ISM2019 (Microscopy)

SINGLE MOLECULE MOTILITY AND LIVE IMAGING MICROSCOPY ANALYSIS REVEALS COMPLEX PHOSPHO-REGULATION PATHWAYS OF A MITOTIC MOTOR PROTEIN

Alina Goldstein-Levitin 1 Nurit Siegler 1 Darya Goldman 1 Mary Popov 1 Himanshu Pandy 1 Ervin Valk 2 Mart Loog 2 Liam Holt 3 Larisa Gheber 1
1Department of Chemistry and Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel
2Institute of Technology, University of Tartu, Tartu, Estonia
3Institute for Systems Genetics, New York University Langone Health, New York, NY, USA

Kinesin-5 motor proteins are homotetrameric proteins carrying two sets of catalytic motor domains on opposite sides of the active complex which allows them to cross-link and slide apart anti-parallel microtubules (MTs) emanating from each pole of the bipolar spindle. S. cerevisiae cells express two kinesin-5 homologues, Cin8 and Kip1, which partially overlap in function, perform essential roles in spindle assembly and elongation and at least one functional kinesin-5 is required to support yeast viability. Recently it was found that the S. cerevisiae kinsesin-5s; Cin8 and Kip1 are phospho-regulated by Cdk1, which governs their localization to the mitotic spindle during mitosis. Cin8 contains five putative Cdk1 phosphorylation sites, three in the motor domain and two in the stalk and tail. Although it was demonstrated that phospho-regulation of Cin8 at its motor domain by Cdk1 regulates its localization, the contribution of each of the three Cdk1 sites was never explored. We first established the role of each of the three Cdk1 sites in the motor domain by employing an analysis method specifically developed for quantifying Cin8 distribution in live cells. We then combined these results to analysis by in vitro single molecule assay conducted on purified mutants of Cin8. Since Cin8 changes its motile properties when walking alone or in clusters, we conducted an advanced intensity analysis to distinguish between the two modes and determine their motile properties. We found that one of the sites, S277, located in a large insert in loop 8, is the major regulator of Cin8 phospho-regulation in vivo, and responsible for most of Cin8 phospho-regulation of localization and functionality in vivo, however the mostly conserved site at position S493 is responsible for regulating Cin8 motility and association to MTs. In addition, we tested the rigidity of phospho-regulation of Cin8, and examined whether phosphorylation at newly created Cdk1 sites can mimic the known phospho-regulation or create new phenotypes. For this purpose, we generated phospho-deficient mutant of Cin8 and introduced new Cdk1 sites by single amino acid replacement and examined them by in vivo microscopy analysis methods we established before. We found that out of 29 novel sites, only two novel sites resulted in phospho-regulation of Cin8, although they were not able to fully recapitulate the original phosphor-regulation of Cin8. This result indicates that phospho-regulation of Cin8 by Cdk1 at this region is rigid and highly dependent on the structural context.









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