Cooperation between Rho-GEF Gef2 and its binding partner Nod1 in the regulation of fission yeast cytokinesis

Cytokinesis is the last step of the cell-division cycle, which requires precise spatial and temporal regulation to ensure genetic stability. Rho guanine nucleotide exchange factors (Rho GEFs) and Rho GTPases are among the key regulators of cytokinesis. We previously found that putative Rho-GEF Gef2 coordinates with Polo kinase Plo1 to control the medial cortical localization of anillin-like protein Mid1 in fission yeast. Here we show that an adaptor protein, Nod1, colocalizes with Gef2 in the contractile ring and its precursor cortical nodes. Like gef2∆, nod1∆ has strong genetic interactions with various cytokinesis mutants involved in division-site positioning, suggesting a role of Nod1 in early cytokinesis. We find that Nod1 and Gef2 interact through the C-termini, which is important for their localization. The contractile-ring localization of Nod1 and Gef2 also depends on the interaction between Nod1 and the F-BAR protein Cdc15, where the Nod1/Gef2 complex plays a role in contractile-ring maintenance and affects the septation initiation network. Moreover, Gef2 binds to purified GTPases Rho1, Rho4, and Rho5 in vitro. Taken together, our data indicate that Nod1 and Gef2 function cooperatively in a protein complex to regulate fission yeast cytokinesis.     

Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis

Rho GTPases, activated by guanine nucleotide exchange factors (GEFs), are essential regulators of polarized cell growth, cytokinesis, and many other cellular processes. However, the regulation of Rho-GEFs themselves is not well understood. Rgf3 is an essential GEF for Rho1 GTPase in fission yeast. We show that Rgf3 protein levels and localization are regulated by arrestin-related protein Art1. art1∆ cells lyse during cell separation with a thinner and defective septum. As does Rgf3, Art1 concentrates to the contractile ring starting at early anaphase and spreads to the septum during and after ring constriction. Art1 localization depends on its C-terminus, and Art1 is important for maintaining Rgf3 protein levels. Biochemical experiments reveal that the Rgf3 C-terminus binds to Art1. Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site. As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells. Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.     

The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast

The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3⁺ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation.     

Septum formation is regulated by the RHO4‐specific exchange factors BUD3 and RGF3 and by the landmark protein BUD4 in Neurospora crassa

Rho GTPases have multiple, yet poorly defined functions during cytokinesis. By screening a Neurospora crassa knock‐out collection for Rho guanine nucleotide exchange factor (GEF) mutants that phenocopy rho‐4 defects (i.e. lack of septa, slow growth, abnormal branching and cytoplasmic leakage), we identified two strains defective in homologues of Bud3p and Rgf3 of budding and fission yeast respectively. The function of these proteins as RHO4‐specific GEFs was determined by in vitro assays. In vivo microscopy suggested that the two GEFs and their target GTPase act as two independent modules during the selection of the septation site and the actual septation process. Furthermore, we determined that the N. crassa homologue of the anillinrelated protein BUD4 is required for septum initiation and that its deficiency leads to typical rho4 defects. Localization of BUD4 as a cortical ring prior to septation initiation was independent of functional BUD3 or RGF3. These data position BUD4 upstream of both RHO4 functions in the septation process and make BUD4 a prime candidate for a cortical marker protein involved in the selection of future septation sites. The persistence of both BUD proteins and of RHO4 at the septal pore suggests additional functions of these proteins at mature septa.     

Fission Yeast Rgf2p Is a Rho1p Guanine Nucleotide Exchange Factor Required for Spore Wall Maturation and for the Maintenance of Cell Integrity in the Absence of Rgf1p

