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1.  B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast 
PLoS Genetics  2010;6(5):e1000935.
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.
Author Summary
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.
doi:10.1371/journal.pgen.1000935
PMCID: PMC2865516  PMID: 20463882
2.  Kinesin-related proteins required for assembly of the mitotic spindle 
The Journal of Cell Biology  1992;118(1):95-108.
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.
PMCID: PMC2289520  PMID: 1618910
3.  Structural basis for the assembly of the mitotic motor Kinesin-5 into bipolar tetramers 
eLife  2014;3:e02217.
Chromosome segregation during mitosis depends upon Kinesin-5 motors, which display a conserved, bipolar homotetrameric organization consisting of two motor dimers at opposite ends of a central rod. Kinesin-5 motors crosslink adjacent microtubules to drive or constrain their sliding apart, but the structural basis of their organization is unknown. In this study, we report the atomic structure of the bipolar assembly (BASS) domain that directs four Kinesin-5 subunits to form a bipolar minifilament. BASS is a novel 26-nm four-helix bundle, consisting of two anti-parallel coiled-coils at its center, stabilized by alternating hydrophobic and ionic four-helical interfaces, which based on mutagenesis experiments, are critical for tetramerization. Strikingly, N-terminal BASS helices bend as they emerge from the central bundle, swapping partner helices, to form dimeric parallel coiled-coils at both ends, which are offset by 90°. We propose that BASS is a mechanically stable, plectonemically-coiled junction, transmitting forces between Kinesin-5 motor dimers during microtubule sliding.
DOI: http://dx.doi.org/10.7554/eLife.02217.001
eLife digest
Successful cell division requires copies of the chromosomes containing the genetic material of a cell to be accurately copied and then separated so that when a cell divides, each new daughter cell contains exactly one copy of each chromosome. If this does not happen, the cell may malfunction or die.
To separate the duplicated chromosomes, a biological machine called the mitotic spindle forms inside the cell. This has two poles, one at each end, with each pole being responsible for gathering together the chromosomes for delivery to each of the daughter cells. Large numbers of long, thin protein tubes called microtubules extend out of each pole. Some microtubules attach to the chromosomes, whilst others are responsible for pushing apart the two poles—and the chromosomes attached to them—to the opposite sides of the cell before it divides.
To move the poles, motor proteins slide pairs of microtubules that are attached to opposite poles over each other. The Kinesin-5 family of motor proteins is particularly important for mitosis, because it is essential for forming the mitotic spindle and for making it work correctly. These motors assemble into motile machines that can apply a force to both of the microtubules in a sliding pair at the same time because they contain motor units at each end connected by a central rod.
The structure of this central rod is crucial for the successful operation of Kinesin-5. Scholey, Nithianantham et al. have now worked out the structure of a region of this filament called the bipolar assembly, or BASS domain. This structure is more complicated than expected: it contains four helixes made of protein that are all intertwined with each other.
In addition, Scholey, Nithianantham et al. found two ‘molecular pockets’ that small molecules can access. By entering the pockets, the molecules could disrupt the structure of the BASS domain, and consequently prevent Kinesin-5 from forming the dual-ended machines required to work properly. As Kinesin-5 is required to build the mitotic spindle, this would interfere with cell division. Targeting molecules into these pockets could therefore potentially form part of an anti-cancer therapy, preventing the rapid cell divisions behind the spread of the disease.
DOI: http://dx.doi.org/10.7554/eLife.02217.002
doi:10.7554/eLife.02217
PMCID: PMC3978770  PMID: 24714498
mitosis; Kinesin-5; microtubule; motor protein; coiled-coil; X-ray structure; D. melanogaster
4.  The Kip3-Like Kinesin KipB Moves along Microtubules and Determines Spindle Position during Synchronized Mitoses in Aspergillus nidulans Hyphae†  
Eukaryotic Cell  2004;3(3):632-645.
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.
doi:10.1128/EC.3.3.632-645.2004
PMCID: PMC420139  PMID: 15189985
5.  Mitotic Spindle Positioning in Saccharomyces cerevisiae Is Accomplished by Antagonistically Acting Microtubule Motor Proteins  
The Journal of Cell Biology  1997;138(5):1041-1053.
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.
PMCID: PMC2136752  PMID: 9281582
6.  Localization and function of Kinesin-5-like proteins during assembly and maintenance of mitotic spindles in Silvetia compressa 
BMC Research Notes  2009;2:106.
