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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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The Kif18A C-terminus targets plus end–directed motors to K-fibers but is not sufficient for accumulation at K-fiber ends. (A) Schematic of Kif18A-tail chimeras. Numbers in parentheses indicate the amino acids used in each fragment. (B) Western blots of lysates transfected with the indicated siRNAs and GFP-tagged transgenes. Purified Kif18A-GFP (30 ng) was included for comparison. Blots were probed with anti-Kif18A (top) and anti-GAPDH (bottom) antibodies. The amount of Kif18A expressed in Kif18A siRNA–treated cells from three independent experiments is 9.8 ± 1.3% relative to controls. Note that Kif18A-770 does not contain the epitope recognized by the anti-Kif18A antibody. An anti-GFP blot of this sample is included in Supplemental Figure S2. (C) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells immunostained with ACA (centromeres, red) and tubulin (blue). (D) Line scans of GFP-tagged kinesin (green trace) relative to tubulin (blue trace) and ACA fluorescence (red trace) along peripheral K-fibers.
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Figure 1: The Kif18A C-terminus targets plus end–directed motors to K-fibers but is not sufficient for accumulation at K-fiber ends. (A) Schematic of Kif18A-tail chimeras. Numbers in parentheses indicate the amino acids used in each fragment. (B) Western blots of lysates transfected with the indicated siRNAs and GFP-tagged transgenes. Purified Kif18A-GFP (30 ng) was included for comparison. Blots were probed with anti-Kif18A (top) and anti-GAPDH (bottom) antibodies. The amount of Kif18A expressed in Kif18A siRNA–treated cells from three independent experiments is 9.8 ± 1.3% relative to controls. Note that Kif18A-770 does not contain the epitope recognized by the anti-Kif18A antibody. An anti-GFP blot of this sample is included in Supplemental Figure S2. (C) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells immunostained with ACA (centromeres, red) and tubulin (blue). (D) Line scans of GFP-tagged kinesin (green trace) relative to tubulin (blue trace) and ACA fluorescence (red trace) along peripheral K-fibers.

Mentions: The Kif18A C-terminal tail is necessary for the motor's accumulation at K-fiber plus ends (Stumpff et al., 2011). To determine whether the Kif18A tail is sufficient to facilitate the concentration of other plus end–directed kinesins at K-fiber ends, we engineered chimeric kinesins consisting of the motor and neck-linker domains of Kif18B (kinesin-8), Kif4A (kinesin-4), and Kif5B (kinesin-1) fused to the Kif18A C-terminus (Figure 1A). Chimeric kinesins were expressed as enhanced green fluorescent protein (EGFP)-fusion proteins in HeLa cells depleted of endogenous Kif18A, and cells with similar GFP fluorescence levels were evaluated (Supplemental Figure S1). Kif18A small interfering RNA (siRNA)–treated cells expressed 9.8 ± 1.3% of the Kif18A detected by Western blot in control-treated cells (Figure 1B). Whereas all of the chimeras localized to K-fibers, only wild-type Kif18A and the Kif18B-18A chimera efficiently concentrated at K-fiber ends (Figure 1, C and D). Of interest, the Kif4A-18A chimera displayed accumulation at the ends of K-fibers on the spindle periphery but not at the ends of those on the spindle interior. In contrast, the Kif5B-18A chimera was uniformly distributed on the spindle (Figure 1, C and D). These data indicate that the Kif18A-tail can target plus end– directed kinesins to K-fibers; however, an element common to the kinesin-8 motors Kif18A and Kif18B is additionally required for efficient accumulation at K-fiber ends.


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

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

The Kif18A C-terminus targets plus end–directed motors to K-fibers but is not sufficient for accumulation at K-fiber ends. (A) Schematic of Kif18A-tail chimeras. Numbers in parentheses indicate the amino acids used in each fragment. (B) Western blots of lysates transfected with the indicated siRNAs and GFP-tagged transgenes. Purified Kif18A-GFP (30 ng) was included for comparison. Blots were probed with anti-Kif18A (top) and anti-GAPDH (bottom) antibodies. The amount of Kif18A expressed in Kif18A siRNA–treated cells from three independent experiments is 9.8 ± 1.3% relative to controls. Note that Kif18A-770 does not contain the epitope recognized by the anti-Kif18A antibody. An anti-GFP blot of this sample is included in Supplemental Figure S2. (C) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells immunostained with ACA (centromeres, red) and tubulin (blue). (D) Line scans of GFP-tagged kinesin (green trace) relative to tubulin (blue trace) and ACA fluorescence (red trace) along peripheral K-fibers.
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Related In: Results  -  Collection

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Figure 1: The Kif18A C-terminus targets plus end–directed motors to K-fibers but is not sufficient for accumulation at K-fiber ends. (A) Schematic of Kif18A-tail chimeras. Numbers in parentheses indicate the amino acids used in each fragment. (B) Western blots of lysates transfected with the indicated siRNAs and GFP-tagged transgenes. Purified Kif18A-GFP (30 ng) was included for comparison. Blots were probed with anti-Kif18A (top) and anti-GAPDH (bottom) antibodies. The amount of Kif18A expressed in Kif18A siRNA–treated cells from three independent experiments is 9.8 ± 1.3% relative to controls. Note that Kif18A-770 does not contain the epitope recognized by the anti-Kif18A antibody. An anti-GFP blot of this sample is included in Supplemental Figure S2. (C) Fluorescence micrographs of the indicated GFP-tagged kinesins (green) in Kif18A-depleted HeLa cells immunostained with ACA (centromeres, red) and tubulin (blue). (D) Line scans of GFP-tagged kinesin (green trace) relative to tubulin (blue trace) and ACA fluorescence (red trace) along peripheral K-fibers.
Mentions: The Kif18A C-terminal tail is necessary for the motor's accumulation at K-fiber plus ends (Stumpff et al., 2011). To determine whether the Kif18A tail is sufficient to facilitate the concentration of other plus end–directed kinesins at K-fiber ends, we engineered chimeric kinesins consisting of the motor and neck-linker domains of Kif18B (kinesin-8), Kif4A (kinesin-4), and Kif5B (kinesin-1) fused to the Kif18A C-terminus (Figure 1A). Chimeric kinesins were expressed as enhanced green fluorescent protein (EGFP)-fusion proteins in HeLa cells depleted of endogenous Kif18A, and cells with similar GFP fluorescence levels were evaluated (Supplemental Figure S1). Kif18A small interfering RNA (siRNA)–treated cells expressed 9.8 ± 1.3% of the Kif18A detected by Western blot in control-treated cells (Figure 1B). Whereas all of the chimeras localized to K-fibers, only wild-type Kif18A and the Kif18B-18A chimera efficiently concentrated at K-fiber ends (Figure 1, C and D). Of interest, the Kif4A-18A chimera displayed accumulation at the ends of K-fibers on the spindle periphery but not at the ends of those on the spindle interior. In contrast, the Kif5B-18A chimera was uniformly distributed on the spindle (Figure 1, C and D). These data indicate that the Kif18A-tail can target plus end– directed kinesins to K-fibers; however, an element common to the kinesin-8 motors Kif18A and Kif18B is additionally required for efficient accumulation at K-fiber 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