this pathway is required to maintain the transformed morphology and motility of a tumor cell. Smurf1 thus provides a link between the control of cell polarity and ubiquitin-mediated RhoA degradation during directional cell movements. Here we further discuss the mechanism by which the spatial control of Smurf1 activity is accomplished and the potential implications of these findings in cancer and development.     

Smurf1: A Link between Cell Polarity and Ubiquitination

Members of the Rho family of small guanosine triphosphatases are well known for their important functions in the dynamic regulation of actin cytoskeleton. We recently found that a HECT domain E3 ubiquitin ligase, called Smurf1, regulates cell polarity and protrusion formation by targeting RhoA for degradation at cellular protrusions. Smurf1 regulates these functions as a partner of protein kinase Czeta, a component of the polarity complex. Furthermore, using siRNA-mediated knockdown, we demonstrated this pathway is required to maintain the transformed morphology and motility of a tumor cell. Smurf1 thus provides a link between the control of cell polarity and ubiquitin-mediated RhoA degradation during directional cell movements. Here we further discuss the mechanism by which the spatial control of Smurf1 activity is accomplished and the potential implications of these findings in cancer and development.     

Gene silencing in Fucus embryos: developmental consequences of RNAi‐mediated cytoskeletal disruption

Brown algae (Phaeophyceae) are an important algal class that play a range of key ecological roles. They are often important components of rocky shore communities. A number of members of the Fucales and Ectocarpales have provided models for the study of multicellular evolution, reproductive biology and polarized development. Indeed the fucoid algae exhibit the unusual feature of inducible embryo polarization, allowing many classical studies of polarity induction. The potential of further studies of brown algae in these important areas has been increasingly hindered by the absence of tools for manipulation of gene expression that would facilitate further mechanistic analysis and gene function studies at a molecular level. The aim of this study was to establish a method that would allow the analysis of gene function through RNAi‐mediated gene knockdown. We show that injection of double‐stranded RNA (dsRNA) corresponding to an α‐tubulin gene into Fucus serratus Linnaeus zygotes induces the loss of a large proportion of the microtubule cytoskeleton, leading to growth arrest and disruption of cell division. Injection of dsRNA targeting β‐actin led to reduced rhizoid growth, enlarged cells and the failure to develop apical hair cells. The silencing effect on actin expression was maintained for 3 months. These results indicate that the Fucus embryo possesses a functional RNA interference system that can be exploited to investigate gene function during embryogenesis.     

Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, regulates the actin cytoskeleton and polarized secretion independently of its DHHC motif

Rho and Rab family GTPases play a key role in cytoskeletal organization and vesicular trafficking, but the exact mechanisms by which these GTPases regulate polarized cell growth are incompletely understood. A previous screen for genes that interact with CDC42, which encodes a Rho GTPase, found SWF1/PSL10. Here, we show Swf1p, a member of the DHHC-CRD family of palmitoyltransferases, localizes to actin cables and cortical actin patches in Saccharomyces cerevisiae. Deletion of SWF1 results in misorganization of the actin cytoskeleton and decreased stability of actin filaments in vivo. Cdc42p localization depends upon Swf1p primarily after bud emergence. Importantly, we revealed that the actin regulating activity of Swf1p is independent of its DHHC motif. A swf1 mutant, in which alanine substituted for the cysteine required for the palmitoylation activity of DHHC-CRD proteins, displayed wild-type actin organization and Cdc42p localization. Bgl2p-marked exocytosis was found wild type in this mutant, although invertase secretion was impaired. These data indicate Swf1p has at least two distinct functions, one of which regulates actin organization and Bgl2p-marked secretion. This report is the first to link the function of a DHHC-CRD protein to Cdc42p and the regulation of the actin cytoskeleton.     

Unique and Overlapping Functions of Formins Frl and DAAM During Ommatidial Rotation and Neuronal Development in Drosophila

The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Interestingly, we also found that frl mutants exhibit an axon growth phenotype in the mushroom body, a center for olfactory learning in the Drosophila brain, which is also affected in a subset of PCP genes. Significantly, Frl cooperates with Cdc42 and another formin, DAAM, during mushroom body formation. This study thus suggests that different formins can cooperate or act independently in distinct tissues, likely integrating various signaling inputs with the regulation of the cytoskeleton. It furthermore highlights the importance and complexity of formin-dependent cytoskeletal regulation in multiple organs and developmental contexts.     

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.     