Schizosaccharomyces pombe Rho1p is essential, directly activates β-1,3-glucan synthase, and participates in the regulation of morphogenesis. In S. pombe, Rho1p is activated by at least three guanine nucleotide exchange factors (GEFs): Rgf1p, Rgf2p, and Rgf3p. In this study we show that Rgf2p is a Rho1p GEF required for sporulation. The rgf2⁺ deletion did not affect forespore membrane formation and the nuclei were encapsulated properly. However, the mutant ascospores appeared dark and immature. The rgf2Δ zygotes were not able to release the ascospores spontaneously, and the germination efficiency was greatly reduced compared to wild-type (wt) spores. This phenotype resembles that of the mutants in bgs2⁺, which encodes a sporulation-specific glucan synthase subunit. In fact, glucan synthase activity was diminished in sporulating rgf2Δ diploids. Rgf2p also plays a role in β-glucan biosynthesis during vegetative growth. Overexpression of rgf2⁺ specifically increased GTP-bound Rho1p, caused changes in cell morphology, and elicited an increase in β-1,3-glucan synthase activity. Moreover, the simultaneous disruption of rgf1⁺ and rgf2⁺ was lethal and both Rgf1p and Rgf2p were able to partially substitute for each other. Our results suggest that Rgf1p and Rgf2p are alternative GEFs with an essential overlapping function in Rho1p activation during vegetative growth.     

Roles of putative Rho-GEF Gef2 in division-site positioning and contractile-ring function in fission yeast cytokinesis

Cytokinesis is crucial for integrating genome inheritance and cell functions. In multicellular organisms, Rho-guanine nucleotide exchange factors (GEFs) and Rho GTPases are key regulators of division-plane specification and contractile-ring formation during cytokinesis, but how they regulate early steps of cytokinesis in fission yeast remains largely unknown. Here we show that putative Rho-GEF Gef2 and Polo kinase Plo1 coordinate to control the medial cortical localization and function of anillin-related protein Mid1. The division-site positioning defects of gef2Δ plo1-ts18 double mutant can be partially rescued by increasing Mid1 levels. We find that Gef2 physically interacts with the Mid1 N-terminus and modulates Mid1 cortical binding. Gef2 localization to cortical nodes and the contractile ring depends on its last 145 residues, and the DBL-homology domain is important for its function in cytokinesis. Our data suggest the interaction between Rho-GEFs and anillins is an important step in the signaling pathways during cytokinesis. In addition, Gef2 also regulates contractile-ring function late in cytokinesis and may negatively regulate the septation initiation network. Collectively, we propose that Gef2 facilitates and stabilizes Mid1 binding to the medial cortex, where the localized Mid1 specifies the division site and induces contractile-ring assembly.     

Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases

Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology–pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of ∼0.3 µM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of βPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Gef1p, a new guanine nucleotide exchange factor for Cdc42p, regulates polarity in Schizosaccharomyces pombe

Schizosaccharomyces pombe cdc42(+) regulates cell morphology and polarization of the actin cytoskeleton. Scd1p/Ral1p is the only described guanine nucleotide exchange factor (GEF) for Cdc42p in S. pombe. We have identified a new GEF, named Gef1p, specifically regulating Cdc42p. Gef1p binds to inactive Cdc42p but not to other Rho GTPases in two-hybrid assays. Overexpression of gef1(+) increases specifically the GTP-bound Cdc42p, and Gef1p is capable of stimulating guanine nucleotide exchange of Cdc42p in vitro. Overexpression of gef1(+) causes changes in cell morphology similar to those caused by overexpression of the constitutively active cdc42G12V allele. Gef1p localizes to the septum. gef1(+) deletion is viable but causes a mild cell elongation and defects in bipolar growth and septum formation, suggesting a role for Gef1p in the control of cell polarity and cytokinesis. The double mutant gef1delta scd1delta is not viable, indicating that they share an essential function as Cdc42p activators. However, both deletion and overexpression of either gef1(+) or scd1(+) causes different morphological phenotypes, which suggest different functions. Genetic evidence revealed a link between Gef1p and the signaling pathway of Shk1/Orb2p and Orb6p. In contrast, no genetic interaction between Gef1p and Shk2p-Mkh1p pathway was observed.     

