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

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.

The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State

“…There is also evidence of communication through the tetrameric coiled-coil domain meaning that mechanical activity of one pair of heads may regulate the behavior of the motors at the opposite end of the molecule (46). Additionally, this model provides open questions on the ionic strength-dependent directional switching of yeast kinesin Cin8 (47,48) and the microtubule depolymerizing properties of yeast kinesin-5 Kip1p (49,50). Finally, phosphorylation may regulate motor activity (51).…”

Control mechanisms of microtubule overlap regions

“…At 3 nM ase1, the sliding velocity was 48% higher than in its absence (p<0.05). A similar decrease in velocity was reported for microtubule sliding by ScCin8 in cell extracts lacking Scase1 (Gerson-Gurwitz et al, 2011). Moreover, spindle elongation rates are lower in ase1∆ cells (Loïodice et al, 2005;Syrovatkina et al, 2013).…”

“…The velocity of gliding was slightly different for the two directions in our standard buffer containing MRB80 and 72mM KCl (1.17±0.33 µm/min; ±SE; N=37 for plus-end directed and 1.49±0.53 µm/ min; N =18 for minus-end directed gliding; p<0.05). Bidirectional gliding was earlier reported for kinesin-5 motors from budding and fission yeasts and similarly to ScCin8, we observed that minus-end-directed motion became more prevalent at higher ionic strength ( Figure 2C) (Gerson-Gurwitz et al, 2011;Roostalu et al, 2011;Fridman et al, 2013;Edamatsu, 2014). Because recombinant N-and C-terminal fusions of GFP and klp9 were not motile we could not simply establish whether individual klp9 motors moved towards microtubule minus-ends like other kinesin-5 motors from yeasts.…”

“…Different types of motors have been proposed to slide bundled microtubules apart. Kinesin-5 is responsible for spindle elongation in many cases (Sawin and Mitchison, 1995;Collins et al, 2014), but also kinesin-8 , kinesin-12, kinesin-14 and dynein are known to induce relative sliding (Fink et al, 2009;Tanenbaum et al, 2009Tanenbaum et al, , 2013Gerson-Gurwitz et al, 2011;Roostalu et al, 2011;Su et al, 2013). Some of these motors are plus end-directed and some are minus end-directed.…”

“…Traditionally, kinesin-5 motor proteins, like eg5, are purified from insect cells (Kapitein et al, 2005). Both kinesin-5 proteins from budding yeast, kip1 and cin8, were purified before in insect cells (Gordon and Roof, 1999;Gerson-Gurwitz et al, 2011;Roostalu et al, 2011) or from yeast cells overexpressing the protein (Gordon and Roof, 1999;Gerson-Gurwitz et al, 2011;Fridman et al, 2013). However, cut7, the other kinesin-5 from S. pombe was purified successfully from E. coli before (Edamatsu, 2014).…”