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

Himanshu Pandey, Sudhir Kumar Singh, Mayan Sadan, Mary Popov, Meenakshi Singh, Geula Davidov, Sayaka Inagaki, Jawdat Al-Bassam, Raz Zarivach, Steven S. Rosenfeld, Larisa Gheber

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Keywords

  • Bi-directional motility
  • Cin8
  • Context-dependent directionality reversal
  • Kinesin-5
  • Motor–microtubule interaction

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Cellular and Molecular Neuroscience
  • Cell Biology

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