Flexible microtubule anchoring modulates the bi-directional motility of the kinesin-5 Cin8.

Two modes of motility have been reported for bi-directional kinesin-5 motors: (a) context-dependent directionality reversal, a mode in which motors undergo persistent minus-end directed motility at the single-molecule level and switch to plus-end directed motility in different assays or under different conditions, such as during MT gliding or antiparallel sliding or as a function of motor clustering; and (b) bi-directional motility, defined as movement in two directions in the same assay, without persistent unidirectional motility. Here, we examine how modulation of motor-microtubule (MT) interactions affects these two modes of motility for the bi-directional kinesin-5, Cin8. We report that the large insert in loop 8 (L8) within the motor domain of Cin8 increases the MT affinity of Cin8 in vivo and in vitro and is required for Cin8 intracellular functions. We consistently found that recombinant purified L8 directly binds MTs and L8 induces single Cin8 motors to behave according to context-dependent directionality reversal and bi-directional motility modes at intermediate ionic strength and according to a bi-directional motility mode in an MT surface-gliding assay under low motor density conditions. We propose that the largely unstructured L8 facilitates flexible anchoring of Cin8 to the MTs. This flexible anchoring enables the direct observation of bi-directional motility in motility assays. Remarkably, although L8-deleted Cin8 variants exhibit a strong minus-end directed bias at the single-molecule level, they also exhibit plus-end directed motility in an MT-gliding assay. Thus, L8-induced flexible MT anchoring is required for bi-directional motility of single Cin8 molecules but is not necessary for context-dependent directionality reversal of Cin8 in an MT-gliding assay.

Characterization of the motility of monomeric kinesin-5/Cin8.

Cin8, the Saccharomyces cerevisiae kinesin-5, has an essential role in mitosis. In in vitro motility assays, tetrameric and dimeric Cin8 constructs showed bidirectional motility in response to ionic strength or Cin8 motor density. However, whether property-switching directionality is present in a monomeric form of Cin8 is unknown. Here we engineered monomeric Cin8 constructs with and without the Cin8-specific ∼99 residues in the loop 8 domain and examined the directionality of these constructs using an in vitro polarity-marked microtubule gliding assay within the range of the motor density or ionic strength. We found that both monomeric constructs showed only plus end-directed activity over the ranges measured, which suggested that minus end-directed motility driven by Cin8 is necessary for at least dimeric forms. Using an in vitro microtubule corkscrewing assay, we also found that monomeric Cin8 corkscrewed microtubules around their longitudinal axes with a constant left-handed pitch. Overall, our results imply that plus-end-directed and left-handed motor activity comprise the intrinsic properties of the Cin8 motor domain as with other monomeric N-kinesins.

A potential physiological role for bi-directional motility and motor clustering of mitotic kinesin-5 Cin8 in yeast mitosis.

The bipolar kinesin-5 Cin8 switches from minus- to plus-end-directed motility under various conditions in vitro The mechanism and physiological significance of this switch remain unknown. Here, we show that under high ionic strength conditions, Cin8 moves towards and concentrates in clusters at the minus ends of stable and dynamic microtubules. Clustering of Cin8 induces a switch from fast minus- to slow plus-end-directed motility and forms sites that capture antiparallel microtubules (MTs) and induces their sliding apart through plus-end-directed motility. In early mitotic cells with monopolar spindles, Cin8 localizes near the spindle poles at microtubule minus ends. This localization is dependent on the minus-end-directed motility of Cin8. In cells with assembled bipolar spindles, Cin8 is distributed along the spindle microtubules. We propose that minus-end-directed motility is required for Cin8 clustering near the spindle poles before spindle assembly. Cin8 clusters promote the capture of microtubules emanating from the neighboring spindle poles and mediate their antiparallel sliding. This activity is essential to maximize microtubule crosslinking before bipolar spindle assembly and to induce the initial separation of the spindle poles.

Three Cdk1 sites in the kinesin-5 Cin8 catalytic domain coordinate motor localization and activity during anaphase.

