Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.
The assembly of a bipolar mitotic spindle is essential for the accurate segregation of sister chromatids during mitosis and, hence, for successful cell division. Spindle assembly depends on the successful duplication of the spindle poles, followed by their separation to opposing ends of the cell. Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle, the relevant CDK targets have not been identified. Motor proteins of the kinesin-5 family generate movement on the microtubules that make up the spindle and are believed to power spindle pole separation. By employing the budding yeast Saccharomyces cerevisiae as a model, we have found evidence that cyclin/CDKs control spindle assembly by phosphorylating the kinesins-5 Kip1 and Cin8. When phosphorylation at a conserved CDK site in the motor domain of Kip1 is blocked, spindle pole separation is greatly diminished but neither protein abundance nor localization is affected. We have also obtained direct evidence by mass spectrometry that Kip1 and Cin8 are phosphorylated in vivo at consensus CDK sites in their tail domains. Our findings suggest that B-cyclin/CDKs regulate spindle assembly by regulating kinesin-5 motor activity.
The roles of two kinesin-related proteins, Kip2p and Kip3p, in
microtubule function and nuclear migration were investigated. Deletion
of either gene resulted in nuclear migration defects similar to those
described for dynein and kar9 mutants. By indirect
immunofluorescence, the cytoplasmic microtubules in
kip2Δwere consistently short or absent throughout the
cell cycle. In contrast, in kip3Δ strains, the
cytoplasmic microtubules were significantly longer than wild type at
telophase. Furthermore, in the kip3Δ cells with
nuclear positioning defects, the cytoplasmic microtubules were
misoriented and failed to extend into the bud. Localization studies
found Kip2p exclusively on cytoplasmic microtubules throughout the cell
cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic
microtubules. Genetic analysis demonstrated that the
kip2Δ kar9Δ double mutants were
synthetically lethal, whereas kip3Δ
kar9Δ double mutants were viable. Conversely,
kip3Δ dhc1Δ double mutants were
synthetically lethal, whereas kip2Δ
dhc1Δ double mutants were viable. We suggest that the
kinesin-related proteins, Kip2p and Kip3p, function in nuclear
migration and that they do so by different mechanisms. We propose that
Kip2p stabilizes microtubules and is required as part of the
dynein-mediated pathway in nuclear migration. Furthermore, we propose
that Kip3p functions, in part, by depolymerizing microtubules and is
required for the Kar9p-dependent orientation of the cytoplasmic
Spindle orientation and nuclear migration are crucial events in cell growth and differentiation of many eukaryotes. Here we show that KIP3, the sixth and final kinesin-related gene in Saccharomyces cerevisiae, is required for migration of the nucleus to the bud site in preparation for mitosis. The position of the nucleus in the cell and the orientation of the mitotic spindle was examined by microscopy of fixed cells and by time-lapse microscopy of individual live cells. Mutations in KIP3 and in the dynein heavy chain gene defined two distinct phases of nuclear migration: a KIP3-dependent movement of the nucleus toward the incipient bud site and a dynein-dependent translocation of the nucleus through the bud neck during anaphase. Loss of KIP3 function disrupts the unidirectional movement of the nucleus toward the bud and mitotic spindle orientation, causing large oscillations in nuclear position. The oscillatory motions sometimes brought the nucleus in close proximity to the bud neck, possibly accounting for the viability of a kip3 null mutant. The kip3 null mutant exhibits normal translocation of the nucleus through the neck and normal spindle pole separation kinetics during anaphase. Simultaneous loss of KIP3 and kinesin-related KAR3 function, or of KIP3 and dynein function, is lethal but does not block any additional detectable movement. This suggests that the lethality is due to the combination of sequential and possibly overlapping defects. Epitope-tagged Kip3p localizes to astral and central spindle microtubules and is also present throughout the cytoplasm and nucleus.
