Accurate chromosome segregation during mitosis relies on the organization of microtubules into a bipolar spindle. Kinesin-5 proteins play an evolutionarily conserved role in establishing spindle bipolarity [1, 2] and clinical trials are currently evaluating inhibitors of human kinesin-5 (i.e. Eg5) for chemotherapeutic potential. However, in mammalian somatic cells Eg5 activity is dispensable for maintenance of bipolar spindles once they are formed [3, 4], suggesting distinct requirements for establishment versus maintenance of spindle bipolarity. By combining Eg5 inhibition with RNA interference of other spindle proteins, we show that mitotic cells deficient in MCAK fail to maintain spindle bipolarity in the absence of Eg5 activity. Collapse of bipolar spindles in MCAK-deficient cells is driven by pole focusing activities and is independent of MCAK function at centromeres, implicating hyperstabilized non-kinetochore microtubules in spindle collapse. Conversely, destabilizing non-kinetochore microtubules in early mitosis reduces the reliance on Eg5 for establishment of spindle bipolarity and renders cells partially resistant to Eg5 inhibitors. Thus, the temporal requirement for microtubule sliding generated by Eg5 activity during bipolar spindle assembly in mammalian cells is regulated by changes in the dynamic behavior of microtubules during mitosis.
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.
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 proper segregation of chromosomes during meiosis or mitosis requires the assembly of well organized spindles. In many organisms, meiotic spindles lack centrosomes. The formation of such acentrosomal spindles seems to involve first assembly or capture of microtubules (MTs) in a random pattern around the meiotic chromosomes and then parallel bundling and bipolar organization by the action of MT motors and other proteins. Here, we describe the structure, distribution, and function of KLP-18, a Caenorhabditis elegans Klp2 kinesin. Previous reports of Klp2 kinesins agree that it concentrates in spindles, but do not provide a clear view of its function. During prometaphase, metaphase, and anaphase, KLP-18 concentrates toward the poles in both meiotic and mitotic spindles. Depletion of KLP-18 by RNA-mediated interference prevents parallel bundling/bipolar organization of the MTs that accumulate around female meiotic chromosomes. Hence, meiotic chromosome segregation fails, leading to haploid or aneuploid embryos. Subsequent assembly and function of centrosomal mitotic spindles is normal except when aberrant maternal chromatin is present. This suggests that although KLP-18 is critical for organizing chromosome-derived MTs into a parallel bipolar spindle, the order inherent in centrosome-derived astral MT arrays greatly reduces or eliminates the need for KLP-18 organizing activity in mitotic spindles.
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.
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
Kinesins and dyneins play important roles during cell division. Using RNA interference (RNAi) to deplete individual (or combinations of) motors followed by immunofluorescence and time-lapse microscopy, we have examined the mitotic functions of cytoplasmic dynein and all 25 kinesins in Drosophila S2 cells. We show that four kinesins are involved in bipolar spindle assembly, four kinesins are involved in metaphase chromosome alignment, dynein plays a role in the metaphase-to-anaphase transition, and one kinesin is needed for cytokinesis. Functional redundancy and alternative pathways for completing mitosis were observed for many single RNAi knockdowns, and failure to complete mitosis was observed for only three kinesins. As an example, inhibition of two microtubule-depolymerizing kinesins initially produced monopolar spindles with abnormally long microtubules, but cells eventually formed bipolar spindles by an acentrosomal pole-focusing mechanism. From our phenotypic data, we construct a model for the distinct roles of molecular motors during mitosis in a single metazoan cell type.
kinesin; spindle; dynein; centrosome; kinetochore
Chromosome segregation during mitosis depends on the action of the mitotic spindle, a self-organizing, bipolar protein machine which uses microtubules (MTs) and their associated motors1,2. Members of the BimC subfamily of kinesin-related MT–motor proteins are believed to be essential for the formation and functioning of a normal bipolar spindle3–14. Here we report that KRP130, a homotetrameric BimC-related kinesin purified from Drosophila melanogaster embryos13, has an unusual ultrastructure. It consists of four kinesin-related polypeptides assembled into a bipolar aggregate with motor domains at opposite ends, analogous to a miniature myosin filament15. Such a bipolar ‘minifilament’ could crosslink spindle MTs and slide them relative to one another. We do not know of any other MT motors that have a bipolar structure.
Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase, which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors.
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.
Kinesin-5 (Eg-5) motor proteins are essential for maintenance of spindle bipolarity in animals. The roles of Kinesin-5 proteins in other systems, such as Arabidopsis, Dictyostelium, and sea urchin are more varied. We are studying Kinesin-5-like proteins during early development in the brown alga Silvetia compressa. Previously, this motor was shown to be needed to assemble a bipolar spindle, similar to animals. This report builds on those findings by investigating the localization of the motor and probing its function in spindle maintenance.
