A Highlights from MBoC Selection: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

Rho GTPases, activated by Rho guanine nucleotide exchange factors (GEFs), are conserved molecular switches for signal transductions that regulate diverse cellular processes, including cell polarization and cytokinesis. The fission yeast Schizosaccharomyces pombe has six Rho GTPases (Cdc42 and Rho1–Rho5) and seven Rho GEFs (Scd1, Rgf1–Rgf3, and Gef1–Gef3). The GEFs for Rho2–Rho5 have not been unequivocally assigned. In particular, Gef3, the smallest Rho GEF, was barely studied. Here we show that Gef3 colocalizes with septins at the cell equator. Gef3 physically interacts with septins and anillin Mid2 and depends on them to localize. Gef3 coprecipitates with GDP-bound Rho4 in vitro and accelerates nucleotide exchange of Rho4, suggesting that Gef3 is a GEF for Rho4. Consistently, Gef3 and Rho4 are in the same genetic pathways to regulate septum formation and/or cell separation. In gef3∆ cells, the localizations of two potential Rho4 effectors—glucanases Eng1 and Agn1—are abnormal, and active Rho4 level is reduced, indicating that Gef3 is involved in Rho4 activation in vivo. Moreover, overexpression of active Rho4 or Eng1 rescues the septation defects of mutants containing gef3∆. Together our data support that Gef3 interacts with the septin complex and activates Rho4 GTPase as a Rho GEF for septation in fission yeast.     

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

Whereas actomyosin and septin ring organization and function in cytokinesis are thoroughly described, little is known regarding the mechanisms by which the actomyosin ring interacts with septins and associated proteins to coordinate cell division. Here we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p colocalizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p coimmunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for relocalization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44∆ cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p.     

Hob3p, the fission yeast ortholog of human BIN3, localizes Cdc42p to the division site and regulates cytokinesis

Cdc42 GTPase is required for polarization in eukaryotic cells, but its spatial regulation is poorly understood. In Schizosaccharomyces pombe, Cdc42p is activated by Scd1p and Gef1p, two guanine-nucleotide exchange factors. Two-hybrid screening identified Hob3p as a Gef1p binding partner. Hob3p is a BAR domain-containing protein ortholog of human Bin3. Hob3p also interacts directly with Cdc42p independently of Gef1p. Hob3p, Cdc42p and Gef1p form a complex, and Hob3p facilitates Gef1p-Cdc42p interaction and activation. Hob3p forms a ring in the division area, similar to that of Gef1p. This localization requires actin polymerization and Cdc15p but is independent of the septation initiation network. Hob3p is required for the concentration of Cdc42p to the division area. The actomyosin ring contraction is slower in hob3Delta than in wild-type cells, and this contributes to its cytokinesis defect. Moreover, this report extends previous evidence that human Bin3 suppresses the cytokinesis phenotype of hob3Delta cells, showing that Bin3 can partially recover the GTP-Cdc42p level and its localization. These results suggest that Hob3p is required to recruit and activate Cdc42p at the cell division site and that this function might be conserved in other eukaryotes.     

Mutational Analysis of Essential Septins Reveals a Role for Septin-Mediated Signaling in Filamentation

Septin proteins are conserved structural proteins that often demarcate regions of cell division. The essential nature of the septin ring, composed of several septin proteins, complicates investigation of the functions of the ring, although careful analysis in the model yeast Saccharomyces cerevisiae has elucidated the role that septins play in the cell cycle. Mutation analysis of nonessential septins in the pathogenic fungus Candida albicans has shown that septins also have vital roles in cell wall regulation (CWR), hyphal formation, and pathogenesis. While mutations in nonessential septins have been useful in establishing phenotypes, the septin defect is so slight that identifying causative associations between septins and downstream effectors has been difficult. In this work, we describe decreased abundance by mRNA perturbation (DAmP) alleles of essential septins, which display a septin defect more severe than the defect observed in deletions of nonessential septins. The septin DAmP alleles have allowed us to genetically separate the roles of septins in hyphal growth and CWR and to identify the cyclic AMP pathway as a pathway that likely acts in a parallel manner with septins in hyphal morphogenesis.     

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.     