Kinesins are motor proteins which are classified into 11 different families. We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans. Relatedness analyses based on the motor domains grouped them into nine families. In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases. The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low. A disruption of kipB demonstrated that it is not essential for vegetative growth. kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules. kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis. Meiosis and ascospore formation were not affected. Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4, suggesting an important but redundant role of KipB in mitosis. KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends.
The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.
kinesin; dynein; motor proteins; microtubules; mitotic spindle
Two Saccharomyces cerevisiae kinesin-related motors, Cin8p and Kip1p, perform an essential role in the separation of spindle poles during spindle assembly and a major role in spindle elongation. Cin8p and Kip1p are also required to prevent an inward spindle collapse prior to anaphase. A third kinesin-related motor, Kar3p, may act antagonistically to Cin8p and Kip1p since loss of Kar3p partially suppresses the spindle collapse in cin8 kip1 mutants. We have tested the relationship between Cin8p and Kar3p by overexpressing both motors using the inducible GAL1 promoter. Overexpression of KAR3 results in a shrinkage of spindle size and a temperature-dependent inhibition of the growth of wild-type cells. Excess Kar3p has a stronger inhibitory effect on the growth of cin8 kip1 mutants and can completely block anaphase spindle elongation in these cells. In contrast, overexpression of CIN8 leads to premature spindle elongation in all cells tested. This is the first direct demonstration of antagonistic motors acting on the intact spindle and suggests that spindle length is determined by the relative activity of Kar3p-like and Cin8p/Kip1p-like motors.
Kinesins from the bipolar (Kinesin-5) family are conserved in eukaryotic organisms and play critical roles during the earliest stages of mitosis to mediate spindle pole body separation and formation of a bipolar mitotic spindle. To date, genes encoding bipolar kinesins have been reported to be essential in all organisms studied. We report the characterization of CaKip1p, the sole member of this family in the human pathogenic yeast Candida albicans. C. albicans Kip1p appears to localize to the mitotic spindle and loss of CaKip1p function interferes with normal progression through mitosis. Inducible excision of CaKIP1 revealed phenotypes unique to C. albicans, including viable homozygous Cakip1 mutants and an aberrant spindle morphology in which multiple spindle poles accumulate in close proximity to each other. Expression of the C. albicans Kip1 motor domain in Escherichia coli produced a protein with microtubule-stimulated ATPase activity that was inhibited by an aminobenzothiazole (ABT) compound in an ATP-competitive fashion. This inhibition results in ‘rigor-like’, tight association with microtubules in vitro. Upon treatment of C. albicans cells with the ABT compound, cells were killed, and terminal phenotype analysis revealed an aberrant spindle morphology similar to that induced by loss of the CaKIP1 gene. The ABT compound discovered is the first example of a fungal spindle inhibitor targeted to a mitotic kinesin. Our results also show that the non-essential nature and implementation of the bipolar motor in C. albicans differs from that seen in other organisms, and suggest that inhibitors of a non-essential mitotic kinesin may offer promise as cidal agents for antifungal drug discovery.
Proper positioning of the mitotic spindle is often essential for cell division and differentiation processes. The asymmetric cell division characteristic of budding yeast, Saccharomyces cerevisiae, requires that the spindle be positioned at the mother–bud neck and oriented along the mother–bud axis. The single dynein motor encoded by the S. cerevisiae genome performs an important but nonessential spindle-positioning role. We demonstrate that kinesin-related Kip3p makes a major contribution to spindle positioning in the absence of dynein. The elimination of Kip3p function in dyn1Δ cells severely compromised spindle movement to the mother–bud neck. In dyn1Δ cells that had completed positioning, elimination of Kip3p function caused spindles to mislocalize to distal positions in mother cell bodies. We also demonstrate that the spindle-positioning defects exhibited by dyn1 kip3 cells are caused, to a large extent, by the actions of kinesin- related Kip2p. Microtubules in kip2Δ cells were shorter and more sensitive to benomyl than wild-type, in contrast to the longer and benomyl-resistant microtubules found in dyn1Δ and kip3Δ cells. Most significantly, the deletion of KIP2 greatly suppressed the spindle localization defect and slow growth exhibited by dyn1 kip3 cells. Likewise, induced expression of KIP2 caused spindles to mislocalize in cells deficient for dynein and Kip3p. Our findings indicate that Kip2p participates in normal spindle positioning but antagonizes a positioning mechanism acting in dyn1 kip3 cells. The observation that deletion of KIP2 could also suppress the inviability of dyn1Δ kar3Δ cells suggests that kinesin-related Kar3p also contributes to spindle positioning.