Background
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.
Findings
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.
Conclusion
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.
doi:10.1186/1756-0500-2-106
PMCID: PMC2706839  PMID: 19527496
7.  Novel Roles for Saccharomyces cerevisiae Mitotic Spindle Motors 
The Journal of Cell Biology  1999;147(2):335-350.
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.
PMCID: PMC2174232  PMID: 10525539
kinesin; dynein; motor proteins; microtubules; mitotic spindle
8.  Interplay of microtubule dynamics and sliding during bipolar spindle formation in mammalian cells 
Current biology : CB  2009;19(24):2108-2113.
Summary
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.
doi:10.1016/j.cub.2009.10.056
PMCID: PMC2805786  PMID: 19931454
9.  Functional Characterisation and Drug Target Validation of a Mitotic Kinesin-13 in Trypanosoma brucei 
PLoS Pathogens  2010;6(8):e1001050.
Mitotic kinesins are essential for faithful chromosome segregation and cell proliferation. Therefore, in humans, kinesin motor proteins have been identified as anti-cancer drug targets and small molecule inhibitors are now tested in clinical studies. Phylogenetic analyses have assigned five of the approximately fifty kinesin motor proteins coded by Trypanosoma brucei genome to the Kinesin-13 family. Kinesins of this family have unusual biochemical properties because they do not transport cargo along microtubules but are able to depolymerise microtubules at their ends, therefore contributing to the regulation of microtubule length. In other eukaryotic genomes sequenced to date, only between one and three Kinesin-13s are present. We have used immunolocalisation, RNAi-mediated protein depletion, biochemical in vitro assays and a mouse model of infection to study the single mitotic Kinesin-13 in T. brucei. Subcellular localisation of all five T. brucei Kinesin-13s revealed distinct distributions, indicating that the expansion of this kinesin family in kinetoplastids is accompanied by functional diversification. Only a single kinesin (TbKif13-1) has a nuclear localisation. Using active, recombinant TbKif13-1 in in vitro assays we experimentally confirm the depolymerising properties of this kinesin. We analyse the biological function of TbKif13-1 by RNAi-mediated protein depletion and show its central role in regulating spindle assembly during mitosis. Absence of the protein leads to abnormally long and bent mitotic spindles, causing chromosome mis-segregation and cell death. RNAi-depletion in a mouse model of infection completely prevents infection with the parasite. Given its essential role in mitosis, proliferation and survival of the parasite and the availability of a simple in vitro activity assay, TbKif13-1 has been identified as an excellent potential drug target.
Author Summary
Kinesins represent a class of mechanochemical enzymes that are able to move along microtubule filaments and transport cargo in a directional manner within the cell. Of particular importance are mitotic kinesins, as they ensure the accurate segregation of chromosomes and therefore cell survival. Such kinesins are involved in building and maintaining the mitotic microtubule-based spindle and in chromosome translocation during mitosis. Mitotic kinesins are potentially excellent drug targets because of their roles in an essential process of cell multiplication. Unregulated cell proliferation is associated with diseases such as cancer, but also many infectious diseases. Therefore, the identification of kinesins essential for the proliferation of parasites in the human host offers an attractive prospect for intervention. In our study we present a comprehensive biochemical and cell biological analysis of a mitotic kinesin in the protozoan parasite Trypanosoma brucei, causative agent of sleeping sickness in Africa. We show that this kinesin is essential for parasite survival not only in cultured cells but also in mice infected with this parasite and therefore establish this kinesin as a potential drug target in parasitic infections.
doi:10.1371/journal.ppat.1001050
PMCID: PMC2924347  PMID: 20808899
10.  Microtubule sliding activity of a kinesin-8 promotes spindle assembly and spindle length control 
Nature cell biology  2013;15(8):948-957.
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.
doi:10.1038/ncb2801
PMCID: PMC3767134  PMID: 23851487
11.  Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake 
The Journal of Cell Biology  2011;193(2):285-294.
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.
doi:10.1083/jcb.201011002
PMCID: PMC3080259  PMID: 21482719
12.  Dynamic Positioning of Mitotic Spindles in Yeast: 
Molecular Biology of the Cell  2000;11(11):3949-3961.
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.
PMCID: PMC15049  PMID: 11071919
13.  Mitotic Spindle Assembly around RCC1-Coated Beads in Xenopus Egg Extracts 
PLoS Biology  2011;9(12):e1001225.