Mid2p stabilizes septin rings during cytokinesis in fission yeast

Septins are filament-forming proteins with a conserved role in cytokinesis. In the fission yeast Schizosaccharomyces pombe, septin rings appear to be involved primarily in cell-cell separation, a late stage in cytokinesis. Here, we identified a protein Mid2p on the basis of its sequence similarity to S. pombe Mid1p, Saccharomyces cerevisiae Bud4p, and Candida albicans Int1p. Like septin mutants, mid2delta mutants had delays in cell-cell separation. mid2delta mutants were defective in septin organization but not contractile ring closure or septum formation. In wild-type cells, septins assembled first during mitosis in a single ring and during septation developed into double rings that did not contract. In mid2delta cells, septins initially assembled in a single ring but during septation appeared in the cleavage furrow, forming a washer or disc structure. FRAP studies showed that septins are stable in wild-type cells but exchange 30-fold more rapidly in mid2delta cells. Mid2p colocalized with septins and required septins for its localization. A COOH-terminal pleckstrin homology domain of Mid2p was required for its localization and function. No genetic interactions were found between mid2 and the related gene mid1. Thus, these studies identify a new factor responsible for the proper stability and function of septins during cytokinesis.     

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.     

Reining in cytokinesis with a septin corral

Septins are a family of conserved GTP-binding proteins that function in cytokinesis in fungi and animals. In budding yeast, septins form scaffolds for assembly of the actomyosin contractile ring at the cleavage plane, a role that does not appear to be conserved in other organisms. The septins form an hourglass-shaped collar at the mother-bud neck, which splits into two rings flanking the division plane at cytokinesis. A recent study(1) demonstrates that these two septin rings constitute diffusion barriers that create a cytokinetic compartment to retain cortical cytokinetic factors in proximity to the cleavage plane.     

Dual Function of Cyk2, a cdc15/PSTPIP Family Protein,in Regulating Actomyosin Ring Dynamics and Septin Distribution

We previously showed that the budding yeast Saccharomyces cerevisiae assembles an actomyosin-based ring that undergoes a contraction-like size change during cytokinesis. To learn more about the biochemical composition and activity of this ring, we have characterized the in vivo distribution and function of Cyk2p, a budding yeast protein that exhibits significant sequence similarity to the cdc15/PSTPIP family of cleavage furrow proteins. Video microscopy of cells expressing green fluorescent protein (GFP)-tagged Cyk2p revealed that Cyk2p forms a double ring that coincides with the septins through most of the cell cycle. During cytokinesis, however, the Cyk2 double ring merges with the actomyosin ring and exhibits a contraction-like size change that is dependent on Myo1p. The septin double ring, in contrast, does not undergo the contraction-like size change but the separation between the two rings increases during cytokinesis. These observations suggest that the septin-containing ring is dynamically distinct from the actomyosin ring and that Cyk2p transits between the two types of structures. Gene disruption of CYK2 does not affect the assembly of the actomyosin ring but results in rapid disassembly of the ring during the contraction phase, leading to incomplete cytokinesis, suggesting that Cyk2p has an important function in modulating the stability of the actomyosin ring during contraction. Overexpression of Cyk2p also blocks cytokinesis, most likely due to a loss of the septins from the bud neck, indicating that Cyk2p may also play a role in regulating the localization of the septins.     

IQGAP-related Rng2p organizes cortical nodes and ensures position of cell division in fission yeast

Correct positioning of the cell division machinery is crucial for genomic stability and cell fate determination. The fission yeast Schizosaccharomyces pombe, like animal cells, divides using an actomyosin ring and is an attractive model to study eukaryotic cytokinesis. In S. pombe, positioning of the actomyosin ring depends on the anillin-related protein Mid1p. Mid1p arrives first at the medial cortex and recruits actomyosin ring components to node-like structures, although how this is achieved is unknown. Here we show that the IQGAP-related protein Rng2p, an essential component of the actomyosin ring, is a key element downstream of Mid1p. Rng2p physically interacts with Mid1p and is required for the organization of other actomyosin ring components into cortical nodes. Failure of localization of Rng2p to the nodes prevents medial retention of Mid1p and leads to actomyosin ring assembly in a node-independent manner at nonmedial locations. We conclude that Mid1p recruits Rng2p to cortical nodes at the division site and that Rng2p, in turn, recruits other components of the actomyosin ring to cortical nodes, thereby ensuring correct placement of the division site.     