Two Saccharomyces cerevisiae genes, CIN8 and KIP1 (a.k.a. CIN9), were identified by their requirement for normal chromosome segregation. Both genes encode polypeptides related to the heavy chain of the microtubule- based force-generating enzyme kinesin. Cin8p was found to be required for pole separation during mitotic spindle assembly at 37 degrees C, although overproduced Kip1p could substitute. At lower temperatures, the activity of at least one of these proteins was required for cell viability, indicating that they perform an essential but redundant function. Cin8p was observed to be a component of the mitotic spindle, colocalizing with the microtubules that lie between the poles. Taken together, these findings suggest that these proteins interact with spindle microtubules to produce an outwardly directed force acting upon the poles.
The mitotic spindle is a complex and dynamic structure. Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis. We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein. Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Δ mutants lack the rapid phase of anaphase B, kip1Δ mutants show defects in the slow phase of anaphase B, and kip3Δ mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell. The kip3Δ and kip1Δ mutants affect the duration of anaphase, but cin8Δ does not.
mitosis; kinesin; microtubule; anaphase; yeast
The Saccharomyces cerevisiae kinesin-related motor Kar3p, though known to be required for karyogamy, plays a poorly defined, nonessential role during vegetative growth. We have found evidence suggesting that Kar3p functions to limit the number and length of cytoplasmic microtubules in a cell cycle–specific manner. Deletion of KAR3 leads to a dramatic increase in cytoplasmic microtubules, a phenotype which is most pronounced from START through the onset of anaphase but less so during late anaphase in synchronized cultures. We have immunolocalized HA-tagged Kar3p to the spindle pole body region, and fittingly, Kar3p was not detected by late anaphase. A microtubule depolymerizing activity may be the major vegetative role for Kar3p. Addition of the microtubule polymerization inhibitors nocodazol or benomyl to the medium or deletion of the nonessential α-tubulin TUB3 gene can mostly correct the abnormal microtubule arrays and other growth defects of kar3 mutants, suggesting that these phenotypes result from excessive microtubule polymerization. Microtubule depolymerization may also be the mechanism by which Kar3p acts in opposition to the anaphase B motors Cin8p and Kip1p. A preanaphase spindle collapse phenotype of cin8 kip1 mutants, previously shown to involve Kar3p, is markedly delayed when microtubule depolymerization is inhibited by the tub2-150 mutation. These results suggest that the Kar3p motor may act to regulate the length and number of microtubules in the preanaphase spindle.
Incomplete spindle disassembly causes lethality in budding yeast. We propose that spindle disassembly is required to reinitiate the spindle cycle during the subsequent mitosis by regenerating the nuclear pool of assembly-competent tubulin.
