Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck

The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.     

Elm1 kinase activates the spindle position checkpoint kinase Kin4

Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.     

The elm1 kinase functions in a mitotic signaling network in budding yeast

In budding yeast, the Clb2 mitotic cyclin initiates a signaling network that negatively regulates polar bud growth during mitosis. This signaling network appears to require the function of a Clb2-binding protein called Nap1, the Cdc42 GTPase, and two protein kinases called Gin4 and Cla4. In this study, we demonstrate that the Elm1 kinase also plays a role in the control of bud growth during mitosis. Cells carrying a deletion of the ELM1 gene undergo a prolonged mitotic delay, fail to negatively regulate polar bud growth during mitosis, and show defects in septin organization. In addition, Elm1 is required in vivo for the proper regulation of both the Cla4 and Gin4 kinases and interacts genetically with Cla4, Gin4, and the mitotic cyclins. Previous studies have suggested that Elm1 may function to negatively regulate the Swe1 kinase. To further understand the functional relationship between Elm1 and Swe1, we have characterized the phenotype of Deltaelm1 Deltaswe1 cells. We found that Deltaelm1 Deltaswe1 cells are inviable at 37 degrees C and that a large proportion of Deltaelm1 Deltaswe1 cells grown at 30 degrees C contain multiple nuclei, suggesting severe defects in cytokinesis. In addition, we found that Elm1 is required for the normal hyperphosphorylation of Swe1 during mitosis. We propose a model in which the Elm1 kinase functions in a mitotic signaling network that controls events required for normal bud growth and cytokinesis, while the Swe1 kinase functions in a checkpoint pathway that delays nuclear division in response to defects in these events.     

A novel pathway that coordinates mitotic exit with spindle position

In budding yeast, the spindle position checkpoint (SPC) delays mitotic exit until the mitotic spindle moves into the neck between the mother and bud. This checkpoint works by inhibiting the mitotic exit network (MEN), a signaling cascade initiated and controlled by Tem1, a small GTPase. Tem1 is regulated by a putative guanine exchange factor, Lte1, but the function and regulation of Lte1 remains poorly understood. Here, we identify novel components of the checkpoint that operate upstream of Lte1. We present genetic evidence in agreement with existing biochemical evidence for the molecular mechanism of a pathway that links microtubule-cortex interactions with Lte1 and mitotic exit. Each component of this pathway is required for the spindle position checkpoint to delay mitotic exit until the spindle is positioned correctly.     

The checkpoint kinase Hsl1p is activated by Elm1p-dependent phosphorylation

Saccharomyces cerevisiae cells growing in the outdoor environment must adapt to sudden changes in temperature and other variables. Many such changes trigger stress responses that delay bud emergence until the cells can adapt. In such circumstances, the morphogenesis checkpoint delays mitosis until a bud has been formed. Mitotic delay is due to the Wee1 family mitotic inhibitor Swe1p, whose degradation is linked to bud emergence by the checkpoint kinase Hsl1p. Hsl1p is concentrated at the mother-bud neck through association with septin filaments, and it was reported that Hsl1p activation involved relief of autoinhibition in response to septin interaction. Here we challenge the previous identification of an autoinhibitory domain and show instead that Hsl1p activation involves the phosphorylation of threonine 273, promoted by the septin-associated kinase Elm1p. We identified elm1 mutants in a screen for defects in Swe1p degradation and show that a phosphomimic T273E mutation in HSL1 bypasses the need for Elm1p in this pathway.     

A novel role for the GTPase-activating protein Bud2 in the spindle position checkpoint

The spindle position checkpoint (SPC) ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP), and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF) for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2(R682A) to function in the checkpoint. Based on assays of heterozygous diploids, bud2(R682A), was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN). Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins.     

Polo-like kinase-1 regulates kinetochore-microtubule dynamics and spindle checkpoint silencing

Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore-microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore-microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore-microtubule attachments and subsequently silencing the spindle checkpoint.     