Bni5p,a septin-interacting protein,is required for normal septin function and cytokinesis in Saccharomyces cerevisiae

In the budding yeast Saccharomyces cerevisiae, the Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Sep7p/Shs1p septins assemble early in the cell cycle in a ring that marks the future cytokinetic site. The septins appear to be major structural components of a set of filaments at the mother-bud neck and function as a scaffold for recruiting proteins involved in cytokinesis and other processes. We isolated a novel gene, BNI5, as a dosage suppressor of the cdc12-6 growth defect. Overexpression of BNI5 also suppressed the growth defects of cdc10-1, cdc11-6, and sep7Delta strains. Loss of BNI5 resulted in a cytokinesis defect, as evidenced by the formation of connected cells with shared cytoplasms, and deletion of BNI5 in a cdc3-6, cdc10-1, cdc11-6, cdc12-6, or sep7Delta mutant strain resulted in enhanced defects in septin localization and cytokinesis. Bni5p localizes to the mother-bud neck in a septin-dependent manner shortly after bud emergence and disappears from the neck approximately 2 to 3 min before spindle disassembly. Two-hybrid, in vitro binding, and protein-localization studies suggest that Bni5p interacts with the N-terminal domain of Cdc11p, which also appears to be sufficient for the localization of Cdc11p, its interaction with other septins, and other critical aspects of its function. Our data suggest that the Bni5p-septin interaction is important for septin ring stability and function, which is in turn critical for normal cytokinesis.     

Cytokinetic nodes in fission yeast arise from two distinct types of nodes that merge during interphase

We investigated the assembly of cortical nodes that generate the cytokinetic contractile ring in fission yeast. Observations of cells expressing fluorescent fusion proteins revealed two types of interphase nodes. Type 1 nodes containing kinase Cdr1p, kinase Cdr2p, and anillin Mid1p form in the cortex around the nucleus early in G2. Type 2 nodes with protein Blt1p, guanosine triphosphate exchange factor Gef2p, and kinesin Klp8p emerge from contractile ring remnants. Quantitative measurements and computer simulations showed that these two types of nodes come together by a diffuse-and-capture mechanism: type 2 nodes diffuse to the equator and are captured by stationary type 1 nodes. During mitosis, cytokinetic nodes with Mid1p and all of the type 2 node markers incorporate into the contractile ring, whereas type 1 nodes with Cdr1p and Cdr2p follow the separating nuclei before dispersing into the cytoplasm, dependent on septation initiation network signaling. The two types of interphase nodes follow parallel branches of the pathway to prepare nodes for cytokinesis.     

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.     

Mlc1p promotes septum closure during cytokinesis via the IQ motifs of the vesicle motor Myo2p

Little is known about the molecular machinery that directs secretory vesicles to the site of cell separation during cytokinesis. We show that in Saccharomyces cerevisiae, the class V myosin Myo2p and the Rab/Ypt Sec4p, that are required for vesicle polarization processes at all stages of the cell cycle, form a complex with each other and with a myosin light chain, Mlc1p, that is required for actomyosin ring assembly and cytokinesis. Mlc1p travels on secretory vesicles and forms a complex(es) with Myo2p and/or Sec4p. Its functional interaction with Myo2p is essential during cytokinesis to target secretory vesicles to fill the mother bud neck. The role of Mlc1p in actomyosin ring assembly instead is dispensable for this process. Therefore, in yeast, as recently shown in mammals, class V myosins associate with vesicles via the formation of a complex with Rab/Ypt proteins. Further more, myosin light chains, via their ability to be transported by secretory vesicles and to interact with class V myosin IQ motifs, can regulate vesicle polarization processes at a specific location and stage of the cell cycle.     

