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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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Kif18A-tail chimeras rapidly accumulate at K-fiber ends in Taxol-treated cells. (A) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells after a 5-min incubation with 10 μM Taxol. Cells were immunostained with ACA (centromeres, red) and tubulin (blue). (B) Line scans of GFP-tagged kinesins (green trace) relative to tubulin (blue trace) and ACA (red trace) along K-fibers in 10 μM Taxol–treated cells. (C) Still images from live-cell analyses of GFP-tagged kinesin relocalization after the addition of Taxol. Taxol was added immediately after the 0-s image was captured.
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Figure 2: Kif18A-tail chimeras rapidly accumulate at K-fiber ends in Taxol-treated cells. (A) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells after a 5-min incubation with 10 μM Taxol. Cells were immunostained with ACA (centromeres, red) and tubulin (blue). (B) Line scans of GFP-tagged kinesins (green trace) relative to tubulin (blue trace) and ACA (red trace) along K-fibers in 10 μM Taxol–treated cells. (C) Still images from live-cell analyses of GFP-tagged kinesin relocalization after the addition of Taxol. Taxol was added immediately after the 0-s image was captured.

Mentions: Given the ability of purified Kif4A to form a steady-state accumulation at the ends of stable microtubules in vitro, the accumulation of Kif4A-18A specifically at the ends of peripheral K-fibers could be explained if peripheral and interior K-fibers display differences in their dynamic instability (Subramanian et al., 2013). Consistent with this, previous studies demonstrated significant reduction in the oscillatory movements of kinetochores, which are closely coupled to K-fiber dynamic instability, on the periphery compared with those in the interior of the spindle (Canman et al., 2002; Cimini et al., 2004; Stumpff et al., 2008). Thus we hypothesized that peripheral K-fibers provide a stable track that allows a steady-state accumulation of Kif4A-18A. To test this idea, we briefly treated Kif18A-depleted HeLa cells expressing GFP-tagged chimeric kinesins with the microtubule-stabilizing drug Taxol (10 μM) and then analyzed kinesin localization. Similar to the previously observed behavior of Kif18A-FL in Taxol-treated cells, the concentration of each of the chimeric kinesins at the ends of K-fibers was increased after Taxol addition (Figure 2, A and B; Stumpff et al., 2011). GFP-chimera signal accumulated to K-fiber ends within 60–90 s of Taxol addition (Figure 2C). Of importance, this rapid accumulation is consistent with efficient plus end–directed movement of active motors to microtubule ends.


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

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

Kif18A-tail chimeras rapidly accumulate at K-fiber ends in Taxol-treated cells. (A) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells after a 5-min incubation with 10 μM Taxol. Cells were immunostained with ACA (centromeres, red) and tubulin (blue). (B) Line scans of GFP-tagged kinesins (green trace) relative to tubulin (blue trace) and ACA (red trace) along K-fibers in 10 μM Taxol–treated cells. (C) Still images from live-cell analyses of GFP-tagged kinesin relocalization after the addition of Taxol. Taxol was added immediately after the 0-s image was captured.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214779&req=5

Figure 2: Kif18A-tail chimeras rapidly accumulate at K-fiber ends in Taxol-treated cells. (A) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells after a 5-min incubation with 10 μM Taxol. Cells were immunostained with ACA (centromeres, red) and tubulin (blue). (B) Line scans of GFP-tagged kinesins (green trace) relative to tubulin (blue trace) and ACA (red trace) along K-fibers in 10 μM Taxol–treated cells. (C) Still images from live-cell analyses of GFP-tagged kinesin relocalization after the addition of Taxol. Taxol was added immediately after the 0-s image was captured.
Mentions: Given the ability of purified Kif4A to form a steady-state accumulation at the ends of stable microtubules in vitro, the accumulation of Kif4A-18A specifically at the ends of peripheral K-fibers could be explained if peripheral and interior K-fibers display differences in their dynamic instability (Subramanian et al., 2013). Consistent with this, previous studies demonstrated significant reduction in the oscillatory movements of kinetochores, which are closely coupled to K-fiber dynamic instability, on the periphery compared with those in the interior of the spindle (Canman et al., 2002; Cimini et al., 2004; Stumpff et al., 2008). Thus we hypothesized that peripheral K-fibers provide a stable track that allows a steady-state accumulation of Kif4A-18A. To test this idea, we briefly treated Kif18A-depleted HeLa cells expressing GFP-tagged chimeric kinesins with the microtubule-stabilizing drug Taxol (10 μM) and then analyzed kinesin localization. Similar to the previously observed behavior of Kif18A-FL in Taxol-treated cells, the concentration of each of the chimeric kinesins at the ends of K-fibers was increased after Taxol addition (Figure 2, A and B; Stumpff et al., 2011). GFP-chimera signal accumulated to K-fiber ends within 60–90 s of Taxol addition (Figure 2C). Of importance, this rapid accumulation is consistent with efficient plus end–directed movement of active motors to microtubule ends.

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