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ABSTRACT Kinesin-5 motors are essential to separate mitotic spindle poles and assemble a bipolar spindle in many organisms. These motors crosslink and slide apart antiparallel microtubules via microtubule plus-end-directed motility. However, kinesin-5 localization is enhanced away from antiparallel overlaps. Increasing evidence suggests this localization occurs due to bidirectional motility or trafficking. The purified fission-yeast kinesin-5 protein Cut7 moves bidirectionally, but bidirectionality has not been shown in cells, and the function of the minus-end-directed movement is unknown. Here, we characterized the motility of Cut7 on bipolar and monopolar spindles and observed movement toward both plus- and minus-ends of microtubules. Notably, the activity of the motor increased at anaphase B onset. Perturbations to microtubule dynamics only modestly changed Cut7 movement, whereas Cut7 mutation reduced movement. These results suggest that the directed motility of Cut7 contributes to the movement of the motor. Comparison of the Cut7 mutant and human Eg5 (also known as KIF11) localization suggest a new hypothesis for the function of minus-end-directed motility and spindle-pole localization of kinesin-5s.
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Kinesin-5/Cut7 C-terminal tail phosphorylation is essential for microtubule sliding force and bipolar mitotic spindle assembly
Kinesin-5 motors play an essential role during mitotic spindle assembly in many organisms: they crosslink antiparallel spindle microtubules, step toward plus ends, and slide the microtubules apart. This activity separates the spindle poles and chromosomes. Kinesin-5s are not only plus-end-directed, but can walk or be carried toward MT minus ends where they show enhanced localization. The kinesin-5 C-terminal tail interacts with and regulates the motor, affecting structure, motility, and sliding force of purified kinesin-535–37 along with motility and spindle assembly in cells. The tail contains phosphorylation sites, particularly in the conserved BimC box. Nine mitotic phosphorylation sites were identified in the kinesin-5 motor of the fission yeast Schizosaccharomyces pombe, suggesting that multi-site phosphorylation may regulate kinesin-5s. Here, we show that mutating all nine sites to either alanine or glutamate causes temperature-sensitive lethality due to a failure of bipolar spindle assembly. We characterize kinesin-5 localization and sliding force in the spindle, based on Cut7-dependent microtubule minus-end protrusions in cells lacking kinesin-14 motors. Imaging and computational modeling show that Cut7p simultaneously moves toward minus ends of protrusion MTs and plus ends of spindle midzone MTs. Phosphorylation mutants show dramatic decreases in protrusions and sliding force. Comparison to a model of force to create protrusions suggests that tail truncation and phosphorylation mutants decrease Cut7p sliding force similarly to tail-truncated human Eg5. Our results show that C-terminal tail phosphorylation is required for kinesin-5/Cut7 sliding force and bipolar spindle assembly in fission yeast.
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- Award ID(s):
- 2133243
- PAR ID:
- 10611137
- Publisher / Repository:
- Cell Press
- Date Published:
- Journal Name:
- Current Biology
- Volume:
- 34
- Issue:
- 20
- ISSN:
- 0960-9822
- Page Range / eLocation ID:
- 4781 to 4793.e6
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Kinesin-5 motor proteins play essential roles during mitosis in most organisms. Their tetrameric structure and plus-end-directed motility allow them to bind to and move along antiparallel microtubules, thereby pushing spindle poles apart to assemble a bipolar spindle. Recent work has shown that the C-terminal tail is particularly important to kinesin-5 function: The tail affects motor domain structure, ATP hydrolysis, motility, clustering, and sliding force measured for purified motors, as well as motility, clustering, and spindle assembly in cells. Because previous work has focused on presence or absence of the entire tail, the functionally important regions of the tail remain to be identified. We have therefore characterized a series of kinesin-5/Cut7 tail truncation alleles in fission yeast. Partial truncation causes mitotic defects and temperature-sensitive growth, while further truncation that removes the conserved BimC motif is lethal. We compared the sliding force generated bycut7mutants using a kinesin-14 mutant background in which some microtubules detach from the spindle poles and are pushed into the nuclear envelope. These Cut7-driven protrusions decreased as more of the tail was truncated, and the most severe truncations produced no observable protrusions. Our observations suggest that the C-terminal tail of Cut7p contributes to both sliding force and midzone localization. In the context of sequential tail truncation, the BimC motif and adjacent C-terminal amino acids are particularly important for sliding force. In addition, moderate tail truncation increases midzone localization, but further truncation of residues N-terminal to the BimC motif decreases midzone localization.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1016/j.cub.2024.08.035