Beads coated with the guanine nucleotide exchange factor RCC1 and a kinesin motor protein are sufficient to induce mitotic spindle assembly in Xenopus egg cytoplasm.
During cell division the genetic material on chromosomes is distributed to daughter cells by a dynamic microtubule structure called the mitotic spindle. Here we establish a reconstitution system to assess the contribution of individual chromosome proteins to mitotic spindle formation around single 10 µm diameter porous glass beads in Xenopus egg extracts. We find that Regulator of Chromosome Condensation 1 (RCC1), the Guanine Nucleotide Exchange Factor (GEF) for the small GTPase Ran, can induce bipolar spindle formation. Remarkably, RCC1 beads oscillate within spindles from pole to pole, a behavior that could be converted to a more typical, stable association by the addition of a kinesin together with RCC1. These results identify two activities sufficient to mimic chromatin-mediated spindle assembly, and establish a foundation for future experiments to reconstitute spindle assembly entirely from purified components.
Author Summary
The mitotic spindle is a bipolar structure that is responsible for separating the two sets of duplicated chromosomes in a dividing cell, thereby delivering one set to each of the two daughter cells. It is built from dynamic filaments called microtubules, as well as hundreds of other components that contribute to the organization and dynamics of the microtubules and to chromosome movement. To understand which proteins are essential for spindle formation and function, we would like to be able to build it from purified components. As a step towards this goal, we coupled individual proteins to inert glass beads (as a substitute for chromosomes), to determine what combination of proteins can induce spindle assembly in a complex cytoplasm derived from frog eggs. We found that a single enzyme called RCC1 is sufficient to activate a pathway that stabilizes and organizes microtubules into a bipolar structure around the bead, but that this bead then oscillated back and forth between the poles of the spindle. By coupling a microtubule-based motor protein together with RCC1 on the bead, we were able to balance the bead in the center of the spindle. Thus, two proteins immobilized on a bead can substitute for a chromosome and induce stable spindle formation.
doi:10.1371/journal.pbio.1001225
PMCID: PMC3246454  PMID: 22215983
14.  Spindle assembly requires complete disassembly of spindle remnants from the previous cell cycle 
Molecular Biology of the Cell  2012;23(2):258-267.
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.
doi:10.1091/mbc.E11-08-0701
PMCID: PMC3258171  PMID: 22090343
15.  KLP-18, a Klp2 Kinesin, Is Required for Assembly of Acentrosomal Meiotic Spindles in Caenorhabditis elegansV⃞ 
Molecular Biology of the Cell  2003;14(11):4458-4469.
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.
doi:10.1091/mbc.E03-05-0283
PMCID: PMC266765  PMID: 12937278
16.  Kinesin-related KIP3 of Saccharomyces cerevisiae Is Required for a Distinct Step in Nuclear Migration  
The Journal of Cell Biology  1997;138(5):1023-1040.
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.
PMCID: PMC2136764  PMID: 9281581
17.  Gamma-Tubulin Is Required for Bipolar Spindle Assembly and for Proper Kinetochore Microtubule Attachments during Prometaphase I in Drosophila Oocytes 
PLoS Genetics  2011;7(8):e1002209.
In many animal species the meiosis I spindle in oocytes is anastral and lacks centrosomes. Previous studies of Drosophila oocytes failed to detect the native form of the germline-specific γ-tubulin (γTub37C) in meiosis I spindles, and genetic studies have yielded conflicting data regarding the role of γTub37C in the formation of bipolar spindles at meiosis I. Our examination of living and fixed oocytes carrying either a null allele or strong missense mutation in the γtub37C gene demonstrates a role for γTub37C in the positioning of the oocyte nucleus during late prophase, as well as in the formation and maintenance of bipolar spindles in Drosophila oocytes. Prometaphase I spindles in γtub37C mutant oocytes showed wide, non-tapered spindle poles and disrupted positioning. Additionally, chromosomes failed to align properly on the spindle and showed morphological defects. The kinetochores failed to properly co-orient and often lacked proper attachments to the microtubule bundles, suggesting that γTub37C is required to stabilize kinetochore microtubule attachments in anastral spindles. Although spindle bipolarity was sometimes achieved by metaphase I in both γtub37C mutants, the resulting chromosome masses displayed highly disrupted chromosome alignment. Therefore, our data conclusively demonstrate a role for γTub37C in both the formation of the anastral meiosis I spindle and in the proper attachment of kinetochore microtubules. Finally, multispectral imaging demonstrates the presences of native γTub37C along the length of wild-type meiosis I spindles.
