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 mo...

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Veröffentlicht in:Biochemical and biophysical research communications 2021-05, Vol.555, p.115-120
Hauptverfasser: Yamagishi, Masahiko, Maruyama, Yohei, Sugawa, Mitsuhiro, Yajima, Junichiro
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Sprache:eng
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Zusammenfassung: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. •Monomeric form of Cin8 kinesin-5 generates only plus end-directed force.•Deletion of the unique insertion in monomeric Cin8 does not cause bi-directionality.•Cin8 directional reversal requires at least linked pairs of motor domains.•Monomeric Cin8 molecules cause microtubules to corkscrew with a left-handed pitch.•Kinesin motor domain might have intrinsic plus-ended and left-handed directionality.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2021.03.134