Interaction of p190RhoGAP with C-terminal Domain of p120-catenin Modulates Endothelial Cytoskeleton and Permeability

p120-catenin is a multidomain intracellular protein, which mediates a number of cellular functions, including stabilization of cell-cell transmembrane cadherin complexes as well as regulation of actin dynamics associated with barrier function, lamellipodia formation, and cell migration via modulation of the activities of small GTPAses. One mechanism involves p120 catenin interaction with Rho GTPase activating protein (p190RhoGAP), leading to p190RhoGAP recruitment to cell periphery and local inhibition of Rho activity. In this study, we have identified a stretch of 23 amino acids within the C-terminal domain of p120 catenin as the minimal sequence responsible for the recruitment of p190RhoGAP (herein referred to as CRAD; catenin-RhoGAP association domain). Expression of the p120-catenin truncated mutant lacking the CRAD in endothelial cells attenuated effects of barrier protective oxidized phospholipid, OxPAPC. This effect was accompanied by inhibition of membrane translocation of p190RhoGAP, increased Rho signaling, as well as suppressed activation of Rac1 and its cytoskeletal effectors PAK1 (p21-activated kinase 1) and cortactin. Expression of p120 catenin-truncated mutant lacking CRAD also delayed the recovery process after thrombin-induced endothelial barrier disruption. Concomitantly, RhoA activation and downstream signaling were sustained for a longer period of time, whereas Rac signaling was inhibited. These data demonstrate a critical role for p120-catenin (amino acids 820–843) domain in the p120-catenin·p190RhoGAP signaling complex assembly, membrane targeting, and stimulation of p190RhoGAP activity toward inhibition of the Rho pathway and reciprocal up-regulation of Rac signaling critical for endothelial barrier regulation.     

Proteomic Analysis of GLUT4 Storage Vesicles Reveals LRP1 to Be an Important Vesicle Component and Target of Insulin Signaling

Insulin stimulates the translocation of intracellular GLUT4 to the plasma membrane where it functions in adipose and muscle tissue to clear glucose from circulation. The pathway and regulation of GLUT4 trafficking are complicated and incompletely understood and are likely to be contingent upon the various proteins other than GLUT4 that comprise and interact with GLUT4-containing vesicles. Moreover, not all GLUT4 intracellular pools are insulin-responsive as some represent precursor compartments, thus posing a biochemical challenge to the purification and characterization of their content. To address these issues, we immunodepleted precursor GLUT4-rich vesicles and then immunopurified GLUT4 storage vesicle (GSVs) from primary rat adipocytes and subjected them to semi-quantitative and quantitative proteomic analysis. The purified vesicles translocate to the cell surface almost completely in response to insulin, the expected behavior for bona fide GSVs. In total, over 100 proteins were identified, about 50 of which are novel in this experimental context. LRP1 (low density lipoprotein receptor-related protein 1) was identified as a major constituent of GSVs, and we show it interacts with the lumenal domains of GLUT4 and other GSV constituents. Its cytoplasmic tail interacts with the insulin-signaling pathway target, AS160 (Akt substrate of 160 kDa). Depletion of LRP1 from 3T3-L1 adipocytes reduces GLUT4 expression and correspondingly results in decreased insulin-stimulated 2-[³H]deoxyglucose uptake. Furthermore, adipose-specific LRP1 knock-out mice also exhibit decreased GLUT4 expression. These findings suggest LRP1 is an important component of GSVs, and its expression is needed for the formation of fully functional GSVs.     

The 14-3-3 protein rad24p modulates function of the cdc14p family phosphatase clp1p/flp1p in fission yeast

Schizosaccharomyces pombe cells divide through the use of an actomyosin-based contractile ring. In response to perturbation of the actomyosin ring, S. pombe cells delay in a "cytokinesis-competent" state characterized by continuous repair and maintenance of the actomyosin ring and a G2 delay. This checkpoint mechanism requires the function of the Cdc14p-family phosphatase Clp1p/Flp1p and the septation initiation network (SIN). In response to cytokinetic defects, Clp1p, normally nucleolar in interphase, is retained in the cytoplasm until completion of cell division in a SIN-dependent manner. Here, we show that a phosphorylated form of Clp1p binds the 14-3-3 protein Rad24p and is retained in the cytoplasm in a Rad24p-dependent manner in response to cytokinesis defects. This physical interaction depends on the function of the SIN component, Sid2p. In the absence of Rad24p, cells are unable to maintain SIN signaling and lose viability upon mild cytokinetic stress. The requirement of Rad24p in this checkpoint is bypassed by ectopic activation of the SIN. Furthermore, SIN-dependent nuclear exclusion of Clp1p is dependent on Rad24p function. We conclude that Rad24p-mediated cytoplasmic retention of Clp1p/Flp1p is important for cell viability upon stress to the division apparatus.     

