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The acentrosomal spindle apparatus has kinetochore fibers organized and converged toward opposite poles; however, mechanisms underlying the organization of these microtubule fibers into an orchestrated bipolar array were largely unknown. Kinesin-14D is one of the four classes of Kinesin-14 motors that are conserved from green algae to flowering plants. In Arabidopsis thaliana, three Kinesin-14D members displayed distinct cell cycle-dependent localization patterns on spindle microtubules in mitosis. Notably, Kinesin-14D1 was enriched on the midzone microtubules of prophase and mitotic spindles and later persisted in the spindle and phragmoplast midzones. The kinesin-14d1 mutant had kinetochore fibers disengaged from each other during mitosis and exhibited hypersensitivity to the microtubule-depolymerizing herbicide oryzalin. Oryzalin-treated kinesin-14d1 mutant cells had kinetochore fibers tangled together in collapsed spindle microtubule arrays. Kinesin-14D1, unlike other Kinesin-14 motors, showed slow microtubule plus end-directed motility, and its localization and function were dependent on its motor activity and the novel malectin-like domain. Our findings revealed a Kinesin-14D1-dependent mechanism that employs interpolar microtubules to regulate the organization of kinetochore fibers for acentrosomal spindle morphogenesis.

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The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.

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Plant cells form acentrosomal spindles with microtubules (MTs) converged toward two structurally undefined poles by employing MT minus end-directed Kinesin-14 motors. To date, it is unclear whether the convergent bipolar MT array assumes unified poles in plant spindles, and if so, how such a goal is achieved. Among six classes of Kinesin-14 motors in Arabidopsis thaliana, the Kinesin-14A motors ATK1 (KatA) and ATK5 share the essential function in spindle morphogenesis. To understand how the two functionally redundant Kinesin-14A motors contributed to the spindle assembly, we had ATK1-GFP and ATK5-GFP fusion proteins expressed in their corresponding null mutants and found that they were functionally comparable to their native forms. Although ATK1 was a nuclear protein and ATK5 cytoplasmic prior to nuclear envelop breakdown, at later mitotic stages, the two motors shared similar localization patterns of uniform association with both spindle and phragmoplast MTs. We found that ATK1 and ATK5 were rapidly concentrated toward unified polar foci when cells were under hyperosmotic conditions. Concomitantly, spindle poles became perfectly focused as if there were centrosome-like MT-organizing centers where ATK1 and ATK5 were highly enriched and at which kinetochore fibers pointed. The separation of ATK1/ATK5-highlighted MTs from those of kinetochore fibers suggested that the motors translocated interpolar MTs. Our protein purification and live-cell imaging results showed that ATK1 and ATK5 are associated with each other in vivo. The stress-induced spindle pole convergence was also accompanied by poleward accumulation of the MT nucleator γ-tubulin. These results led to the conclusion that the two Kinesin-14A motors formed oligomeric motor complexes that drove MT translocation toward the spindle pole to establish acentrosomal spindles with convergent poles.

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The maize abnormal chromosome 10 (Ab10) haplotype encodes a meiotic drive system that converts heterochromatic knobs into centromere-like bodies that are preferentially segregated through female meiosis. Ab10 was first described in the 1940s and has been intensively studied. Here I provide a comprehensive review of the literature, starting from the discovery of knobs and Ab10, preceding through the classic literature, and finishing with molecular structure and mechanisms. The defining features of the Ab10 haplotype are its two specialized kinesins, Kinesin driver and TR-1 kinesin, that activate neocentromeres at knobs containing different classes of the tandem repeat. In most Ab10 haplotypes, the two kinesin/knob systems cooperate to promote maximum meiotic drive. However, recent interpretations suggest that each kinesin/knob system can function as an independent meiotic driver and that in some cases they compete with each other. Ab10 is present at low frequencies throughout the genus Zea and has significantly expanded genome size by promoting the formation of knobs throughout the genome.