Specific inhibition of Elm1 kinase activity reveals functions required for early G1 events

In budding yeast, the Elm1 kinase is required for coordination of cell growth and cell division at G(2)/M. Elm1 is also required for efficient cytokinesis and for regulation of Swe1, the budding yeast homolog of the Wee1 kinase. To further characterize Elm1 function, we engineered an ELM1 allele that can be rapidly and selectively inhibited in vivo. We found that inhibition of Elm1 kinase activity during G(2) results in a phenotype similar to the phenotype caused by deletion of the ELM1 gene, as expected. However, inhibition of Elm1 kinase activity earlier in the cell cycle results in a prolonged G(1) delay. The G(1) requirement for Elm1 kinase activity occurs before bud emergence, polarization of the septins, and synthesis of G(1) cyclins. Inhibition of Elm1 kinase activity during early G(1) also causes defects in the organization of septins, and inhibition of Elm1 kinase activity in a strain lacking the redundant G(1) cyclins CLN1 and CLN2 is lethal. These results demonstrate that the Elm1 kinase plays an important role in G(1) events required for bud emergence and septin organization.     

Asymmetric recruitment of dynein to spindle poles and microtubules promotes proper spindle orientation in yeast

The orientation of the mitotic spindle plays a key role in determining whether a polarized cell will divide symmetrically or asymmetrically. In most cell types, cytoplasmic dynein plays a critical role in spindle orientation. However, how dynein directs opposite spindle poles toward distinct and predetermined cell ends is poorly understood. Here, we show that dynein distributes preferentially to the spindle pole bodies (SPB) and astral microtubules (MTs) proximal to the bud in metaphase yeast cells. Dynein asymmetry depended on the bud neck kinases Elm1, Hsl1, and Gin4, on the spindle pole components Cnm67 and Cdk1, and on the B-type cyclins Clb1 and Clb2. Furthermore, phenotypic and genetic studies both indicated that dynein is unable to orient the spindle when it localizes to both poles and associated microtubules. Together, our data indicate that proper orientation of the spindle requires dynein to act on a single spindle pole.     

Knockdown of B-Raf impairs spindle formation and the mitotic checkpoint in human somatic cells

It is well established that B-Raf signaling through the MAP kinase (ERK) pathways plays a prominent role in regulating cell proliferation but how it does this is not completely understood. Here, we show that B-Raf serves a physiological role during mitosis in human somatic cells. Knockdown of B-Raf using short interfering RNA (siRNA) resulted in pleiotropic spindle abnormalities and misaligned chromosomes in over 80% of the mitotic cells analyzed. A second B-Raf siRNA gave similar results suggesting these effects are specific to down-regulating B-Raf protein. In agreement with these findings, a portion of B-Raf was detected at the spindle structures including the spindle poles and kinetochores. Knockdown of C-Raf (Raf-1) had no detectable effects on spindle formation or chromosome alignment. Activation of the spindle assembly checkpoint was found to be dependent on B-Raf as evident by the inability of checkpoint proteins Bub1 and Mad2 to localize to unattached kinetochores in HeLa cells treated with B-Raf siRNA. Consistent with this, live-cell imaging microscopy showed that B-Raf-depleted cells exited mitosis earlier than control non-depleted cells. Finally, we provide evidence that B-Raf signaling promotes phosphorylation and kinetochore localization of the mitotic checkpoint kinase Mps1. Blocking B-Raf expression, ERK activity, or phosphorylation at Ser-821 residue perturbed Mps1 localization at unattached kinetochores. Thus, our data implicates a mitotic role for B-Raf in regulating spindle formation and the spindle checkpoint in human somatic cells.     

Kinetochore-localized BUB-1/BUB-3 complex promotes anaphase onset in C. elegans

The conserved Bub1/Bub3 complex is recruited to the kinetochore region of mitotic chromosomes, where it initiates spindle checkpoint signaling and promotes chromosome alignment. Here we show that, in contrast to the expectation for a checkpoint pathway component, the BUB-1/BUB-3 complex promotes timely anaphase onset in Caenorhabditis elegans embryos. This activity of BUB-1/BUB-3 was independent of spindle checkpoint signaling but required kinetochore localization. BUB-1/BUB-3 inhibition equivalently delayed separase activation and other events occurring during mitotic exit. The anaphase promotion function required BUB-1’s kinase domain, but not its kinase activity, and this function was independent of the role of BUB-1/BUB-3 in chromosome alignment. These results reveal an unexpected role for the BUB-1/BUB-3 complex in promoting anaphase onset that is distinct from its well-studied functions in checkpoint signaling and chromosome alignment, and suggest a new mechanism contributing to the coordination of the metaphase-to-anaphase transition.     