Anti-Eg5 antibodies were used to investigate localization of Kinesin-5-like proteins in brown algal zygotes. In interphase zygotes, localization was predominantly within the nucleus. As zygotes entered mitosis, these motor proteins strongly associated with spindle poles and, to a lesser degree, with the polar microtubule arrays and the spindle midzone. In order to address whether Kinesin-5-like proteins are required to maintain spindle bipolarity, we applied monastrol to synchronized zygotes containing bipolar spindles. Monastrol is a cell-permeable chemical inhibitor of the Kinesin-5 class of molecular motors. We found that inhibition of motor function in pre-formed spindles induced the formation of multipolar spindles and short bipolar spindles.
Based upon these localization and inhibitor studies, we conclude that Kinesin-5-like motors in brown algae are more similar to the motors of animals than those of plants or protists. However, Kinesin-5-like proteins in S. compressa serve novel roles in spindle formation and maintenance not observed in animals.
Kinesins are a diverse superfamily of motor proteins that drive organelles and other microtubule-based movements in eukaryotic cells. These motors play important roles in multiple events during both interphase and cell division. Dictyostelium discoideum contains 13 kinesin motors, 12 of which are grouped into nine families, plus one orphan. Functions for 11 of the 13 motors have been previously investigated; we address here the activities of the two remaining kinesins, both isoforms with central motor domains. Kif6 (of the kinesin-13 family) appears to be essential for cell viability. The partial knockdown of Kif6 with RNA interference generates mitotic defects (lagging chromosomes and aberrant spindle assemblies) that are consistent with kinesin-13 disruptions in other organisms. However, the orphan motor Kif9 participates in a completely novel kinesin activity, one that maintains a connection between the microtubule-organizing center (MTOC) and nucleus during interphase. kif9 null cell growth is impaired, and the MTOC appears to disconnect from its normally tight nuclear linkage. Mitotic spindles elongate in a normal fashion in kif9− cells, but we hypothesize that this kinesin is important for positioning the MTOC into the nuclear envelope during prophase. This function would be significant for the early steps of cell division and also may play a role in regulating centrosome replication.
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 kinesin-5 Saccharomyces cerevisiae homologue Cin8 is shown here to be differentially phosphorylated during late anaphase at Cdk1-specific sites located in its motor domain. Wild-type Cin8 binds to the early-anaphase spindles and detaches from the spindles at late anaphase, whereas the phosphorylation-deficient Cin8-3A mutant protein remains attached to a larger region of the spindle and spindle poles for prolonged periods. This localization of Cin8-3A causes faster spindle elongation and longer anaphase spindles, which have aberrant morphology. By contrast, the phospho-mimic Cin8-3D mutant exhibits reduced binding to the spindles. In the absence of the kinesin-5 homologue Kip1, cells expressing Cin8-3D exhibit spindle assembly defects and are not viable at 37°C as a result of spindle collapse. We propose that dephosphorylation of Cin8 promotes its binding to the spindle microtubules before the onset of anaphase. In mid to late anaphase, phosphorylation of Cin8 causes its detachment from the spindles, which reduces the spindle elongation rate and aids in maintaining spindle morphology.
Cdk1; Cin8; Kinesin-5; Microtubules; Mitosis
We identified two new Saccharomyces cerevisiae kinesin-related genes, KIP1 and KIP2, using polymerase chain reaction primers corresponding to highly conserved regions of the kinesin motor domain. Both KIP proteins are expressed in vivo, but deletion mutations conferred no phenotype. Moreover, kip1 kip2 double mutants and a triple mutant with kinesin- related kar3 had no synthetic phenotype. Using a genetic screen for mutations that make KIP1 essential, we identified another gene, KSL2, which proved to be another kinesin-related gene, CIN8. KIP1 and CIN8 are functionally redundant: double mutants arrested in mitosis whereas the single mutants did not. The microtubule organizing centers of arrested cells were duplicated but unseparated, indicating that KIP1 or CIN8 is required for mitotic spindle assembly. Consistent with this role, KIP1 protein was found to colocalize with the 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.
In Saccharomyces cerevisiae, chromosome congression clusters kinetochores on either side of the spindle equator at metaphase. Many organisms require one or more kinesin-8 molecular motors to achieve chromosome alignment. The yeast kinesin-8, Kip3, has been well studied in vitro but a role in chromosome congression has not been reported. We investigated Kip3's role in this process using semi-automated, quantitative fluorescence microscopy and time-lapse imaging and found that Kip3 is required for congression. Deletion of KIP3 increases inter-kinetochore distances and increases the variability in the position of sister kinetochores along the spindle axis during metaphase. Kip3 does not regulate spindle length and is not required for kinetochore-microtubule attachment. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule lengths.