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Cells form a bipolar spindle during mitosis to ensure accurate chromosome segregation. Proper spindle architecture is established by a set of kinesin motors and microtubule-associated proteins. In most eukaryotes, kinesin-5 motors are essential for this process, and genetic or chemical inhibition of their activity leads to the emergence of monopolar spindles and cell death. However, these deficiencies can be rescued by simultaneous inactivation of kinesin-14 motors, as they counteract kinesin-5. We conducted detailed genetic analyses in fission yeast to understand the mechanisms driving spindle assembly in the absence of kinesin-5. Here, we show that deletion of the dri1 gene, which encodes a putative RNA-binding protein, can rescue temperature sensitivity caused by cut7-22, a fission yeast kinesin-5 mutant. Interestingly, kinesin-14/Klp2 levels on the spindles in the cut7 mutants were significantly reduced by the dri1 deletion, although the total levels of Klp2 and the stability of spindle microtubules remained unaffected. Moreover, RNA-binding motifs of Dri1 are essential for its cytoplasmic localization and function. We have also found that a portion of Dri1 is spatially and functionally sequestered by chaperone-based protein aggregates upon mild heat stress and limits cell division at high temperatures. We propose that Dri1 might be involved in post-transcriptional regulation through its RNA-binding ability to promote the loading of Klp2 on the spindle microtubules.

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Maize heterochromatic knobs cheat female meiosis by forming neocentromeres that bias their segregation into the future egg cell. In this issue of Genes & Development, Swentowsky and colleagues (pp. 1239-1251) show that two types of knobs, those composed of 180-bp and TR1 sequences, recruit their own novel and divergent kinesin-14 family members to form neocentromeres.

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Kinesin-14s are microtubule-based motor proteins that play important roles in mitotic spindle assembly [1]. Ncd-type kinesin-14s are a subset of kinesin-14 motors that exist as homodimers with an N-terminal microtubule-binding tail, a coiled-coil central stalk (central stalk), a neck, and two identical C-terminal motor domains. To date, no Ncd-type kinesin-14 has been found to naturally exhibit long-distance minus-end-directed processive motility on single microtubules as individual homodimers. Here, we show that GiKIN14a from Giardia intestinalis [2] is an unconventional Ncd-type kinesin-14 that uses its N-terminal microtubule-binding tail to achieve minus-end-directed processivity on single microtubules over micrometer distances as a homodimer. We further find that although truncation of the N-terminal tail greatly reduces GiKIN14a processivity, the resulting tailless construct GiKIN14a-Δtail is still a minimally processive motor and moves its center of mass via discrete 8-nm steps on the microtubule. In addition, full-length GiKIN14a has significantly higher stepping and ATP hydrolysis rates than does GiKIN14a-Δtail. Inserting a flexible polypeptide linker into the central stalk of full-length GiKIN14a nearly reduces its ATP hydrolysis rate to that of GiKIN14a-Δtail. Collectively, our results reveal that the N-terminal tail of GiKIN14a is a de facto dual regulator of motility and reinforce the notion of the central stalk as a key mechanical determinant of kinesin-14 motility [3].

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A maize chromosome variant called abnormal chromosome 10 (Ab10) converts knobs on chromosome arms into neocentromeres, causing their preferential segregation to egg cells in a process known as meiotic drive. We previously demonstrated that the gene Kinesin driver (Kindr) on Ab10 encodes a kinesin-14 required to mobilize neocentromeres made up of the major tandem repeat knob180. Here we describe a second kinesin-14 gene, TR-1 kinesin (Trkin), that is required to mobilize neocentromeres made up of the minor tandem repeat TR-1. Trkin lies in a 4-Mb region of Ab10 that is not syntenic with any other region of the maize genome and shows extraordinary sequence divergence from Kindr and other kinesins in plants. Despite its unusual structure, Trkin encodes a functional minus end-directed kinesin that specifically colocalizes with TR-1 in meiosis, forming long drawn out neocentromeres. TRKIN contains a nuclear localization signal and localizes to knobs earlier in prophase than KINDR. The fact that TR-1 repeats often co-occur with knob180 repeats suggests that the current role of the TRKIN/TR-1 system is to facilitate the meiotic drive of the KINDR/knob180 system.

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Minus-end directed, non-processive kinesin-14 Ncd is a dimeric protein with C-terminally located motor domains (heads). Generation of the power-stroke by Ncd consists of a lever-like rotation of a long superhelical 'stalk' segment while one of the kinesin's heads is bound to the microtubule. The last ∼30 amino acids of Ncd head play a crucial but still poorly understood role in this process. Here, we used accelerated molecular dynamics simulations to explore the conformational dynamics of several systems built upon two crystal structures of Ncd, the asymmetrical T436S mutant in pre-stroke/post-stroke conformations of two partner subunits and the symmetrical wild-type protein in pre-stroke conformation of both subunits. The results revealed a new conformational state forming following the inward motion of the subunits and stabilized with several hydrogen bonds to residues located on the border or within the C-terminal linker, i.e. a modeled extension of the C-terminus by residues 675-683. Forming of this new, compact Ncd conformation critically depends on the length of the C-terminus extending to at least residue 681. Moreover, the associative motion leading to the compact conformation is accompanied by a partial lateral rotation of the stalk. We propose that the stable compact conformation of Ncd may represent an initial state of the working stroke.

