Motility of Single Molecules and Clusters of Bi-directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

Himanshu Pandey, Tatiana Zvagelsky, Mary Popov, Mayan Sadan, Neta Yanir, Alina Goldstein-Levitin, Nurit Siegler, Shira Hershfinkel, Yahel Abraham, Roy Abraham, Levi A. Gheber, Larisa Gheber

Research output: Contribution to journalArticlepeer-review


The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Original languageAmerican English
Article numbere63425
JournalJournal of Visualized Experiments
Issue number180
StatePublished - 1 Feb 2022

All Science Journal Classification (ASJC) codes

  • General Chemical Engineering
  • General Immunology and Microbiology
  • General Biochemistry,Genetics and Molecular Biology
  • General Neuroscience


Dive into the research topics of 'Motility of Single Molecules and Clusters of Bi-directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells'. Together they form a unique fingerprint.

Cite this