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.
Highlights
Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis
Centralspindlin is a heterotetrameric protein consisting of a dimer of the kinesin-6 motor subunit MKLP1, and a dimer of the non-motor subunit, CYK44,7
We report that dimeric C. elegans MKLP1 drives a left-handed helical motion around the microtubule with minimum protofilament switching to the right side while the left-handed helical motions driven by monomers or by the heterotetramers with CYK4 are less persistent, showing more frequent off-axis motion to the right side
Summary
Centralspindlin, a complex of the MKLP1 kinesin-6 and CYK4 GAP subunits, plays key roles in metazoan cytokinesis. In many kinesins with an N-terminal motor domain (Nkinesins), the ~15 amino acid neck-linker, which links the microtubule-binding catalytic core to the neck coiled coil, has been shown to play crucial roles in the mechanochemical transduction of the chemical energy released by ATP hydrolysis to the mechanical works such as alternate stepping on a microtubule[1,2]. For the functions of centralspindlin in cytokinesis, its timely and sharp accumulation to the center of the central spindle and the midbody is essential[12,13,14,15] This relies on its plus-end directed motility, which is modulated by the CYK-binding to the neck of MKLP1 and by further assembly of the heterotetramers into multimeric clusters. We discuss the implications of these observations for the efficient motility in the crowded mitotic spindle
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