Multicopy Suppression Screen in the <Emphasis Type="Italic">msb3 msb4 Saccharomyces cerevisiae</Emphasis> Double Mutant,Affected in Ypt/RabGAP Activity

The Msb3p and Msb4p proteins of Saccharomyces cerevisiae are members of the Ypt/Rab-specific GTPase-activating protein (GAP) family. They are essential to vesicular trafficking and involved in the regulation of exocytosis and in the organization of the actin cytoskeleton, but their exact biological roles have yet to be determined. The msb3 msb4 yeast double mutation causes growth inhibition in the presence of DMSO and/or caffeine, affects the organization of the actin cytoskeleton, produces a random budding pattern in diploid cells, and affects segregation of the nucleus. To find cell components that interact genetically with the products of the MSB3 and MSB4 genes, we screened a genomic library for multicopy suppressor genes restoring normal growth of the double mutant in the presence of DMSO and caffeine. Six genes were identified, and the extent to which each gene corrects specific growth defects of the msb3 msb4 mutant is described. The encoded suppressors were classified on the basis of functional features into four groups: vesicular transport proteins (Sec7p, Vps35p, and Uso1p), a protein involved in cell division (Sap155p), a molecular chaperon (Ssz1p), and a protein associated with the 25S proteasome (Cic1p).     

Role of Auxin in Induction of Polarity in Fucus vesiculosus Zygotes

We studied the effects of auxin (indole-3-acetic acid) on formation of the primary polarity axis in zygotes of the brown algae Fucus vesiculosusL. Within the first 2.5 h after fertilization, the zygotes release this phytohormone in the ambient medium. The treatment of developing zygotes with the inhibitor of indole-3-acetic acid transport from the cell 2,3,5-triiodobenzoic acid at 5 mg/l arrests the auxin secretion and leads to its accumulation in the cells. This causes a significant delay in zygote polarization. The treatment of zygotes with the exogenous indole-3-acetic acid at 1 mg/l stimulates cell polarization and formation of a rhizoid protuberance. When auxin was added to the medium with triiodobenzoic acid, the inhibitory effect of the latter was eliminated. It has been proposed that the content of indole-3-acetic acid in the ambient medium is a key factor in the induction of polarity of the F. vesiculosus zygotes.     

Localization of AtROP4 and AtROP6 and interaction with the guanine nucleotide dissociation inhibitor AtRhoGDI1 from Arabidopsis

The small GTPases of the Rho family play a key role in actin cytoskeletal organization. In plants, a novel Rho subfamily, called ROP (Rho of plants), has been found. In Arabidopsis, 12 ROP GTPases have been identified which differ mainly at their C-termini. To test the localization of two members of this subfamily (AtROP4 and AtROP6), we have generated translational fusions with the green fluorescent protein (GFP). Microscopic analysis of transiently transfected BY2 cells revealed a predominant localization of AtROP4 in the perinuclear region, while AtROP6 was localized almost exclusively to the plasma membrane. Swapping of the AtROP4 and AtROP6 C-termini produced a change in localization. As RhoGDIs are known to bind to the C-terminus of GTPases of the Rho family, we searched for ArabidopsisRhoGDI genes. We identified the AtRhoGDI1gene and mapped it to chromosome 3. AtRhoGDI1 encodes a 22.5 kDa protein which contains highly conserved amino acids in the isoprene binding pocket and exhibits 29% to 37% similarity to known mammalian RhoGDI homologues. The AtRhoGDI1 gene was expressed in all tissues studied. Using the yeast two-hybrid system, we showed specific interaction of AtRhoGDI1 with both AtROP4 and AtROP6 as well as with their GTP-locked mutants, but not with a GTPase of the RAB family. Recombinant GST-AtRhoGDI1 could bind GFP-AtROP4 from transgenic tobacco BY2 cell extracts, confirming the interaction observed with the two-hybrid system.these authors contributed equally to the work     

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.     

Mammalian Rho GTPases: new insights into their functions from in vivo studies

Rho GTPases are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking and cytokinesis. These proteins are conserved from plants and yeast to mammals, and function by interacting with and stimulating various downstream targets, including actin nucleators, protein kinases and phospholipases. The roles of Rho GTPases have been extensively studied in different mammalian cell types using mainly dominant negative and constitutively active mutants. The recent availability of knockout mice for several members of the Rho family reveals new information about their roles in signalling to the cytoskeleton and in development.     

Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae

Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization. There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p. Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p). Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins. Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle. In a small-budded cell, Bni1p moved along the bud cortex. This dynamic localization of Bni1p coincided with the apparent site of bud growth. A bni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck. Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p. These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton.     

The role of Rho family proteins in controlling the migration of crawling cells

Migration of crawling cells (amoebae and some kinds of the tissue cells) is a process related to the dynamic reorganization of actomyosin cytoskeleton. That reorganization engages actin polymerization and de-polymerization, branching of actin network and interaction of myosin II with actin filaments. All those cytoskeleton changes lead to the cell progression, contraction and shifting of the uropod and the cell adhesion. Numerous external stimuli, which activate various surface receptors and signal transduction pathways, can promote migration. Rho family proteins play an important role in the regulation of actin cytoskeleton organization. The most known members of this family are Rho, Rac and Cdc42 proteins, present in all mammalian tissue cells. These proteins control three different stages of cell migration: progression of the frontal edge, adhesion which stabilizes the frontal area, and de-adhesion and shifting of the uropod. Cdc42 and Rac control cell polarization, lamellipodium formation and expansion, organization of focal complexes. Rho protein regulates contractile activity of actomyosin cytoskeleton outside the frontal area, and thus contraction and de-adhesion of the uropod.