A unique kinesin-8 surface loop provides specificity for chromosome alignment.
Bottom Line: To address this question, we engineered chimeric kinesins that contain the Kif4A, Kif18B (kinesin-8), or Kif5B (kinesin-1) motor domain fused to the C-terminal tail of Kif18A.Mutational studies of Kif18A indicate that this control depends on both its C-terminus and a unique, positively charged surface loop, called loop2, within the motor domain.These data support a model in which microtubule-attenuating kinesins are molecularly "tuned" to control the dynamics of specific subsets of spindle microtubules.
Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405.Show MeSH
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Mentions: Although the localization of these chimeras was similar in Taxol-treated cells, it was not clear whether their behavior at K-fiber ends was comparable. To address this question, we used a fluorescence recovery after photobleaching (FRAP) assay to examine the kinetics of chimeric kinesins at K-fiber ends in Taxol-treated cells. For these experiments, a small region of interest containing GFP-kinesin was photobleached near a monomeric red fluorescent protein (mRFP)-CENP-B labeled centromere, and fluorescence recovery was visualized at 2-s intervals (Figure 3A). Wild-type Kif18A and the Kif18B-18A chimera displayed only a small mobile fraction at K-fiber ends under these conditions, consistent with previous studies of Kif18A turnover (Stumpff et al., 2011; Figure 3B and Table 1). In contrast, the Kif4A-18A and Kif5B-18A chimeras displayed significantly higher mobile fractions at K-fiber ends (Figure 3B and Table 1). A Kif18A truncation mutant (Kif18A-770) that lacks the microtubule-binding regions at the C-terminal end of the tail accumulates at K-fibers ends specifically in Taxol-treated cells (Supplemental Figure S2). Kif18A-770 displays an increase in the mobile fraction at K-fiber ends but does not fully recover (Figure 3B, Supplemental Figure S2, and Table 1). These data indicate that Kif18A's stable association at K-fiber ends requires both the C-terminal tail and a kinesin-8 motor–specific element that is shared between Kif18A and Kif18B. Furthermore, the behavior of Kif4A-18A and Kif5B-18A at the ends of Taxol-stabilized K-fibers is consistent with these motors forming a steady-state accumulation, similar to that observed for purified Kif4A (Subramanian et al., 2013).
Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405.