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Microtubule mechanical properties are essential for understanding basic cellular processes, including cell motility and division, but the forces that result in microtubule rupture or breakage have not yet been measured directly. These forces are essential to understand the mechanical properties of the cytoskeleton and responses by cells to both normal conditions and stress caused by injury or disease. Here we estimate the force required to rupture a microtubule by analyzing kinesin-14 Ncd motor-induced microtubule breakage in ensemble motility assays. We model the breakage events as caused by Ncd motors pulling or pushing on single microtubules that are clamped at one end by other motors attached to the glass surface. The number of pulling or pushing Ncd motors is approximated from the length of the microtubule bound to the surface and the forces produced by the pulling or pushing motors are estimated from forces produced by the Ncd motor in laser-trap assays, reported by others. Our analysis provides an estimate, to the first approximation, of ~ 500 pN for the minimal force required to rupture a 13-pf microtubule. The value we report is close to the forces estimated from microtubule stretching/fragmentation experiments and overlaps with the forces applied by AFM in microtubule indentation assays that destabilize microtubules and break microtubule protofilaments. It is also consistent with the forces required to disrupt protein noncovalent bonds in force spectroscopy experiments. These findings are relevant to microtubule deformation and breakage caused by cellular tension in vivo.

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KIFC1 (also called HSET or kinesin-14a) is best known as a multifunctional motor protein essential for mitosis. The present studies are the first to explore KIFC1 in terminally postmitotic neurons. Using RNA interference to partially deplete KIFC1 from rat neurons (from animals of either gender) in culture, pharmacologic agents that inhibit KIFC1, and expression of mutant KIFC1 constructs, we demonstrate critical roles for KIFC1 in regulating axonal growth and retraction as well as growth cone morphology. Experimental manipulations of KIFC1 elicit morphological changes in the axon as well as changes in the organization, distribution, and polarity orientation of its microtubules. Together, the results indicate a mechanism by which KIFC1 binds to microtubules in the axon and slides them into alignment in an ATP-dependent fashion and then cross-links them in an ATP-independent fashion to oppose their subsequent sliding by other motors.SIGNIFICANCE STATEMENT Here, we establish that KIFC1, a molecular motor well characterized in mitosis, is robustly expressed in neurons, where it has profound influence on the organization of microtubules in a number of different functional contexts. KIFC1 may help answer long-standing questions in cellular neuroscience such as, mechanistically, how growth cones stall and how axonal microtubules resist forces that would otherwise cause the axon to retract. Knowledge about KIFC1 may help researchers to devise strategies for treating disorders of the nervous system involving axonal retraction given that KIFC1 is expressed in adult neurons as well as developing neurons.

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A common mitotic defect observed in cancer cells that possess supernumerary (more than two) centrosomes is multipolar spindle formation [1, 2]. Such structures are resolved into a bipolar geometry by minus-end-directed motor proteins, such as cytoplasmic dynein and the kinesin-14 HSET [3-8]. HSET is also thought to antagonize plus-end-directed kinesin-5 Eg5 to balance spindle forces [4, 5, 7, 9]. However, the biomechanics of this force opposition are unclear, as HSET has previously been defined as a non-processive motor [10-16]. Here, we use optical trapping to elucidate the mechanism of force generation by HSET. We show that a single HSET motor has a processive nature with the ability to complete multiple steps while trapped along a microtubule and when unloaded can move in both directions for microns. Compared to other kinesins, HSET has a relatively weak stall force of 1.1 pN [17, 18]. Moreover, HSET's tail domain and its interaction with the E-hook of tubulin are necessary for long-range motility. In vitro polarity-marked bundle assays revealed that HSET selectively generates force in anti-parallel bundles on the order of its stall force. When combined with varied ratios of Eg5, HSET adopts Eg5's directionality while acting as an antagonizing force brake, requiring at least a 10-fold higher Eg5 concentration to surpass HSET's sliding force. These results reveal HSET's ability to change roles within the spindle from acting as an adjustable microtubule slider and force regulator to a processive motor that aids in minus end focusing.

