Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry

EMBO J. 2011 Nov 18;30(24):4942-54. doi: 10.1038/emboj.2011.403.

Abstract

Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (∼25 nm/s) towards the midzone, but occasionally also faster (∼55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Gene Deletion
  • Kinesin / chemistry
  • Kinesin / genetics
  • Kinesin / metabolism*
  • Microscopy, Fluorescence
  • Microtubules / metabolism*
  • Microtubules / ultrastructure
  • Movement
  • Osmolar Concentration
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae / ultrastructure
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spindle Apparatus / metabolism
  • Spindle Apparatus / ultrastructure

Substances

  • CIN8 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Kinesin