The Rho1p Exchange Factor Rgf1p Signals Upstream from the Pmk1 Mitogen-activated Protein Kinase Pathway in Fission Yeast

The Schizosaccharomyces pombe exchange factor Rgf1p specifically regulates Rho1p during polarized growth. Rgf1p activates the β-glucan synthase (GS) complex containing the catalytic subunit Bgs4p and is involved in the activation of growth at the second end, a transition that requires actin reorganization. In this work, we investigated Rgf1p signaling and observed that Rgf1p acted upstream from the Pck2p-Pmk1p MAPK signaling pathway. We noted that Rgf1p and calcineurin play antagonistic roles in Cl⁻ homeostasis; rgf1Δ cells showed the vic phenotype (viable in the presence of immunosuppressant and chlorine ion) and were unable to grow in the presence of high salt concentrations, both phenotypes being characteristic of knockouts of the MAPK components. In addition, mutations that perturb signaling through the MAPK pathway resulted in defective cell integrity (hypersensitivity to caspofungin and β-glucanase). Rgf1p acts by positively regulating a subset of stimuli toward the Pmk1p-cell integrity pathway. After osmotic shock and cell wall damage HA-tagged Pmk1p was phosphorylated in wild-type cells but not in rgf1Δ cells. Finally, we provide evidence to show that Rgf1p regulates Pmk1p activation in a process that involves the activation of Rho1p and Pck2p, and we demonstrate that Rgf1p is unique in this signaling process, because Pmk1p activation was largely independent of the other two Rho1p-specific GEFs, Rgf2p and Rgf3p.     

Specific recognition of Rac2 and Cdc42 by DOCK2 and DOCK9 guanine nucleotide exchange factors

Recognition of cognate Rho GTPases by guanine-nucleotide exchange factors (GEF) is fundamental to Rho GTPase signaling specificity. Two main GEF families use either the Dbl homology (DH) or the DOCK homology region 2 (DHR-2) catalytic domain. How DHR-2-containing GEFs distinguish between the GTPases Rac and Cdc42 is not known. To determine how these GEFs specifically recognize the two Rho GTPases, we studied the amino acid sequences in Rac2 and Cdc42 that are crucial for activation by DOCK2, a Rac-specific GEF, and DOCK9, a distantly related Cdc42-specific GEF. Two elements in the N-terminal regions of Rac2 and Cdc42 were found to be essential for specific interactions with DOCK2 and DOCK9. One element consists of divergent amino acid residues in the switch 1 regions of the GTPases. Significantly, these residues were also found to be important for GTPase recognition by Rac-specific DOCK180, DOCK3, and DOCK4 GEFs. These findings were unexpected because the same residues were shown previously to interact with GTPase effectors rather than GEFs. The other element comprises divergent residues in the beta3 strand that are known to mediate specific recognition by DH domain containing GEFs. Remarkably, Rac2-to-Cdc42 substitutions of four of these residues were sufficient for Rac2 to be specifically activated by DOCK9. Thus, DOCK2 and DOCK9 specifically recognize Rac2 and Cdc42 through their switch 1 as well as beta2-beta3 regions and the mode of recognition via switch 1 appears to be conserved among diverse Rac-specific DHR-2 GEFs.     

Tyrosine Phosphorylation of Dbl Regulates GTPase Signaling

Rho GTPases are molecular “switches” that cycle between “on” (GTP-bound) and “off” (GDP-bound) states and regulate numerous cellular activities such as gene expression, protein synthesis, cytoskeletal rearrangements, and metabolic responses. Dysregulation of GTPases is a key feature of many diseases, especially cancers. Guanine nucleotide exchange factors (GEFs) of the Dbl family are activated by mitogenic cell surface receptors and activate the Rho family GTPases Cdc42, Rac1, and RhoA. The molecular mechanisms that regulate GEFs from the Dbl family are poorly understood. Our studies reveal that Dbl is phosphorylated on tyrosine residues upon stimulation by growth factors and that this event is critical for the regulated activation of the GEF. These findings uncover a novel layer of complexity in the physiological regulation of this protein.     