The bipolar kinesin-5 motors perform essential functions in mitotic spindle dynamics. We previously demonstrated that phosphorylation of at least one of the Cdk1 sites in the catalytic domain of the Saccharomyces cerevisiae kinesin-5 Cin8 (S277, T285, S493) regulates its localization to the anaphase spindle. The contribution of these three sites to phospho-regulation of Cin8, as well as the timing of such contributions, remains unknown. Here, we examined the function and spindle localization of phospho-deficient (serine/threonine to alanine) and phospho-mimic (serine/threonine to aspartic acid) Cin8 mutants. In vitro, the three Cdk1 sites undergo phosphorylation by Clb2-Cdk1. In cells, phosphorylation of Cin8 affects two aspects of its localization to the anaphase spindle, translocation from the spindle-pole bodies (SPBs) region to spindle microtubules (MTs) and the midzone, and detachment from the mitotic spindle. We found that phosphorylation of S277 is essential for the translocation of Cin8 from SPBs to spindle MTs and the subsequent detachment from the spindle. Phosphorylation of T285 mainly affects the detachment of Cin8 from spindle MTs during anaphase, while phosphorylation at S493 affects both the translocation of Cin8 from SPBs to the spindle and detachment from the spindle. Only S493 phosphorylation affected the anaphase spindle elongation rate. We conclude that each phosphorylation site plays a unique role in regulating Cin8 functions and postulate a model in which the timing and extent of phosphorylation of the three sites orchestrates the anaphase function of Cin8.

Yeast Kinesin-5 Motor Protein CIN8 Promotes Accurate Chromosome Segregation.

Accurate chromosome segregation depends on bipolar chromosome-microtubule attachment and tension generation on chromosomes. Incorrect chromosome attachment results in chromosome missegregation, which contributes to genome instability. The kinetochore is a protein complex that localizes at the centromere region of a chromosome and mediates chromosome-microtubule interaction. Incorrect chromosome attachment leads to checkpoint activation to prevent anaphase onset. Kinetochore detachment activates the spindle assembly checkpoint (SAC), while tensionless kinetochore attachment relies on both the SAC and tension checkpoint. In budding yeast Saccharomyces cerevisiae, kinesin-5 motor proteins Cin8 and Kip1 are needed to separate spindle pole bodies for spindle assembly, and deletion of CIN8 causes lethality in the absence of SAC. To study the function of Cin8 and Kip1 in chromosome segregation, we constructed an auxin-inducible degron (AID) mutant, cin8-AID. With this conditional mutant, we first confirmed that cin8-AID kip1∆ double mutants were lethal when Cin8 is depleted in the presence of auxin. These cells arrested in metaphase with unseparated spindle pole bodies and kinetochores. We further showed that the absence of either the SAC or tension checkpoint was sufficient to abolish the cell-cycle delay in cin8-AID mutants, causing chromosome missegregation and viability loss. The tension checkpoint-dependent phenotype in cells with depleted Cin8 suggests the presence of tensionless chromosome attachment. We speculate that the failed spindle pole body separation in cin8 mutants could increase the chance of tensionless syntelic chromosome attachments, which depends on functional tension checkpoint for survival.

Synthetic-Evolution Reveals Narrow Paths to Regulation of the Saccharomyces cerevisiae Mitotic Kinesin-5 Cin8.

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions, but some substrates maintain precise phosphosite positions over billions of years. Here, we examined the phosphoregulation of the kinesin-5, Cin8, using synthetic Cdk1-sites. We first analyzed the three native Cdk1 sites within the catalytic motor domain. Any single site conferred regulation, but to different extents. Synthetic sites were then systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites, 8 disrupted function; 19 were neutral, similar to the phospho-deficient variant; and only two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one was immediately adjacent to a native Cdk1 site. Only one novel site position resulted in phospho-regulation. This site was sampled elsewhere in evolution, but the synthetic version was inefficient in S. cerevisiae. This study shows that a single phosphorylation site can modulate complex spindle dynamics, but likely requires further evolution to optimally regulate the precise reaction cycle of a mitotic motor.

Successive Kinesin-5 Microtubule Crosslinking and Sliding Promote Fast, Irreversible Formation of a Stereotyped Bipolar Spindle.

Separation of duplicated spindle poles is the first step in forming the mitotic spindle. Kinesin-5 crosslinks and slides anti-parallel microtubules (MTs), but it is unclear how these two activities contribute to the first steps in spindle formation. In this study, we report that in monopolar spindles, the duplicated spindle poles snap apart in a fast and irreversible step that produces a nascent bipolar spindle. Using mutations in Kinesin-5 that inhibit microtubule sliding, we show that the fast, irreversible pole separation is primarily driven by microtubule crosslinking. Electron tomography revealed microtubule pairs in monopolar spindles have short overlaps that intersect at high angles and are unsuited for ensemble Kinesin-5 sliding. However, maximal extension of a subset of anti-parallel microtubule pairs approaches the length of nascent bipolar spindles and is consistent with a Kinesin-5 crosslinking-driven transition. Nonetheless, microtubule sliding by Kinesin-5 contributes to stabilizing the nascent spindle and setting its stereotyped equilibrium length.

A Kinesin-5, Cin8, Recruits Protein Phosphatase 1 to Kinetochores and Regulates Chromosome Segregation.

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8's kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.