Incomplete mitotic spindle disassembly causes lethality in budding yeast. To determine why spindle disassembly is required for cell viability, we used live-cell microscopy to analyze a double mutant strain containing a conditional mutant and a deletion mutant compromised for the kinesin-8 and anaphase-promoting complex-driven spindle-disassembly pathways (td-kip3 and doc1Δ, respectively). Under nonpermissive conditions, spindles in td-kip3 doc1Δ cells could break apart but could not disassemble completely. These cells could exit mitosis and undergo cell division. However, the daughter cells could not assemble functional, bipolar spindles in the ensuing mitosis. During the formation of these dysfunctional spindles, centrosome duplication and separation, as well as recruitment of key midzone-stabilizing proteins all appeared normal, but microtubule polymerization was nevertheless impaired and these spindles often collapsed. Introduction of free tubulin through episomal expression of α- and β-tubulin or introduction of a brief pulse of the microtubule-depolymerizing drug nocodazole allowed spindle assembly in these td-kip3 doc1Δ mutants. Therefore we propose that spindle disassembly is essential for regeneration of the intracellular pool of assembly-competent tubulin required for efficient spindle assembly during subsequent mitoses of daughter cells.
The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507–519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Δ mutants. The vik1Δ and kar3Δ mutations, but not cik1Δ, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.
kinesin; microtubules; molecular motors; cytoskeleton; Saccharomyces cerevisiae
Accurate chromosome segregation during mitosis requires biorientation of sister chromatids on the microtubules (MT) of the mitotic spindle. Chromosome–MT binding is mediated by kinetochores, which are multiprotein structures that assemble on centromeric (CEN) DNA. The simple CENs of budding yeast are among the best understood, but the roles of kinesin motor proteins at yeast kinetochores have yet to be determined, despite evidence of their importance in higher eukaryotes. We show that all four nuclear kinesins in Saccharomyces cerevisiae localize to kinetochores and function in three distinct processes. Kip1p and Cin8p, which are kinesin-5/BimC family members, cluster kinetochores into their characteristic bilobed metaphase configuration. Kip3p, a kinesin-8,-13/KinI kinesin, synchronizes poleward kinetochore movement during anaphase A. The kinesin-14 motor Kar3p appears to function at the subset of kinetochores that become detached from spindle MTs. These data demonstrate roles for structurally diverse motors in the complex processes of chromosome segregation and reveal important similarities and intriguing differences between higher and lower eukaryotes.
The Saccharomyces cerevisiae kinesin-related gene products Cin8p and Kip1p function to assemble the bipolar mitotic spindle. The cytoplasmic dynein heavy chain homologue Dyn1p (also known as Dhc1p) participates in proper cellular positioning of the spindle. In this study, the roles of these motor proteins in anaphase chromosome segregation were examined. While no single motor was essential, loss of function of all three completely halted anaphase chromatin separation. As combined motor activity was diminished by mutation, both the velocity and extent of chromatin movement were reduced, suggesting a direct role for all three motors in generating a chromosome-separating force. Redundancy for function between different types of microtubule-based motor proteins was also indicated by the observation that cin8 dyn1 double- deletion mutants are inviable. Our findings indicate that the bulk of anaphase chromosome segregation in S. cerevisiae is accomplished by the combined actions of these three motors.
In eukaryotes, mitosis requires the activation of cdc2 kinase via association with cyclin B and dephosphorylation of the threonine 14 and tyrosine 15 residues. It is known that in the budding yeast Saccharomyces cerevisiae, a homologous kinase, Cdc28, mediates the progression through M phase, but it is not clear what specific mitotic function its activation by the dephosphorylation of an equivalent tyrosine (Tyr-19) serves. We report here that cells expressing cdc28-E19 (in which Tyr-19 is replaced by glutamic acid) perform Start-related functions, complete DNA synthesis, and exhibit high levels of Clb2-associated kinase activity but are unable to form bipolar spindles. The failure of these cells to form mitotic spindles is due to their inability to segregate duplicated spindle pole bodies (SPBs), a phenotype strikingly similar to that exhibited by a previously reported mutant defective in both kinesin-like motor proteins Cin8 and Kip1. We also find that the overexpression of SWE1, the budding-yeast homolog of wee1, also leads to a failure to segregate SPBs. These results imply that dephosphorylation of Tyr-19 is required for the segregation of SPBs. The requirement of Tyr-19 dephosphorylation for spindle assembly is also observed under conditions in which spindle formation is independent of mitosis, suggesting that the involvement of Cdc28/Clb kinase in SPB separation is direct. On the basis of these results, we propose that one of the roles of Tyr-19 dephosphorylation is to promote SPB separation.
