Limits...
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.

Show MeSH
Kinesin-8 loop2 is required for mitotic chromosome alignment and spindle length regulation. (A) Schematic of experimental design. Representative images show control and Kif18A siRNA–treated cells immunostained with ACA (centromeres, green) and γ-tubulin (red) antibodies. (B) Representative plot of centromere (ACA) fluorescence distribution as a function of position along the pole-to-pole axis. Fluorescence data were fitted with a Gaussian function, and the full-width at half-maximum (FWHM) was calculated as a metric for centromere alignment. (C, D) Graphs of average FWHM (C) and average spindle length (D) calculated from cells transfected with the indicated siRNAs and transgenes. **p < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4214779&req=5

Figure 6: Kinesin-8 loop2 is required for mitotic chromosome alignment and spindle length regulation. (A) Schematic of experimental design. Representative images show control and Kif18A siRNA–treated cells immunostained with ACA (centromeres, green) and γ-tubulin (red) antibodies. (B) Representative plot of centromere (ACA) fluorescence distribution as a function of position along the pole-to-pole axis. Fluorescence data were fitted with a Gaussian function, and the full-width at half-maximum (FWHM) was calculated as a metric for centromere alignment. (C, D) Graphs of average FWHM (C) and average spindle length (D) calculated from cells transfected with the indicated siRNAs and transgenes. **p < 0.01.

Mentions: To determine whether loop2 is required for Kif18A's regulation of K-fiber lengths during mitosis, we assayed the ability of our chimeric kinesins and loop2 mutants to facilitate chromosome alignment and regulate spindle length in cells depleted of endogenous Kif18A. Cells depleted of Kif18A were transfected with GFP-tagged kinesins and then treated ∼18 h later with MG132 (20 μM), which prevents anaphase entry (Figure 6A). Cells were then fixed and immunofluorescently stained with antibodies against centromeric (ACA) and centrosomal (γ-tubulin) proteins. Under these conditions, the majority of cells transfected with scrambled control siRNAs and a GFP control plasmid had well-aligned kinetochores. In contrast, cells transfected with Kif18A siRNAs and a GFP control plasmid displayed unaligned kinetochores (Figure 6A).


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

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

Kinesin-8 loop2 is required for mitotic chromosome alignment and spindle length regulation. (A) Schematic of experimental design. Representative images show control and Kif18A siRNA–treated cells immunostained with ACA (centromeres, green) and γ-tubulin (red) antibodies. (B) Representative plot of centromere (ACA) fluorescence distribution as a function of position along the pole-to-pole axis. Fluorescence data were fitted with a Gaussian function, and the full-width at half-maximum (FWHM) was calculated as a metric for centromere alignment. (C, D) Graphs of average FWHM (C) and average spindle length (D) calculated from cells transfected with the indicated siRNAs and transgenes. **p < 0.01.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Kinesin-8 loop2 is required for mitotic chromosome alignment and spindle length regulation. (A) Schematic of experimental design. Representative images show control and Kif18A siRNA–treated cells immunostained with ACA (centromeres, green) and γ-tubulin (red) antibodies. (B) Representative plot of centromere (ACA) fluorescence distribution as a function of position along the pole-to-pole axis. Fluorescence data were fitted with a Gaussian function, and the full-width at half-maximum (FWHM) was calculated as a metric for centromere alignment. (C, D) Graphs of average FWHM (C) and average spindle length (D) calculated from cells transfected with the indicated siRNAs and transgenes. **p < 0.01.
Mentions: To determine whether loop2 is required for Kif18A's regulation of K-fiber lengths during mitosis, we assayed the ability of our chimeric kinesins and loop2 mutants to facilitate chromosome alignment and regulate spindle length in cells depleted of endogenous Kif18A. Cells depleted of Kif18A were transfected with GFP-tagged kinesins and then treated ∼18 h later with MG132 (20 μM), which prevents anaphase entry (Figure 6A). Cells were then fixed and immunofluorescently stained with antibodies against centromeric (ACA) and centrosomal (γ-tubulin) proteins. Under these conditions, the majority of cells transfected with scrambled control siRNAs and a GFP control plasmid had well-aligned kinetochores. In contrast, cells transfected with Kif18A siRNAs and a GFP control plasmid displayed unaligned kinetochores (Figure 6A).

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