Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment

Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends. In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis. We find that Kar9 localizes to the prospective daughter cell spindle pole. Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud. Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell. Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud. Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole. In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud. Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.     

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.     

The MEN mediates the effects of the spindle assembly checkpoint on Kar9-dependent spindle pole body inheritance in budding yeast

Many asymmetrically dividing cells segregate the poles of the mitotic spindle non-randomly between their two daughters. In budding yeast, the protein Kar9 localizes almost exclusively to the astral microtubules emanating from the old spindle pole body (SPB) and promotes its movement toward the bud. Thereby, Kar9 orients the spindle relative to the division axis. Here, we show that beyond perturbing Kar9 distribution, activation of the spindle assembly checkpoint (SAC) randomizes SPB inheritance. Inactivation of the B-type cyclin Clb5 led to a SAC-dependent defect in Kar9 orientation and SPB segregation. Furthermore, unlike the Clb4-dependent pathway, the Clb5- and SAC-dependent pathways functioned genetically upstream of the mitotic exit network (MEN) in SPB specification and Kar9-dependent SPB inheritance. Together, our study indicates that Clb5 functions in spindle assembly and that the SAC controls the specification and inheritance of yeast SPBs through inhibition of the MEN.     

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.     

A cell cycle checkpoint monitors cell morphogenesis in budding yeast

Checkpoint controls are regulatory pathways that inhibit cell cycle progression in cells that have not faithfully completed a prior step in the cell cycle. In the budding yeast Saccharomyces cerevisiae, DNA replication and spindle assembly are monitored by checkpoint controls that prevent nuclear division in cells that have failed to complete these processes. During the normal cell cycle, bud formation is temporally coincident with DNA replication and spindle assembly, and the nucleus divides along the mother-bud axis in mitosis. In this report, we show that inhibition of bud formation also causes a dramatic delay in nuclear division. This allows cells to recover from a transient disruption of cell polarity without becoming binucleate. The delay occurs after DNA replication and spindle assembly, and results from delayed activation of the master cell cycle regulatory kinase, Cdc28. Cdc28 activation is inhibited by phosphorylation of Cdc28 on tyrosine 19, and by delayed accumulation of the B-type cyclins Clb1 and Clb2. These results suggest the existence of a novel checkpoint that monitors cell morphogenesis in budding yeast.     

Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae.

The previously described CLB1 and CLB2 genes encode a closely related pair of B-type cyclins. Here we present the sequences of another related pair of B-type cyclin genes, which we term CLB3 and CLB4. Although CLB1 and CLB2 mRNAs rise in abundance at the time of nuclear division, CLB3 and CLB4 are turned on earlier, rising early in S phase and declining near the end of nuclear division. When all possible single and multiple deletion mutants were constructed, some multiple mutations were lethal, whereas all single mutants were viable. All lethal combinations included the clb2 deletion, whereas the clb1 clb3 clb4 triple mutant was viable, suggesting a key role for CLB2. The inviable multiple clb mutants appeared to have a defect in mitosis. Conditional clb mutants arrested as large budded cells with a G2 DNA content but without any mitotic spindle. Electron microscopy showed that the spindle pole bodies had duplicated but not separated, and no spindle had formed. This suggests that the Clb/Cdc28 kinase may have a relatively direct role in spindle formation. The two groups of Clbs may have distinct roles in spindle formation and elongation.     

Distinct mechanisms control the stability of the related S-phase cyclins Clb5 and Clb6

The yeast S-phase cyclins Clb5 and Clb6 are closely related proteins that are synthesized late in G1. Although often grouped together with respect to function, Clb5 and Clb6 exhibit differences in their ability to promote S-phase progression. DNA replication is significantly slowed in clb5Delta mutants but not in clb6Delta mutants. We have examined the basis for the differential functions of Clb5 and Clb6 and determined that unlike Clb5, which is stable until mitosis, Clb6 is degraded rapidly at the G1/S border. N-terminal deletions of CLB6 were hyperstabilized, suggesting that the sequences responsible for directing the destruction of Clb6 reside in the N terminus. Clb6 lacks the destruction box motif responsible for the anaphase promoting complex-mediated destruction of Clb5 but contains putative Cdc4 degron motifs in the N terminus. Clb6 was hyperstabilized in cdc34-3 and cdc4-3 mutants at restrictive temperatures and when S/T-P phosphorylation sites in the N terminus were mutated to nonphosphorylatable residues. Efficient degradation of Clb6 requires the activities of both Cdc28 and Pho85. Finally, hyperstabilized Clb6 expressed from the CLB6 promoter rescued the slow S-phase defect exhibited by clb5Delta cells. Taken together, these findings suggest that the SCF(Cdc4) ubiquitin ligase complex regulates Clb6 turnover and that the functional differences exhibited by Clb5 and Clb6 arise from the distinct mechanisms controlling their stability.     