Kinesin-related Cin8p is the most important spindle-pole-separating motor in Saccharomyces cerevisiae but is not essential for cell viability. We identified 20 genes whose products are specifically required by cell deficient for Cin8p. All are associated with mitotic roles and represent at least four different functional pathways. These include genes whose products act in two spindle motor pathways that overlap in function with Cin8p, the kinesin-related Kip1p pathway and the cytoplasmic dynein pathway. In addition, genes required for mitotic spindle checkpoint function and for normal microtubule stability were recovered. Mutant alleles of eight genes caused phenotypes similar to dyn1 (encodes the dynein heavy chain), including a spindle-positioning defect. We provide evidence that the products of these genes function in concept with dynein. Among the dynein pathway gene products, we found homologues of the cytoplasmic dynein intermediate chain, the p150Glued subunit of the dynactin complex, and human LIS-1, required for normal brain development. These findings illustrate the complex cellular interactions exhibited by Cin8p, a member of a conserved spindle motor family.
Molecular motors play critical roles in the formation of mitotic spindles, either through controlling the stability of individual microtubules, or by cross-linking and sliding microtubule arrays. Kinesin-8 motors are best known for their regulatory roles in controlling microtubule dynamics. They contain microtubule-destabilizing activities, and restrict spindle length in a wide variety of cell types and organisms. Here, we report for the first time on an anti-parallel microtubule-sliding activity of the budding yeast kinesin-8, Kip3. The in vivo importance of this sliding activity was established through the identification of complementary Kip3 mutants that separate the sliding activity and microtubule destabilizing activity. In conjunction with kinesin-5/Cin8, the sliding activity of Kip3 promotes bipolar spindle assembly and the maintenance of genome stability. We propose a “slide-disassemble” model where Kip3’s sliding and destabilizing activity balance during pre-anaphase. This facilitates normal spindle assembly. However, Kip3’s destabilizing activity dominates in late anaphase, inhibiting spindle elongation and ultimately promoting spindle disassembly.
Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans.
We performed individual disruptions of the kinesin genes, kif4, kif8, kif10, and kif11. None of the motors encoded by these genes are essential for development or viability of Dictyostelium. Removal of Kif4 (kinesin-7; CENP-E family) significantly impairs the rate of cell growth and, when combined with a previously characterized dynein inhibition, results in dramatic defects in mitotic spindle assembly. Kif8 (kinesin-4; chromokinesin family) and Kif10 (kinesin-8; Kip3 family) appear to cooperate with dynein to organize the interphase radial microtubule array.
The results reported here extend the number of kinesin gene disruptions in Dictyostelium, to now total 10, among the 13 isoforms. None of these motors, individually, are required for short-term viability. In contrast, homologs of at least six of the 10 kinesins are considered essential in humans. Our work underscores the functional redundancy of motor isoforms in basal organisms while highlighting motor specificity in more complex metazoans. Since motor disruption in Dictyostelium can readily be combined with other motility insults and stresses, this organism offers an excellent system to investigate functional interactions among the kinesin motor family.
We have used anti-peptide antibodies raised against highly conserved regions of the kinesin motor domain to identify kinesin-related proteins in the fission yeast Schizosaccharomyces pombe. Here we report the identification of a new kinesin-related protein, which we have named pkl1. Sequence homology and domain organization place pkl1 in the Kar3/ncd subfamily of kinesin-related proteins. Bacterially expressed pkl1 fusion proteins display microtubule-stimulated ATPase activity, nucleotide-sensitive binding, and bundling of microtubules. Immunofluorescence studies with affinity-purified antibodies indicate that the pkl1 protein localizes to the nucleus and the mitotic spindle. Pkl1 null mutants are viable but have increased sensitivity to microtubule-disrupting drugs. Disruption of pkl1+ suppresses mutations in another kinesin-related protein, cut7, which is known to act in the spindle. Overexpression of pkl1 to very high levels causes a similar phenotype to that seen in cut7 mutants: V-shaped and star-shaped microtubule structures are observed, which we interpret to be spindles with unseparated spindle poles. These observations suggest that pkl1 and cut7 provide opposing forces in the spindle. We propose that pkl1 functions as a microtubule-dependent motor that is involved in microtubule organization in the mitotic spindle.