The checkpoint-dependent nuclear accumulation of Rho1p exchange factor Rgf1p is important for tolerance to chronic replication stress

Guanine nucleotide exchange factors control many aspects of cell morphogenesis by turning on Rho-GTPases. The fission yeast exchange factor Rgf1p (Rho gef1) specifically regulates Rho1p during polarized growth and localizes to cortical sites. Here we report that Rgf1p is relocalized to the cell nucleus during the stalled replication caused by hydroxyurea (HU). Import to the nucleus is mediated by a nuclear localization sequence at the N-terminus of Rgf1p, whereas release into the cytoplasm requires two leucine-rich nuclear export sequences at the C-terminus. Moreover, Rgf1p nuclear accumulation during replication arrest depends on the 14-3-3 chaperone Rad24p and the DNA replication checkpoint kinase Cds1p. Both proteins control the nuclear accumulation of Rgf1p by inhibition of its nuclear export. A mutant, Rgf1p-9A, that substitutes nine serine potential phosphorylation Cds1p sites for alanine fails to accumulate in the nucleus in response to replication stress, and this correlates with a severe defect in survival in the presence of HU. In conclusion, we propose that the regulation of Rgf1p could be part of the mechanism by which Cds1p and Rad24p promote survival in the presence of chronic replication stress. It will be of general interest to understand whether the same is true for homologues of Rgf1p in budding yeast and higher eukaryotes.     

Phosphorylation-dependent inhibition of Cdc42 GEF Gef1 by 14-3-3 protein Rad24 spatially regulates Cdc42 GTPase activity and oscillatory dynamics during cell morphogenesis

Active Cdc42 GTPase, a key regulator of cell polarity, displays oscillatory dynamics that are anticorrelated at the two cell tips in fission yeast. Anticorrelation suggests competition for active Cdc42 or for its effectors. Here we show how 14-3-3 protein Rad24 associates with Cdc42 guanine exchange factor (GEF) Gef1, limiting Gef1 availability to promote Cdc42 activation. Phosphorylation of Gef1 by conserved NDR kinase Orb6 promotes Gef1 binding to Rad24. Loss of Rad24–Gef1 interaction increases Gef1 protein localization and Cdc42 activation at the cell tips and reduces the anticorrelation of active Cdc42 oscillations. Increased Cdc42 activation promotes precocious bipolar growth activation, bypassing the normal requirement for an intact microtubule cytoskeleton and for microtubule-dependent polarity landmark Tea4-PP1. Further, increased Cdc42 activation by Gef1 widens cell diameter and alters tip curvature, countering the effects of Cdc42 GTPase-activating protein Rga4. The respective levels of Gef1 and Rga4 proteins at the membrane define dynamically the growing area at each cell tip. Our findings show how the 14-3-3 protein Rad24 modulates the availability of Cdc42 GEF Gef1, a homologue of mammalian Cdc42 GEF DNMBP/TUBA, to spatially control Cdc42 GTPase activity and promote cell polarization and cell shape emergence.     

Gef1p and Scd1p,the Two GDP-GTP exchange factors for Cdc42p,form a ring structure that shrinks during cytokinesis in Schizosaccharomyces pombe

Fission yeast Cdc42p, a small GTPase of the Rho family, is essential for cell proliferation and maintenance of the rod-like cell morphology. Scd1/Ral1p is a GDP-GTP exchange factor (GEF) for Cdc42p. This study and a parallel study by others establish that Gef1p is another GEF for Cdc42p. Deletions of gef1 and scd1 are synthetically lethal, generating round dead cells, and hence mimic the phenotype of cdc42 deletion. Gef1p is localized mainly to the cell division site. Scd1p is also there, but it is also detectable in other parts of the cell, including the nucleus, growing ends, and the tips of conjugation tubes. Gef1p and Scd1p form a ring structure at the cell division site, which shrinks during cytokinesis following the contraction of the actomyosin ring. Formation of the Gef1p/Scd1p ring apparently depends on the integrity of the actomyosin ring. In turn, recruitment of Cdc42p to the cell division site follows the shrinking Gef1p/Scd1p ring; the Cdc42p accumulates like a closing iris. These observations suggest that Gef1p and Scd1p may have a role in mediating between contraction of the actomyosin ring and formation of the septum, by recruiting active Cdc42p to the septation site.     