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.     

Specialization and targeting of B-type cyclins.

The B-type cyclins of S. cerevisiae are diversified with respect to time of expression during the cell cycle as well as biological function. We replaced the early-expressed CLB5 coding sequence with the late-expressed CLB2 coding sequence, at the CLB5 locus. CLB5::CLB2 exhibited almost no rescue of clb5-specific replication defects, although it could rescue clb1 clb2 lethality, and in synchronized cells Clb2p-associated kinase activity from CLB5::CLB2 rose early in the cell cycle, similar to that of Clb5p. Mutagenesis of a potential substrate-targeting domain of CLB5 reduced biological activity without reducing Clb5p-associated kinase activity. Thus, Clb5p may have targeting domains required for CLB5-specific biological activity.     

Enhanced Cell Polarity in Mutants of the Budding Yeast Cyclin-dependent Kinase Cdc28p

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.     

Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p

Alignment of the mitotic spindle with the axis of cell division is an essential process in Saccharomyces cerevisiae that is mediated by interactions between cytoplasmic microtubules and the cell cortex. We found that a cortical protein, the yeast formin Bni1p, was required for spindle orientation. Two striking abnormalities were observed in bni1Delta cells. First, the initial movement of the spindle pole body (SPB) toward the emerging bud was defective. This phenotype is similar to that previously observed in cells lacking the kinesin Kip3p and, in fact, BNI1 and KIP3 were found to be in the same genetic pathway. Second, abnormal pulling interactions between microtubules and the cortex appeared to cause preanaphase spindles in bni1Delta cells to transit back and forth between the mother and the bud. We therefore propose that Bni1p may localize or alter the function of cortical microtubule-binding sites in the bud. Additionally, we present evidence that other bipolar bud site determinants together with cortical actin are also required for spindle orientation.     

Spindle polarity in S. cerevisiae: MEN can tell

Spatial coordination between the axis of the spindle and the division plane is critical in asymmetric cell divisions. In the budding yeast S. cerevisiae, orientation of the mitotic spindle responds to two intertwined programs dictating the position of the spindle poles: one providing the blueprint for built-in pole asymmetry, the other sequentially confining microtubule-cortex interactions to the bud and the bud neck. The first program sets a temporal asymmetry to limit astral microtubules to a single pole prior to spindle pole separation. The second enforces this polarity by allowing these early formed microtubules to undergo capture at the bud cell cortex while stopping newly formed microtubules once cortical capture shifts to the bud neck. The remarkable precision of this integrated program results in an invariant pattern of spindle pole inheritance in which the "old" spindle pole is destined to the bud. An additional layer of asymmetry is superimposed to couple successful chromosomal segregation between the mother and the bud with mitotic exit. This is based on the asymmetric localization to the committed daughter-bound pole of signaling components of the mitotic exit network. This system operates irrespective of intrinsic spindle polarity to ensure that it is always the pole translocating into the bud that carries the signal to regulate mitotic exit.     

Loss of CDC5 function in Saccharomyces cerevisiae leads to defects in Swe1p regulation and Bfa1p/Bub2p-independent cytokinesis

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G(2)/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1Delta defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.     

Spindle Polarity in S. cerevisiae: MEN Can Tell

Spatial coordination between the axis of the spindle and the division plane is critical in asymmetric cell divisions. In the budding yeast S. cerevisiae, orientation of the mitotic spindle responds to two intertwined programs dictating the position of the spindle poles: one providing the blueprint for built-in pole asymmetry, the other sequentially confining microtubule-cortex interactions to the bud and the bud neck. The first program sets a temporal asymmetry to limit astral microtubules to a single pole prior to spindle pole separation. The second enforces this polarity by allowing these early formed microtubules to undergo capture at the bud cell cortex while stopping newly formed microtubules once cortical capture shifts to the bud neck. The remarkable precision of this integrated program results in an invariant pattern of spindle pole inheritance in which the "old" spindle pole is destined to the bud. An additional layer of asymmetry is superimposed to couple successful chromosomal segregation between the mother and the bud with mitotic exit. This is based on the asymmetric localization to the committed daughter-bound pole of signaling components of the mitotic exit network. This system operates irrespective of intrinsic spindle polarity to ensure that it is always the pole translocating into the bud that carries the signal to regulate mitotic exit.

Key Words:

Cell cycle, Polarity, checkpoint, Microtubule, Cortical cues     

Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.     

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999     

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.     