During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus-ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely-conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus-end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.
To investigate the relationship between structure and function of kinesin-like proteins, we have identified by polymerase chain reaction (PCR) a new kinesin-like protein in the filamentous fungus Aspergillus nidulans, which we have designated KLPA. DNA sequence analysis showed that the predicted KLPA protein contains a COOH terminal kinesin-like motor domain. Despite the structural similarity of KLPA to the KAR3 and NCD kinesin-like proteins of Saccharomyces cerevisiae and Drosophila melanogaster, which also posses COOH-terminal kinesin-like motor domains, there are no significant sequence similarities between the nonmotor or tail portions of these proteins. Nevertheless, expression studies in S. cerevisiae showed that klpA can complement a null mutation in KAR3, indicating that primary amino acid sequence conservation between the tail domains of kinesin-like proteins is not necessarily required for conserved function. Chromosomal deletion of the klpA gene exerted no observable mutant phenotype, suggesting that in A. nidulans there are likely to be other proteins functionally redundant with KLPA. Interestingly, the temperature sensitive phenotype of a mutation in another gene, bimC, which encodes a kinesin-like protein involved in mitotic spindle function in A. nidulans, was suppressed by deletion of klpA. We hypothesize that the loss of KLPA function redresses unbalanced forces within the spindle caused by mutation in bimC, and that the KLPA and BIMC kinesin-like proteins may play opposing roles in spindle function.
Chromosome passenger complexes and bipolar kinesins act together to coordinate spindle elongation, spindle breakdown, and mitotic exit.
During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)–dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.
In the budding yeast Saccharomyces cerevisiae,
movement of the mitotic spindle to a predetermined cleavage plane at
the bud neck is essential for partitioning chromosomes into the mother
and daughter cells. Astral microtubule dynamics are critical to the
mechanism that ensures nuclear migration to the bud neck. The nucleus
moves in the opposite direction of astral microtubule growth in the
mother cell, apparently being “pushed” by microtubule contacts at
the cortex. In contrast, microtubules growing toward the neck and
within the bud promote nuclear movement in the same direction of
microtubule growth, thus “pulling” the nucleus toward the bud neck.
Failure of “pulling” is evident in cells lacking Bud6p, Bni1p,
Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss
of asymmetry in spindle pole body segregation into the bud. The
cytoplasmic motor protein, dynein, is not required for nuclear movement
to the neck; rather, it has been postulated to contribute to spindle
elongation through the neck. In the absence of KAR9,
dynein-dependent spindle oscillations are evident before anaphase
onset, as are postanaphase dynein-dependent pulling forces that exceed
the velocity of wild-type spindle elongation threefold. In addition,
dynein-mediated forces on astral microtubules are sufficient to
segregate a 2N chromosome set through the neck in the absence of
spindle elongation, but cytoplasmic kinesins are not. These
observations support a model in which spindle polarity determinants
(BUD6, BNI1, KAR9) and
cytoplasmic kinesin (KIP3) provide directional cues for
spindle orientation to the bud while restraining the spindle to the
neck. Cytoplasmic dynein is attenuated by these spindle polarity
determinants and kinesin until anaphase onset, when dynein directs
spindle elongation to distal points in the mother and bud.
The depolymerase activity of Kip3 suppresses spindle microtubule polymerization to limit spindle midzone length and prevent microtubule buckling in response to sliding forces.
Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.