A Vps21 endocytic module regulates autophagy

In autophagy, the double-membrane autophagosome delivers cellular components for their degradation in the lysosome. The conserved Ypt/Rab GTPases regulate all cellular trafficking pathways, including autophagy. These GTPases function in modules that include guanine-nucleotide exchange factor (GEF) activators and downstream effectors. Rab7 and its yeast homologue, Ypt7, in the context of such a module, regulate the fusion of both late endosomes and autophagosomes with the lysosome. In yeast, the Rab5-related Vps21 is known for its role in early- to late-endosome transport. Here we show an additional role for Vps21 in autophagy. First, vps21∆ mutant cells are defective in selective and nonselective autophagy. Second, fluorescence and electron microscopy analyses show that vps21∆ mutant cells accumulate clusters of autophagosomal structures outside the vacuole. Third, cells with mutations in other members of the endocytic Vps21 module, including the GEF Vps9 and factors that function downstream of Vps21, Vac1, CORVET, Pep12, and Vps45, are also defective in autophagy and accumulate clusters of autophagosomes. Finally, Vps21 localizes to PAS. We propose that the endocytic Vps21 module also regulates autophagy. These findings support the idea that the two pathways leading to the lysosome—endocytosis and autophagy—converge through the Vps21 and Ypt7 GTPase modules.     

Role of guanine nucleotide exchange factors for Rho family GTPases in the regulation of cell morphology and actin cytoskeleton in fission yeast

Rho GTPases regulate fundamental processes including cell morphology and migration in various organisms. Guanine nucleotide exchange factor (GEF) has a crucial role in activating small GTPase by exchange GDP for GTP. In fission yeast Schizosaccharomyces pombe, six members of the Rho small GTPase family were identified and reported to be involved in cell morphology and polarized cell growth. We identified seven genes encoding Rho GEF domain from genome sequence and analyzed. Overexpressions of identified genes in cell lead to change of morphology, suggesting that all of them are involved in the regulation of cell morphology. Although all of null mutants were viable, two of seven null cells had morphology defects and five of seven displayed altered actin cytoskeleton arrangements. Most of the double mutants were viable and biochemical analysis revealed that each of GEFs bound to several small G proteins. These data suggest that identified Rho GEFs are involved in the regulation of cell morphology and share signals via small GTPase Rho family.     

Paxillin kinase linker (PKL) regulates Vav2 signaling during cell spreading and migration

The Rho family of GTPases plays an important role in coordinating dynamic changes in the cell migration machinery after integrin engagement with the extracellular matrix. Rho GTPases are activated by guanine nucleotide exchange factors (GEFs) and negatively regulated by GTPase-activating proteins (GAPs). However, the mechanisms by which GEFs and GAPs are spatially and temporally regulated are poorly understood. Here the activity of the proto-oncogene Vav2, a GEF for Rac1, RhoA, and Cdc42, is shown to be regulated by a phosphorylation-dependent interaction with the ArfGAP PKL (GIT2). PKL is required for Vav2 activation downstream of integrin engagement and epidermal growth factor (EGF) stimulation. In turn, Vav2 regulates the subsequent redistribution of PKL and the Rac1 GEF β-PIX to focal adhesions after EGF stimulation, suggesting a feedforward signaling loop that coordinates PKL-dependent Vav2 activation and PKL localization. Of interest, Vav2 is required for the efficient localization of PKL and β-PIX to the leading edge of migrating cells, and knockdown of Vav2 results in a decrease in directional persistence and polarization in migrating cells, suggesting a coordination between PKL/Vav2 signaling and PKL/β-PIX signaling during cell migration.     

The Rac1- and RhoG-specific GEF domain of Trio targets filamin to remodel cytoskeletal actin

Rho GTPases control actin reorganization and many other cellular functions. Guanine nucleotide-exchange factors (GEFs) activate Rho GTPases by promoting their exchange of GDP for GTP. Trio is a unique Rho GEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG/Rac1 and RhoA, respectively. Dbl-homology (DH) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology (PH) domains localize Rho GEFs near their downstream targets. Here we show that Trio GEFD1 interacts through its PH domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells. Trio GEFD1 induces actin-based ruffling in filamin-expressing, but not filamin-deficient, cells and in cells transfected with a filamin construct that lacks the Trio-binding domain. In addition, Trio GEFD1 exchange activity is not affected by filamin binding. Our results indicate that filamin, as a molecular target of Trio, may be a scaffold for the spatial organization of Rho-GTPase-mediated signalling pathways.