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Mitotic kinesin-14 homodimers that contain an N-terminal nonmotor microtubule-binding tail contribute to spindle organization by preferentially crosslinking two different spindle microtubules rather than interacting with a single microtubule to generate processive motility. However, the mechanism underlying such selective motility behavior remains poorly understood. Here, we show that when a flexible polypeptide linker is inserted into the coiled-coil central stalk, two homodimeric mitotic kinesin-14s of distinct motility-the processive plus-end-directed KlpA from Aspergillus nidulans [1] and the nonprocessive minus-end-directed Ncd from Drosophila melanogaster [2]-both switch to become processive minus-end-directed motors. Our results demonstrate that the polypeptide linker introduces greater conformational flexibility into the central stalk. Importantly, we find that the linker insertion significantly weakens the ability of Ncd to preferentially localize between and interact with two microtubules. Collectively, our results reveal that besides the canonical role of enabling dimerization, the central stalk also functions as a mechanical component to determine the motility of homodimeric mitotic kinesin-14 motors. We suggest that the central stalk is an evolutionary design that primes these kinesin-14 motors for nontransport roles within the mitotic spindle.

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Many human cancer cells contain more than two centrosomes, yet these cancer cells can form pseudo-bipolar spindles through the mechanism, called centrosome clustering, and survive, instead of committing lethal multipolar mitoses. Kinesin-14/HSET, a minus end-directed motor, plays a crucial role in centrosome clustering. Accordingly, HSET is deemed to be a promising chemotherapeutic target to selectively kill cancer cells. Recently, three HSET inhibitors (AZ82, CW069 and SR31527) have been reported, but their specificity and efficacy have not been evaluated rigorously. This downside partly stems from the lack of robust systems for the assessment of these drugs. Yeasts and filamentous fungi provide not only powerful models for basic and applied biology but also versatile tools for drug discovery and evaluation. Here we show that these three inhibitors on their own are cytotoxic to fission yeast, suggesting that they have off-targets in vivo except for kinesin-14. Nonetheless, intriguingly, AZ82 can neutralize otherwise toxic overproduced HSET; this includes a substantial reduction in the percentage of HSET-driven abnormal mitotic cells and partial suppression of its lethality. SR31527 also displays modest neutralizing activity, while we do not detect such activity in CW069. As an experimental proof-of-principle study, we have treated HSET-overproducing fission yeast cells with extracts prepared from various plant species and found activities that rescue HSET-driven lethality in those from Chamaecyparis pisifera and Toxicodendron trichocarpum. This methodology of protein overproduction in fission yeast, therefore, provides a convenient, functional assay system by which to screen for not only selective human kinesin-14 inhibitors but also those against other molecules of interest.

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C-terminus kinesin motor KIFC1 is known for centrosome clustering in cancer cells with supernumerary centrosomes. KIFC1 crosslinks and glides on microtubules (MT) to assist normal bipolar spindle formation to avoid multi-polar cell division, which might be fatal. Testis cancer is the most common human cancer among young men. However, the gene expression profiles of testis cancer is still not complete and the expression of the C-terminus kinesin motor KIFC1 in testis cancer has not yet been examined. We found that KIFC1 is enriched in seminoma tissues in both mRNA level and protein level, and is specifically enriched in the cells that divide actively. Cell experiments showed that KIFC1 may be essential in cell division, but not essential in metastasis. Based on subcellular immuno-florescent staining results, we also described the localization of KIFC1 during cell cycle. By expressing ΔC-FLAG peptide in the cells, we found that the tail domain of KIFC1 might be essential for the dynamic disassociation of KIFC1, and the motor domain of KIFC1 might be essential for the degradation of KIFC1. Our work provides a new perspective for seminoma research.

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The Golgi apparatus is the central organelle along the eukaryotic secretory and endocytic pathway. In non-polarized mammalian cells, the Golgi complex is usually located proximal to the nucleus at the cell center and is closely associated with the microtubule organizing center. Microtubule networks are essential in the organization and central localization of the Golgi apparatus, but the molecular basis underlying these processes are poorly understood. Here we reveal that minus end-directed kinesin-14 KIFC1 proteins are required for the structural integrity and positioning of the Golgi complex in non-polarized mammalian cells. Remarkably, we found that the motor domain of kinesin-14 KIFC1 regulates the recognition and binding of the Golgi and KIFC1 also statically binds to the microtubules via its tail domain. These findings reveal a new stationary binding model that kinesin-14 KIFC1 proteins function as crosslinkers between the Golgi apparatus and the microtubules and contribute to the central positioning and structural maintenance of the Golgi apparatus.

