REGULATION OF BIDIRECTIONAL MOTILITY OF Kinesin-5 Cin8 BY Loop8, REVEALED BY SINGLE MOLECULE MOTILITY ASSAY AND LIVE CELL IMAGING
Bipolar kinesin-5 motor proteins play central roles in establishing and maintaining the mitotic spindle during cell division. With two pairs of catalytic motor heads located at the opposite ends of tetrameric complex, kinesin-5 motors crosslink and slide apart antiparallel microtubules (MTs) while walking towards the plus-end of the MTs. Recent studies have demonstrated that some yeast kinesin-5 motors, including the S. cerevisiae Cin8 can move bidirectionally along microtubules, switching directionality under different conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011).
Previous study from our group suggested that a uniquely large Loop8 in the motor domain of Cin8 plays a role in the regulation of Cin8 directionality. In vivo Cin8(Δloop8) exhibited reduced midzone-directed motility and efficiency to support spindle elongation (Gerson-Gurwitz et al., 2011). We have found that Cin8 motility varies with its oligomeric state. We employed sensitive microscopy experiments to identify and analyze different oligomers of Cin8 separately. Using this technique and single molecule fluorescence motility essay for GFP labelled Cin8 (Cin8-GFP), we have explored the role of Loop8 in Cin8 motor domain. We found that deletion of the large Loop8 affects Cin8 binding to MTs, its motility and clustering characteristics. We have also made mutation at conserved position of Loop8 interface and found change in motility compared to wild type Cin8. Furthermore, we have cloned and recombinantly purified Loop8 constructs of Cin8 and performed biophysical and biochemical studies analyzing Loop8 binding to MTs. Overall this study reveals the mechanism by which Loop8 regulates the bidirectional motility of Cin8.
Düselder, A., Fridman, V., Thiede, C., Wiesbaum, A., Goldstein, A., Klopfenstein, D.R., Zaitseva, O., Janson, M.E., Gheber, L. and Schmidt, C.F., 2015. Deletion of the tail domain of the kinesin-5 Cin8 affects its directionality. Journal of Biological Chemistry, 290(27), pp.16841-16850.
Fridman, A. Gerson-Gurwitz, O. Shapira, N. Movshovich, S. Lakämper, C.F. Schmidt, and L. Gheber 2013. Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo. Journal of Cell Science 5;126:4147-59.
Gerson-Gurwitz A, Thiede C, Movshovich N, Fridman V, Podolskaya M, Danieli T, Lakämper S, Klopfenstein DR, Schmidt CF, Gheber L. 2011. Directionality of individual kinesin-5 Cin8 motors is modulated by loop 8, ionic strength and microtubule geometry. Embo J. 30:4942-4954.