Abstract
Centralspindlin is a critical regulator of cytokinesis in animal cells. It is a tetramer consisting of ZEN-4/MKLP1, a kinesin-6 motor, and CYK-4/MgcRacGAP, a Rho GTPase-activating protein. At anaphase, centralspindlin localizes to a narrow region of antiparallel microtubule overlap and initiates central spindle assembly. Central spindle assembly requires complex formation between ZEN-4 and CYK-4. However, the structural consequences of CYK-4 binding to ZEN-4 are unclear as are the mechanisms of microtubule bundling. Here we investigate whether CYK-4 binding induces a conformational change in ZEN-4. Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4. Additionally, our data indicate that each neck linker is similarly structured in the bound and unbound states. CYK-4 binding decreases the rate of ZEN-4-mediated microtubule gliding. These results constrain models for the molecular organization of centralspindlin.
Highlights
Cytokinesis requires formation of the centralspindlin complex
Characterization of the structure and conformational dynamics of the minimal interacting regions between ZEN-4 and CYK-4 by continuous wave EPR and double electron-electron resonance (DEER) spectroscopy reveals that CYK-4 binding dramatically stabilizes the relative positions of the neck linker regions of ZEN-4
Using continuous wave electron paramagnetic resonance (CW-EPR)3 and double electron-electron resonance (DEER) spectroscopy methods, we demonstrate that CYK-4 binding results in a conformational change in the neck linker region of ZEN-4
Summary
Cytokinesis requires formation of the centralspindlin complex. Results: The neck linker regions in ZEN-4 are highly mobile when free, but their mobility is greatly restricted by CYK-4 binding. Bundling of the antiparallel microtubules that comprise the central spindle requires centralspindlin, a heterotetrameric complex comprised of two proteins: a kinesin-6 family motor protein, ZEN-4/MKLP1, and a Rho GTPase-activating protein, CYK-4/MgcRacGAP [2]. Using continuous wave electron paramagnetic resonance (CW-EPR) and double electron-electron resonance (DEER) spectroscopy methods, we demonstrate that CYK-4 binding results in a conformational change in the neck linker region of ZEN-4 These changes reduce the rate of microtubule gliding by ZEN-4, and they are likely important for antiparallel microtubule bundling in vivo. These structural changes place strong constraints on potential models for centralspindlin function in central spindle assembly
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