Negative regulation of G1 and G2 by S-phase cyclins of Saccharomyces cerevisiae.

Cell cycle progression in the budding yeast Saccharomyces cerevisiae is controlled by the Cdc28 protein kinase, which is sequentially activated by different sets of cyclins. Previous genetic analysis has revealed that two B-type cyclins, Clb5 and Clb6, have a positive role in DNA replication. In the present study, we show, in addition, that these cyclins negatively regulate G1- and G2-specific functions. The consequences of this negative regulation were most apparent in clb6 mutants, which had a shorter pre-Start G1 phase as well as a shorter G2 phase than congenic wild-type cells. As a consequence, clb6 mutants grew and proliferated more rapidly than wild-type cells. It was more difficult to assess the role of Clb5 in G1 and G2 by genetic analysis because of the extreme prolongation of S phase in clb5 mutants. Nevertheless, both Clb5 and Clb6 were shown to be responsible for down-regulation of the protein kinase activities associated with Cln2, a G1 cyclin, and Clb2, a mitotic cyclin, in vivo. These observations are consistent with the observed cell cycle phase accelerations associated with the clb6 mutant and are suggestive of similar functions for Clb5. Genetic evidence suggested that the inhibition of mitotic cyclin-dependent kinase activities was dependent on and possibly mediated through the CDC6 gene product. Thus, Clb5 and Clb6 may stabilize S phase by promoting DNA replication while inhibiting other cell cycle activities.     

Novel functional dissection of the localization-specific roles of budding yeast polo kinase Cdc5p

Budding yeast polo kinase Cdc5p localizes to the spindle pole body (SPB) and to the bud-neck and plays multiple roles during M-phase progression. To dissect localization-specific mitotic functions of Cdc5p, we tethered a localization-defective N-terminal kinase domain of Cdc5p (Cdc5pDeltaC) to the SPB or to the bud-neck with components specifically localizing to one of these sites and characterized these mutants in a cdc5Delta background. Characterization of a viable, SPB-localizing, CDC5DeltaC-CNM67 mutant revealed that it is defective in timely degradation of Swe1p, a negative regulator of Cdc28p. Loss of BFA1, a negative regulator of mitotic exit, rescued the lethality of a neck-localizing CDC5DeltaC-CDC12 or CDC5DeltaC-CDC3 mutant but yielded severe defects in cytokinesis. These data suggest that the SPB-associated Cdc5p activity is critical for both mitotic exit and cytokinesis, whereas the bud neck-localized Cdc5p is required for proper Swe1p regulation. Interestingly, a cdc5Delta bfa1Delta swe1Delta triple mutant is viable but grows slowly, whereas cdc5Delta cells bearing both CDC5DeltaC-CNM67 and CDC5DeltaC-CDC12 grow well with only a mild cell cycle delay. Thus, SPB- and the bud-neck-localized Cdc5p control most of the critical Cdc5p functions and downregulation of Bfa1p and Swe1p at the respective locations are two critical factors that require Cdc5p.     

Functions of the mitotic B-type cyclins CLB1, CLB2, and CLB3 at mitotic exit antagonized by the CDC14 phosphatase

In the budding yeast Saccharomyces cerevisiae, cell cycle progression and cytokinesis at mitotic exit are proposed to be linked by CDC14 phosphatase antagonizing the function of mitotic B-type cyclin (CLBs). We have isolated a temperature-sensitive mutant, cdc14(A280V), with a mutation in the conserved phosphatase domain. Prolonged arrest in the cdc14(A280V) mutant partially uncoupled cell cycle progression from the completion of cytokinesis as measured by bud re-emergence, in the form of elongated apical projections, and DNA re-replication. In contrast to previous mitotic exit mutants, cdc14(A280V) mutants displayed a strong bias for the first apical projection to form in the mother cell body. Using cdc14(A280V) mutant phenotypes, the functions of the B-type cyclins at mitotic exit were investigated. The preference in mother-daughter apical projection formation was observed to be independent of any individual CLB function. However, cdc14(A280V)clb1Δ cells displayed a pronounced increase in apical projections, while cdc14(A280V)clb3Δ cells were observed to form round cellular chains. While cdc14(A280V) cells arrested at mitotic exit, both cdc14(A280V)clb1Δ and cdc14(A280V)clb3Δ cells completed cytokinesis, but failed cell separation. cdc14(A280V)clb2Δ cells displayed a defect in actin ring assembly. These observations differentiate the functions of CLB1, CLB2, and CLB3 at mitotic exit, and are consistent with the hypothesis that CLB activities are antagonized by the CDC14 phosphatase in order to couple cell cycle progression with cytokinesis at mitotic exit.