Cin8; cluster; GFP-tubulin; kinesin-5; kinesin-8; kinetochore; Kip3; metaphase; microtubule; mitosis; spindle
In Saccharomyces cerevisiae, chromosome congression clusters kinetochores on either side of the spindle equator at metaphase. Many organisms require one or more kinesin-8 molecular motors to achieve chromosome alignment. the yeast kinesin-8, Kip3, has been well studied in vitro but a role in chromosome congression has not been reported. We investigated Kip3's role in this process using semi-automated, quantitative fluorescence microscopy and time-lapse imaging and found that Kip3 is required for congression. Deletion of KIP3 increases inter-kinetochore distances and increases the variability in the position of sister kinetochores along the spindle axis during metaphase. Kip3 does not regulate spindle length and is not required for kinetochore-microtubule attachment. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule lengths.
Cin8; cluster; GFP-tubulin; kinesin-5; kinesin-8; kinetochore; Kip3; metaphase; microtubule; mitosis; spindle
The mitotic apparatus of the early sea urchin embryo is the archetype example of a centrosome-dominated, large aster spindle organized via the centriole of the fertilizing sperm. In this study we tested the hypothesis that artificially activated sea urchin eggs possess the capacity to assemble the anastral, bipolar spindles present in many acentrosomal systems. Control fertilized Lytechinus pictus embryos and ammonia-activated eggs were immunolabeled for tubulin, centrosomal material, the spindle pole structuring protein NuMA and the mitotic kinesins MKLP1/Kinesin-6, Eg5/Kinesin-5 and KinI/Kinesin-13. Confocal imaging showed that a subset of ammonia-activated eggs contained bipolar "mini-spindles" that were anastral, displayed metaphase and anaphase-like stages, labeled for centrosomal material, NuMA and the three mitotic kinesins, and were observed in living eggs using polarization optics. These results suggest that spindle structural and motor proteins have the ability to organize bipolar, anastral spindles in sea urchin eggs activated in the absence of the paternal centriole.
spindle; anastral; sea urchin; centrosome; kinesin
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
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 bni1Δ 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 bni1Δ 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.
mitotic spindle apparatus; cell division; microtubule; actin; Saccharomyces cerevisiae
Kinesin-5 proteins are essential for formation of a bipolar mitotic spindle in most, and perhaps all, eukaryotic cells. Several Kinesin-5 proteins, notably the human version, HsEg5, are targets of a constantly expanding group of small-molecule inhibitors, which hold promise both as tools to probe mechanochemical transduction and as anti-cancer agents. Although most such compounds are selective for HsEg5 and closely related Kinesin-5 proteins, some, such as NSC 622124, exhibit activity against at least one kinesin from outside the Kinesin-5 family. Here we show NSC 622124, despite identification in a screen that yielded inhibitors now known to target the HsEg5 monastrol-binding site, does not compete with 14C-monastrol for binding to HsEg5, and is able to inhibit the basal and microtubule-stimulated ATPase activity of the monastrol-insensitive Kinesin-5, KLP61F. NSC 622124 competes with microtubules, but not ATP, for interaction with HsEg5, and disrupts the microtubule binding of HsEg5, KLP61F and Kinesin-1. Proteolytic degradation of an HsEg5•NSC622124 complex revealed that segments of the α3 and α5 helices map to the inhibitor-binding site. Overall, our results demonstrate that NSC 622124 targets the conserved microtubule-binding site of kinesin proteins. Further, unlike compounds previously reported to target the kinesin microtubule-binding site, NSC 622124 does not produce any enhancement of basal ATPase activity, and thus acts solely as a negative regulator through interaction with a site traditionally viewed as a binding region for positive regulators (i.e., microtubules). Our work emphasizes the concept that microtubule-dependent motor proteins may be controlled at multiple sites by both positive and negative effectors.
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.
Motor proteins have been implicated in various aspects of mitosis, including spindle assembly and chromosome segregation. Here, we show that acentrosomal Arabidopsis cells that are mutant for the kinesin, ATK1, lack microtubule accumulation at the predicted spindle poles during prophase and have reduced spindle bipolarity during prometaphase. Nonetheless, all abnormalities are rectified by anaphase and chromosome segregation appears normal. We conclude that ATK1 is required for normal microtubule accumulation at the spindle poles during prophase and possibly functions in spindle assembly during prometaphase. Because aberrant spindle morphology in these mutants is resolved by anaphase, we postulate that mitotic plant cells contain an error-correcting mechanism. Moreover, ATK1 function seems to be dosage-dependent, because cells containing one wild-type allele take significantly longer to proceed to anaphase as compared with cells containing two wild-type alleles.