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A unique kinesin-8 surface loop provides specificity for chromosome alignment.

Kim H, Fonseca C, Stumpff J - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405.

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A kinesin-8–specific activity is required for stable association with K-fiber ends. (A) Stills from FRAP assays performed in live, Taxol-treated, Kif18A-depleted HeLa cells expressing the indicated GFP-tagged kinesin. Fluorescence intensity was measured before (−2 s) and after (0–60 s) a brief pulse with a 405-nm laser focused to the region indicated by the green circle. (B) Plot of relative GFP fluorescence as a function of time after a bleaching event for the indicated kinesins. Dashed lines are single-exponential fits to the data. Note that the Kif18B-18A and Kif18A-FL data did not fit a single exponential. Quantified data from FRAP experiments are reported in Table 1. Representative images of Kif18A-770 are included in Supplemental Figure S2.
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Figure 3: A kinesin-8–specific activity is required for stable association with K-fiber ends. (A) Stills from FRAP assays performed in live, Taxol-treated, Kif18A-depleted HeLa cells expressing the indicated GFP-tagged kinesin. Fluorescence intensity was measured before (−2 s) and after (0–60 s) a brief pulse with a 405-nm laser focused to the region indicated by the green circle. (B) Plot of relative GFP fluorescence as a function of time after a bleaching event for the indicated kinesins. Dashed lines are single-exponential fits to the data. Note that the Kif18B-18A and Kif18A-FL data did not fit a single exponential. Quantified data from FRAP experiments are reported in Table 1. Representative images of Kif18A-770 are included in Supplemental Figure S2.

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).


A unique kinesin-8 surface loop provides specificity for chromosome alignment.

Kim H, Fonseca C, Stumpff J - Mol. Biol. Cell (2014)

A kinesin-8–specific activity is required for stable association with K-fiber ends. (A) Stills from FRAP assays performed in live, Taxol-treated, Kif18A-depleted HeLa cells expressing the indicated GFP-tagged kinesin. Fluorescence intensity was measured before (−2 s) and after (0–60 s) a brief pulse with a 405-nm laser focused to the region indicated by the green circle. (B) Plot of relative GFP fluorescence as a function of time after a bleaching event for the indicated kinesins. Dashed lines are single-exponential fits to the data. Note that the Kif18B-18A and Kif18A-FL data did not fit a single exponential. Quantified data from FRAP experiments are reported in Table 1. Representative images of Kif18A-770 are included in Supplemental Figure S2.
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Related In: Results  -  Collection

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Figure 3: A kinesin-8–specific activity is required for stable association with K-fiber ends. (A) Stills from FRAP assays performed in live, Taxol-treated, Kif18A-depleted HeLa cells expressing the indicated GFP-tagged kinesin. Fluorescence intensity was measured before (−2 s) and after (0–60 s) a brief pulse with a 405-nm laser focused to the region indicated by the green circle. (B) Plot of relative GFP fluorescence as a function of time after a bleaching event for the indicated kinesins. Dashed lines are single-exponential fits to the data. Note that the Kif18B-18A and Kif18A-FL data did not fit a single exponential. Quantified data from FRAP experiments are reported in Table 1. Representative images of Kif18A-770 are included in Supplemental Figure S2.
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).

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, VT 05405.

Show MeSH
Related in: MedlinePlus