Bud6 directs sequential microtubule interactions with the bud tip and bud neck during spindle morphogenesis in Saccharomyces cerevisiae

In budding yeast, spindle polarity relies on a precise temporal program of cytoplasmic microtubule-cortex interactions throughout spindle assembly. Loss of Clb5-dependent kinase activity under conditions of attenuated Cdc28 function disrupts this program, resulting in diploid-specific lethality. Here we show that polarity loss is tolerated by haploids due to a more prominent contribution of microtubule-neck interactions to spindle orientation inherent to haploids. These differences are mediated by the relative partition of Bud6 between the bud tip and bud neck, distinguishing haploids from diploids. Bud6 localizes initially to the bud tip and accumulates at the neck concomitant with spindle assembly. bud6Delta mutant phenotypes are consistent with Bud6's role as a cortical cue for cytoplasmic microtubule capture. Moreover, mutations that affect Bud6 localization and partitioning disrupt the sequential program of microtubule-cortex interactions accordingly. These data support a model whereby Bud6 sequentially cues microtubule capture events at the bud tip followed by capture events at the bud neck, necessary for correct spindle morphogenesis and polarity.     

Function of Cdc2p-dependent Bub1p phosphorylation and Bub1p kinase activity in the mitotic and meiotic spindle checkpoint

Cdc2p is a cyclin-dependent kinase (CDK) essential for both mitotic and meiotic cell cycle progression in fission yeast. We have found that the spindle checkpoint kinase Bub1p becomes phosphorylated by Cdc2p during spindle damage in mitotic cells. Cdc2p directly phosphorylates Bub1p in vitro at the CDK consensus sites. A Bub1p mutant that cannot be phosphorylated by Cdc2p is checkpoint defective, indicating that Cdc2p-dependent Bub1p phosphorylation is required to activate the checkpoint after spindle damage. The kinase activity of Bub1p is required, but is not sufficient, for complete spindle checkpoint function. The role of Bub1p in maintaining centromeric localization of Rec8p during meiosis I is entirely dependent upon its kinase activity, suggesting that Bub1p kinase activity is essential for establishing proper kinetochore function. Finally, we show that there is a Bub1p-dependent meiotic checkpoint, which is activated in recombination mutants.     

Chk1 is required for spindle checkpoint function

The spindle checkpoint delays anaphase onset in cells with mitotic spindle defects. Here, we show that Chk1, a component of the DNA damage and replication checkpoints, protects vertebrate cells against spontaneous chromosome missegregation and is required to sustain anaphase delay when spindle function is disrupted by taxol, but not when microtubules are completely depolymerized by nocodazole. Spindle checkpoint failure in Chk1-deficient cells correlates with decreased Aurora-B kinase activity and impaired phosphorylation and kinetochore localization of BubR1. Furthermore, Chk1 phosphorylates Aurora-B and enhances its catalytic activity in vitro. We propose that Chk1 augments spindle checkpoint signaling and is required for optimal regulation of Aurora-B and BubR1 when kinetochores produce a weakened signal. In addition, Chk1-deficient cells exhibit increased resistance to taxol. These results suggest a mechanism through which Chk1 could protect against tumorigenesis through its role in spindle checkpoint signaling.     

Regulation of the localization of Dbf2 and mob1 during cell division of saccharomyces cerevisiae.

The mitotic exit network (MEN) governs Cdk inactivation. In budding yeast, MEN consists of the protein phosphatase Cdc14, the ras-like GTPase Tem1, protein kinases Cdc15, Cdc5, Dbf2 and Dbf2-binding protein Mob1. Tem1, Dbf2, Cdc5 and Cdc15 have been reported to be localized at the spindle pole body (SPB). Here we report changes of the localization of Dbf2 and Mob1 during cell division. Dbf2 and Mob1 localize to the SPBs in anaphase and then moves to the bud neck, just prior to actin ring assembly, consistent with their role in cytokinesis. The neck localization, but not SPB localization, of Dbf2 was inhibited by the Bub2 spindle checkpoint. Cdc14 is the downstream target of Dbf2 in Cdk inactivation, but we found that the neck localization of DbP2 and Mob1 was dependent on the Cdc14 activity, suggesting that Dbf2 and Mob1 function in cytokinesis at the end of the mitotic signaling cascade.     

