Abstract:
S. cerevisiae cells express two kinesin-5 motors Cin8 and Kip1. These motors share a bipolar homotetrameric structure involved in mitotic spindle dynamics by crosslinking and sliding apart antiparallel microtubules. Interestingly, recent studies from our and other groups demonstrated the bidirectional motility for yeast kinesin-5 motors, Cin8 and Kip1, that can switch directionality under a number of conditions (Duselder et al., 2015; Fridman et al., 2013; Gerson-Gurwitz et al., 2011). These findings were in contrast to the previous dogma stating that N-terminal kinesin motors are exclusively plus-end directed. We aim to understand the structural and biochemical features of yeast kinesin-5 motors that promote directional switching of motility. To investigate the mechanistic basis for the bidirectional motility of Cin8, we cloned and purified the dimeric constructs of Cin8 and Kip1. For Cin8, the constructs include the native dimer and the Loop 8 (L8) deletion variant. The deletion of Loop 8 construct induces a strong bias towards minus-end directed motility and affects the stoichiometric microtubule binding (Bell et al., 2017; Gerson-Gurwitz et al., 2011). Further we are using Cryo–Electron microscopy (Cryo-EM), which uniquely allows for high-resolution 3D reconstructions of Cin8 bound to microtubules in the presence of different nucleotides. We present preliminary results of decoration of microtubules by Cin8 and Kip1. We believe that the Cryo-EM studies will reveal the unique structural elements involved in the bidirectional motility of Cin8 and Kip1.
References:
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