Author Summary
Proper chromosome segregation during cell division is essential. Missegregation of mitotic chromosomes leads to cell death or cancer, and chromosome missegregation during meiosis leads to miscarriage and birth defects. Cells utilize a bipolar microtubule-based structure known as the meiotic or mitotic spindle to segregate chromosomes. Because proper bipolar spindle formation is critically important for chromosome segregation, cells have many redundant mechanisms to ensure that this structure is properly formed. In most animal cells, centrosomes containing γ-tubulin protein complexes help organize and shape the bipolar spindle. Since meiosis I spindles in oocytes lack centrosomes, the mechanisms by which a meiotic bipolar spindle is assembled are not fully understood. In Drosophila oocytes it was not clear whether γ-tubulin played a role in bipolar spindle assembly or if it was even present on the meiotic spindle. We demonstrate that γ-tubulin plays vital roles in bipolar spindle formation and maintenance, as well as in aligning the chromosomes on the oocyte spindle. Additionally, we show that γ-tubulin is present on the bipolar spindle in Drosophila oocytes. More importantly, we demonstrate that γ-tubulin plays a critical role in the formation of the kinetochore microtubules that are required to properly orient chromosomes on the meiotic spindle.
doi:10.1371/journal.pgen.1002209
PMCID: PMC3154956  PMID: 21852952
18.  Control of Mitotic Spindle Position by the Saccharomyces cerevisiae Formin Bni1p  
The Journal of Cell Biology  1999;144(5):947-961.
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.
PMCID: PMC2148193  PMID: 10085293
mitotic spindle apparatus; cell division; microtubule; actin; Saccharomyces cerevisiae
19.  The roles of microtubule-based motor proteins in mitosis 
The Journal of Cell Biology  2003;162(6):1003-1016.
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.
doi:10.1083/jcb.200303022
PMCID: PMC2172859  PMID: 12975346
kinesin; spindle; dynein; centrosome; kinetochore
20.  Phosphorylation by Cdk1 Increases the Binding of Eg5 to Microtubules In Vitro and in Xenopus Egg Extract Spindles 
PLoS ONE  2008;3(12):e3936.
Background
Motor proteins from the kinesin-5 subfamily play an essential role in spindle assembly during cell division of most organisms. These motors crosslink and slide microtubules in the spindle. Kinesin-5 motors are phosphorylated at a conserved site by Cyclin-dependent kinase 1 (Cdk1) during mitosis. Xenopus laevis kinesin-5 has also been reported to be phosphorylated by Aurora A in vitro.
Methodology/Principal Findings
We investigate here the effect of these phosphorylations on kinesin-5 from Xenopus laevis, called Eg5. We find that phosphorylation at threonine 937 in the C-terminal tail of Eg5 by Cdk1 does not affect the velocity of Eg5, but strongly increases its binding to microtubules assembled in buffer. Likewise, this phosphorylation promotes binding of Eg5 to microtubules in Xenopus egg extract spindles. This enhancement of binding elevates the amount of Eg5 in spindles above a critical level required for bipolar spindle formation. We find furthermore that phosphorylation of Xenopus laevis Eg5 by Aurora A at serine 543 in the stalk is not required for spindle formation.
Conclusions/Significance
These results show that phosphorylation of Eg5 by Cdk1 has a direct effect on the interaction of this motor with microtubules. In egg extract, phosphorylation of Eg5 by Cdk1 ensures that the amount of Eg5 in the spindle is above a level that is required for spindle formation. This enhanced targeting to the spindle appears therefore to be, at least in part, a direct consequence of the enhanced binding of Eg5 to microtubules upon phosphorylation by Cdk1. These findings advance our understanding of the regulation of this essential mitotic motor protein.
doi:10.1371/journal.pone.0003936
PMCID: PMC2592692  PMID: 19079595
21.  Kinesin-5 motors are required for organization of spindle microtubules in Silvetia compressa zygotes 
BMC Plant Biology  2006;6:19.
Background
Monastrol, a chemical inhibitor specific to the Kinesin-5 family of motor proteins, was used to examine the functional roles of Kinesin-5 proteins during the first, asymmetric cell division cycle in the brown alga Silvetia compressa.