Direct phosphorylation and activation of a Nim1-related kinase Gin4 by Elm1 in budding yeast

In budding yeast, Gin4, a Nim1-related kinase, plays an important role in proper organization of the septin ring at the mother-bud neck, a filamentous structure that is critical for diverse cellular processes including mitotic entry and cytokinesis. How Gin4 kinase activity is regulated is not known. Here we showed that a neck-associated Ser/Thr kinase Elm1, which is important for septin assembly, is critical for proper modification of Gin4 and its physiological substrate Shs1. In vitro studies with purified recombinant proteins demonstrated that Elm1 directly phosphorylates and activates Gin4, which in turn phosphorylates Shs1. Consistent with these observations, acute inhibition of Elm1 activity abolished mitotic Gin4 phosphorylation and Gin4-dependent Shs1 modification in vivo. In addition, a gin4 mutant lacking the Elm1-dependent phosphorylation sites exhibited an impaired localization to the bud-neck and, as a result, induced a significant growth defect with an elongated bud morphology. Thus, Elm1 regulates the septin assembly-dependent cellular events by directly phosphorylating and activating the Gin4-dependent pathway(s).     

Regulation of kinetochore recruitment of two essential mitotic spindle checkpoint proteins by Mps1 phosphorylation

Mps1 is a protein kinase that plays essential roles in spindle checkpoint signaling. Unattached kinetochores or lack of tension triggers recruitment of several key spindle checkpoint proteins to the kinetochore, which delays anaphase onset until proper attachment or tension is reestablished. Mps1 acts upstream in the spindle checkpoint signaling cascade, and kinetochore targeting of Mps1 is required for subsequent recruitment of Mad1 and Mad2 to the kinetochore. The mechanisms that govern recruitment of Mps1 or other checkpoint proteins to the kinetochore upon spindle checkpoint activation are incompletely understood. Here, we demonstrate that phosphorylation of Mps1 at T12 and S15 is required for Mps1 recruitment to the kinetochore. Mps1 kinetochore recruitment requires its kinase activity and autophosphorylation at T12 and S15. Mutation of T12 and S15 severely impairs its kinetochore association and markedly reduces recruitment of Mad2 to the kinetochore. Our studies underscore the importance of Mps1 autophosphorylation in kinetochore targeting and spindle checkpoint signaling.     

Disappearance of the budding yeast Bub2-Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit

Budding yeast spindle position checkpoint is engaged by misoriented spindles and prevents mitotic exit by inhibiting the G protein Tem1 through the GTPase-activating protein (GAP) Bub2/Bfa1. Bub2 and Bfa1 are found on both duplicated spindle pole bodies until anaphase onset, when they disappear from the mother-bound spindle pole under unperturbed conditions. In contrast, when spindles are misoriented they remain symmetrically localized at both SPBs. Thus, symmetric localization of Bub2/Bfa1 might lead to inhibition of Tem1, which is also present at SPBs. Consistent with this hypothesis, we show that a Bub2 version symmetrically localized on both SPBs throughout the cell cycle prevents mitotic exit in mutant backgrounds that partially impair it. This effect is Bfa1 dependent and can be suppressed by high Tem1 levels. Bub2 removal from the mother-bound SPB requires its GAP activity, which in contrast appears to be dispensable for Tem1 inhibition. Moreover, it correlates with the passage of one spindle pole through the bud neck because it needs septin ring formation and bud neck kinases.     

The surveillance mechanism of the spindle position checkpoint in yeast

The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother-bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions.     

Septins have a dual role in controlling mitotic exit in budding yeast

In Saccharomyces cerevisiae, the spindle position checkpoint ensures that cells do not exit mitosis until the mitotic spindle moves into the mother/bud neck and thus guarantees that each cell receives one nucleus [1-6]. Mitotic exit is controlled by the small G protein Tem1p. Tem1p and its GTPase activating protein (GAP) Bub2p/Bfa1p are located on the daughter-bound spindle pole body. The GEF Lte1p is located in the bud. This segregation helps keep Tem1p in its inactive GDP state until the spindle enters the neck. However, the checkpoint functions without Lte1p and apparently senses cytoplasmic microtubules in the mother/bud neck [7-9]. To investigate this mechanism, we examined mutants defective for septins, which compose a ring at the neck [10]. We found that the septin mutants sep7Delta and cdc10Delta are defective in the checkpoint. When movement of the spindle into the neck was delayed, mitotic exit occurred, inappropriately leaving both nuclei in the mother. In sep7Delta and cdc10Delta mutants, Lte1p is mislocalized to the mother. In sep7Delta, but not cdc10Delta, mutants, inappropriate mitotic exit depends on Lte1p. These results suggest that septins serve as a diffusion barrier for Lte1p, and that Cdc10p is needed for the septin ring to serve as a scaffold for a putative microtubule sensor.