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Microtubule-organizing centers (MTOCs) nucleate microtubules that can grow autonomously in any direction. To generate bundles of parallel microtubules originating from a single MTOC, the growth of multiple microtubules needs to coordinated, but the underlying mechanism is unknown. Here, we show that a conserved two-component system consisting of the plus-end tracker EB1 and the minus-end-directed molecular motor Kinesin-14 is sufficient to promote parallel microtubule growth. The underlying mechanism relies on the ability of Kinesin-14 to guide growing plus ends along existing microtubules. The generality of this finding is supported by yeast, Drosophila, and human EB1/Kinesin-14 pairs. We demonstrate that plus-end guiding involves a directional switch of the motor due to a force applied via a growing microtubule end. The described mechanism can account for the generation of parallel microtubule networks required for a broad range of cellular functions such as spindle assembly or cell polarization.

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Globozoospermia is a rare (prevalence of <0.1%) but severe male infertility condition. In our previous study, we found that robust KIFC1 immunostaining was detected in the human elongating/elongated spermatids during human acrosomogenesis. However, the relationship between the decreased expression of KIFC1 and human globozoospermia remains largely unknown. Testicular biopsies of 30 globozoospermia and 30 obstructive azoospermia patients who underwent infertility evaluation and treatment were utilized in this study. Reverse transcriptase polymerase chain reaction (RT-PCR), Western blots, immunohistochemistry, an in vivo model, and intratesticular injection of small inhibitory RNA (siRNA) against the Kifc1 gene were employed, and sperm abnormalities were evaluated by hematoxylin and eosin (H&E) staining and immunocytochemistry. We revealed that the testicular level of KIFC1 mRNA in globozoospermia was significantly reduced compared with that in obstructive azoospermia, and the KIFC1 protein was barely detectable in testicular specimens in 30% (9 of 30) of patients with globozoospermia. Furthermore, knockdown of the Kifc1 gene in mice increased the percentage of sperm with globozoospermic defects (26.5%). Decreased KIFC1 expression was mainly observed in the testes of patients with globozoospermia at the spermatid stage, which may be useful for counseling and management of such patients.

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End-binding proteins are capable of tracking the plus-ends of growing microtubules (MTs). The motor protein Ncd, a member of the kinesin-14 family, interacts with EB1 protein and becomes a non-autonomous tip-tracker. Here, we attempted to find out whether at least for Ncd, the efficient EB1-mediated tip-tracking involves the interaction of the kinesin with the MT surface. We prepared a series of Ncd tail mutants in which the MT-binding sites were altered or eliminated. Using TIRF microscopy, we characterized their behavior as tip-trackers and measured the dwell times of single molecules of EB1 and Ncd tail or its mutated forms. The mutated forms of Ncd tail exhibited tip-tracking in the presence of EB1 and the effectiveness of this process was proportional to the affinity of the mutant's tail to MT. Even though the interaction of Ncd with EB1 was weak (Kd∼9µM) the half saturating concentration of EB1 for tip-tracking was 7nM. The dwell time of Ncd tail in the presence of EB1 was ∼1s. The dwell time of EB1 alone was shorter (∼0.3s) and increased considerably in the presence of a large excess of Ncd tail. We demonstrated that tip-tracking of kinesin-14 occurs through several concurrent mechanisms: binding of kinesin only to EB1 located at the MT end, interaction of the kinesin molecules with a composite site formed by EB1 and the MT tip, and probably surface diffusion of the tail along MT. The second mechanism seems to play a crucial role in efficient tip-tracking.

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The classic maize mutant divergent spindle-1 (dv1) causes failures in meiotic spindle assembly and a decrease in pollen viability. By analyzing two independent dv1 alleles we demonstrate that this phenotype is caused by mutations in a member of the kinesin-14A subfamily, a class of C-terminal, minus-end directed microtubule motors. Further analysis demonstrates that defects in early spindle assembly are rare, but that later stages of spindle organization promoting the formation of finely focused spindle poles are strongly dependent on Dv1. Anaphase is error-prone in dv1 lines but not severely so, and the majority of cells show normal chromosome segregation. Live-cell imaging of wild type and mutant plants carrying CFP-tagged ß-tubulin confirm that meiosis in dv1 lines fails primarily at the pole-sharpening phase of spindle assembly. These data indicate that plant kinesin-14A proteins help to enforce bipolarity by focusing spindle poles and that this stage of spindle assembly is not required for transition through the spindle checkpoint but improves the accuracy of chromosome segregation.