Smad Ubiquitylation Regulatory Factor 1/2 (Smurf1/2) Promotes p53 Degradation by Stabilizing the E3 Ligase MDM2

The tumor suppressor p53 protein is tightly regulated by a ubiquitin-proteasomal degradation mechanism. Several E3 ubiquitin ligases, including MDM2 (mouse double minute 2), have been reported to play an essential role in the regulation of p53 stability. However, it remains unclear how the activity of these E3 ligases is regulated. Here, we show that the HECT-type E3 ligase Smurf1/2 (Smad ubiquitylation regulatory factor 1/2) promotes p53 degradation by enhancing the activity of the E3 ligase MDM2. We provide evidence that the role of Smurf1/2 on the p53 stability is not dependent on the E3 activity of Smurf1/2 but rather is dependent on the activity of MDM2. We find that Smurf1/2 stabilizes MDM2 by enhancing the heterodimerization of MDM2 with MDMX, during which Smurf1/2 interacts with MDM2 and MDMX. We finally provide evidence that Smurf1/2 regulates apoptosis through p53. To our knowledge, this is the first report to demonstrate that Smurf1/2 functions as a factor to stabilize MDM2 protein rather than as a direct E3 ligase in regulation of p53 degradation.     

Anillin-related protein Mid1p coordinates the assembly of the cytokinetic contractile ring in fission yeast

In fission yeast cells cortical nodes containing the protein Blt1p and several kinases appear early in G2, mature into cytokinetic nodes by adding anillin Mid1p, myosin-II, formin Cdc12p, and other proteins, and condense into a contractile ring by movements that depend on actin and myosin-II. Previous studies concluded that cells without Mid1p lack cytokinetic nodes and assemble rings unreliably from myosin-II strands but left open questions. Why do strands form outside the equatorial region? Why is ring assembly unreliable without Mid1p? We found in Δmid1 cells that Cdc12p accumulates in cytokinetic nodes scattered in the cortex and produces actin filaments that associate with myosin-II, Rng2p, and Cdc15p to form strands located between the nodes. Strands incorporate nodes, and in ∼67% of cells, strands slowly close into rings that constrict without the normal ∼25-min maturation period. Ring assembly is unreliable and slow without Mid1p because the scattered Cdc12p nodes generate strands spread widely beyond the equator, and growing strands depend on random encounters to merge with other strands into a ring. We conclude that orderly assembly of the contractile ring in wild-type cells depends on Mid1p to recruit myosin-II, Rng2p, and Cdc15p to nodes and to place cytokinetic nodes around the cell equator.     

The nuclear kinase Lsk1p positively regulates the septation initiation network and promotes the successful completion of cytokinesis in response to perturbation of the actomyosin ring in Schizosaccharomyces pombe

Cytokinesis in fission yeast requires the function of an actomyosin-based contractile ring whose constriction is dependent on a signaling module termed the septation initiation network (SIN). In response to minor perturbation of the ring, the duration of SIN signaling is extended concurrently with a delay in nuclear cycle progression. These mechanisms require the conserved phosphatase Clp1p/Flp1p and facilitate the successful completion of cytokinesis, thereby increasing cellular viability. To isolate novel components of this cytokinesis monitoring system, we screened a genome-wide bank of protein kinase deletion mutants and identified Lsk1p, a nuclear-localized protein kinase. Similar to clp1Δ mutants, and in contrast to wild type, lsk1Δ cells are unable to maintain the integrity of the actomyosin ring upon treatment with low doses (0.3 μM) of latrunculin A. However, unlike clp1Δ mutants, lsk1Δ cells are competent to delay nuclear cycle progression after cytokinetic failure. In addition, lsk1Δ mutants suppress the lethal, multiseptate phenotype conferred by hyperactivation of the SIN, demonstrating that Lsk1p is a positive regulator of this module. In this report, we demonstrate that Lsk1p acts in parallel to Clp1p to promote actomyosin ring stability upon checkpoint activation. Our studies also establish that actomyosin ring maintenance and nuclear cycle delay in response to cytokinetic perturbation can be genetically resolved into independent pathways.