CYK4 relaxes the bias in the off-axis motion by MKLP1 kinesin-6

Abstract

Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. CYK4-binding to the long neck region of MKLP1 restricts the configuration of the two MKLP1 motor domains in the centralspindlin. However, it is unclear how the CYK4-binding modulates the interaction of MKLP1 with a microtubule. Here, we performed three-dimensional nanometry of a microbead coated with multiple MKLP1 molecules on a freely suspended microtubule. We found that beads driven by dimeric MKLP1 exhibited persistently left-handed helical trajectories around the microtubule axis, indicating torque generation. By contrast, centralspindlin, like monomeric MKLP1, showed similarly left-handed but less persistent helical movement with occasional rightward movements. Analysis of the fluctuating helical movement indicated that the MKLP1 stochastically makes off-axis motions biased towards the protofilament on the left. CYK4-binding to the neck domains in MKLP1 enables more flexible off-axis motion of centralspindlin, which would help to avoid obstacles along crowded spindle microtubules.

The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

“…Gliding motility assays, as well as motility assays on freely suspended microtubules, showed a counterclockwise rotation for the plus-end-directed motor protein kinesin-8 (Kip3) 36,40 , while another study found that kinesin-8 can switch protofilaments in both directions 38 . Several other motor proteins also exhibit rotational movements, including kinesin-1 33,39 , kinesin-2 35 , cytoplasmic dynein 37 , and kinesin-6 (MKLP1) 42 . However, in contrast to this large body of knowledge on chiral motor stepping in vitro , the role of motor proteins and their asymmetric stepping in the generation of torques within microtubule bundles in vivo and consequently spindle twist is unknown.…”

Coupled yawing and orbital rotation in kinesin motility revealed by polarization measurement of gold nanorods

“…Previous studies proposed that sideward stepping of kinesins and dynein allows them to bypass roadblocks on a microtubule [6][7][8]11,13,35 . We performed Monte Carlo simulations of bypassing of a roadblock with various multiple-motor motilities, especially changing the two parameters: the off-axis asymmetry, 𝛼𝛼 𝑌𝑌 , and the torque generation, 𝜃𝜃 step (Fig.…”

“…Wild-type kinesin-2 and -8, which have longer neck-linker domains than kinesin-1, and kinesin-6, which has an atypical neck region, do not track protofilaments. All these kinesins move towards microtubule plus ends along left-handed helical trajectories, as shown in a bead assay 6,11 , a microtubule surface-gliding assay 8,[11][12][13] , and a single-molecule tracking assay 8 . Furthermore, teams of non-processive dimeric kinesin-14 Ncd [14][15][16] , which moves towards microtubule minus…”

“…ends, as well as teams of truncated single-headed kinesins-1 17,18 , -3 19 , -5 4,20 and -6 11 motors have all been shown to drive corkscrewing motions of microtubules in surfacegliding assays. The handedness of these motions is consistent.…”

“…Whilst plus-end directed kinesins move along a left-handed helix, minus-ended kinesins move along a righthanded helix. These helical motilities imply that the stroke (the unitary impulse of force and movement generated by the motor domains) has an off axis (torque) component, driven either by conformational changes in the motor domain or by the directionally biased selection of microtubule binding sites 6,8,11,15,17,19 (or potentially both). The underlying mechanisms and functional relevance of the torque component remain elusive.…”