Abstract
KIF18A (kinesin-8) is required for mammalian mitotic chromosome alignment. KIF18A confines chromosome movement to the mitotic spindle equator by accumulating at the plus-ends of kinetochore microtubule bundles (K-fibers), where it functions to suppress K-fiber dynamics. It is not understood how the motor accumulates at K-fiber plus-ends, a difficult feat requiring the motor to navigate protein dense microtubule tracks. Our data indicate that KIF18A's relatively long neck linker is required for the motor's accumulation at K-fiber plus-ends. Shorter neck linker (sNL) variants of KIF18A display a deficiency in accumulation at the ends of K-fibers at the center of the spindle. Depletion of K-fiber-binding proteins reduces the KIF18A sNL localization defect, whereas their overexpression reduces wild-type KIF18A's ability to accumulate on this same K-fiber subset. Furthermore, single-molecule assays indicate that KIF18A sNL motors are less proficient in navigating microtubules coated with microtubule-associated proteins. Taken together, these results support a model in which KIF18A's neck linker length permits efficient navigation of obstacles to reach K-fiber ends during mitosis.
Highlights
Kinesin motor proteins are responsible for building and maintaining the mitotic spindle (Sawin et al, 1992; Tanenbaum et al, 2009), transporting, aligning, and orienting chromosomes at the metaphase plate (Levesque & Compton, 2001; Kapoor et al, 2006; Stumpff et al, 2008), and scaffolding the spindle midzone for cytokinesis (Kurasawa et al, 2004)
To investigate the importance of KIF18A neck linker length for its ability to accumulate at K-fiber plus-ends in mitotic cells, we created a panel of Shorter neck linker (sNL) KIF18A variants (Fig 1A)
Because coiled-coil prediction algorithms are inaccurate at pinpointing the beginning of coiled-coil domains and altering the sequence of the kinesin neck linker can affect the coiled-coil (Phillips et al, 2016), we created a series of four sNL variants, taking care to keep the highly conserved N362 in frame
Summary
Kinesin motor proteins are responsible for building and maintaining the mitotic spindle (Sawin et al, 1992; Tanenbaum et al, 2009), transporting, aligning, and orienting chromosomes at the metaphase plate (Levesque & Compton, 2001; Kapoor et al, 2006; Stumpff et al, 2008), and scaffolding the spindle midzone for cytokinesis (Kurasawa et al, 2004). To investigate the importance of KIF18A neck linker length for its ability to accumulate at K-fiber plus-ends in mitotic cells, we created a panel of sNL KIF18A variants (Fig 1A).
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