Results
Monastrol treatment had no effect on developing zygotes prior to entry into mitosis. After mitosis entry, monastrol treatment led to formation of monasters and cell cycle arrest in a dose dependent fashion. These findings indicate that Kinesin-5 motors maintain spindle bipolarity, and are consistent with reports in animal cells. At low drug concentrations that permitted cell division, spindle position was highly displaced from normal, resulting in abnormal division planes. Strikingly, application of monastrol also led to formation of numerous cytasters throughout the cytoplasm and multipolar spindles, uncovering a novel effect of monastrol treatment not observed in animal cells.
Conclusion
We postulate that monastrol treatment causes spindle poles to break apart forming cytasters, some of which capture chromosomes and become supernumerary spindle poles. Thus, in addition to maintaining spindle bipolarity, Kinesin-5 members in S. compressa likely organize microtubules at spindle poles. To our knowledge, this is the first functional characterization of the Kinesin-5 family in stramenopiles.
doi:10.1186/1471-2229-6-19
PMCID: PMC1564386  PMID: 16945151
22.  A Mutation in γ-Tubulin Alters Microtubule Dynamics and Organization and Is Synthetically Lethal with the Kinesin-like Protein Pkl1pV⃞ 
Molecular Biology of the Cell  2000;11(4):1225-1239.
Mitotic segregation of chromosomes requires spindle pole functions for microtubule nucleation, minus end organization, and regulation of dynamics. γ-Tubulin is essential for nucleation, and we now extend its role to these latter processes. We have characterized a mutation in γ-tubulin that results in cold-sensitive mitotic arrest with an elongated bipolar spindle but impaired anaphase A. At 30°C cytoplasmic microtubule arrays are abnormal and bundle into single larger arrays. Three-dimensional time-lapse video microscopy reveals that microtubule dynamics are altered. Localization of the mutant γ-tubulin is like the wild-type protein. Prediction of γ-tubulin structure indicates that non-α/β-tubulin protein–protein interactions could be affected. The kinesin-like protein (klp) Pkl1p localizes to the spindle poles and spindle and is essential for viability of the γ-tubulin mutant and in multicopy for normal cell morphology at 30°C. Localization and function of Pkl1p in the mutant appear unaltered, consistent with a redundant function for this protein in wild type. Our data indicate a broader role for γ-tubulin at spindle poles in regulating aspects of microtubule dynamics and organization. We propose that Pkl1p rescues an impaired function of γ-tubulin that involves non-tubulin protein–protein interactions, presumably with a second motor, MAP, or MTOC component.
PMCID: PMC14843  PMID: 10749926
23.  TOGp, the Human Homolog of XMAP215/Dis1, Is Required for Centrosome Integrity, Spindle Pole Organization, and Bipolar Spindle Assembly 
Molecular Biology of the Cell  2004;15(4):1580-1590.
The XMAP215/Dis1 MAP family is thought to regulate microtubule plus-end assembly in part by antagonizing the catastrophe-promoting function of kin I kinesins, yet XMAP215/Dis1 proteins localize to centrosomes. We probed the mitotic function of TOGp (human homolog of XMAP215/Dis1) using siRNA. Cells lacking TOGp assembled multipolar spindles, confirming results of Gergely et al. (2003. Genes Dev. 17, 336–341). Eg5 motor activity was necessary to maintain the multipolar morphology. Depletion of TOGp decreased microtubule length and density in the spindle by ∼20%. Depletion of MCAK, a kin I kinesin, increased MT lengths and density by ∼20%, but did not disrupt spindle morphology. Mitotic cells lacking both TOGp and MCAK formed bipolar and monopolar spindles, indicating that TOGp and MCAK contribute to spindle bipolarity, without major effects on MT stability. TOGp localized to centrosomes in the absence of MTs and depletion of TOGp resulted in centrosome fragmentation. TOGp depletion also disrupted MT minus-end focus at the spindle poles, detected by localizations of NuMA and the p150 component of dynactin. The major functions of TOGp during mitosis are to focus MT minus ends at spindle poles, maintain centrosome integrity, and contribute to spindle bipolarity.
doi:10.1091/mbc.E03-07-0544
PMCID: PMC379257  PMID: 14718566
24.  Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways. 
Molecular Biology of the Cell  1997;8(6):1035-1050.
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.
Images
PMCID: PMC305712  PMID: 9201714
25.  The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization 
The Journal of Cell Biology  2014;204(6):965-975.
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.
doi:10.1083/jcb.201312039
PMCID: PMC3998799  PMID: 24616221

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