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, 27 (20), 3021-3030

Spatial Regulation of Astral Microtubule Dynamics by Kif18B in PtK Cells

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Spatial Regulation of Astral Microtubule Dynamics by Kif18B in PtK Cells

Claire E Walczak et al. Mol Biol Cell.

Abstract

The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end-directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle.

Figures

FIGURE 1:
FIGURE 1:
Kif18B knockdown causes a decrease in the percentage of metaphase cells. Cells transfected with (A) control, (B) Kif18B-1, or (C) Kif18B-2 siRNAs were fixed and stained for MTs (red) and DNA (blue). Representative deconvolved images from the indicated mitotic stages. Scale bar, 10 μm. (D) Distribution of mitotic stages quantified from six experiments in which 100 cells were counted per experiment, plotted as mean ± SD. *p < 0.05.
FIGURE 2:
FIGURE 2:
Kif18B knockdown does not affect mitotic progression but causes poor chromosome alignment in metaphase. Selected frames from phase-contrast time-lapse images of cells transfected with (A) control or (B) Kif18B-2 siRNAs and then imaged for 2 h at 30-s intervals. Scale bar, 10 μm. (C) Percentage of cells with indicated phenotype in control (20 cells) or Kif18B RNAi (30 cells). (D) Average timing between mitotic stages. Dots represent individual measurements, and bar and whiskers represent mean ± SD.
FIGURE 3:
FIGURE 3:
Kif18B knockdown increases MT polymer levels. Cells transfected with (A) control or (B) Kif18B-1 siRNAs were fixed and stained for MTs (red) and DNA (blue). Color-coded boxes were drawn to indicate the quantification of spindle (red), spindle pole (green), and astral MTs (cyan). Scale bar, 10 μm. (C–E) Average fluorescence intensity measurements calculated for control and Kif18B RNAi cells in regions corresponding to (C) the spindle, (D) the spindle poles, and (E) the astral MTs. (F) Spindle length from the same cells analyzed for MT fluorescence intensity in A–E. Dot plots for a total of 60 control spindles and 54 Kif18B RNAi spindles, with bars and whiskers corresponding to mean ± SD. Three experiments.
FIGURE 4:
FIGURE 4:
Kif18B knockdown increases astral MT length but not kinetochore-fiber MT length. Cells were transfected with (A) control or (B) Kif18B-1 siRNAs and treated with monastrol 3 h before fixation. Cells were stained for MTs (red), DNA (blue), and kinetochores (green). Scale bar, 10 μm. (C) Monopolar spindle area from 55 control and 56 Kif18B RNAi spindles in three independent experiments. Dots represent individual measurements, and bar and whiskers represent mean ± SD. Frequency distribution of (D) astral MT length and (E) kinetochore MT length from three independent experiments in which eight length measurements were averaged per spindle.
FIGURE 5:
FIGURE 5:
Depolymerization of the actin cytoskeleton does not suppress the long astral MTs caused by Kif18B knockdown. (A) Cells transfected with control or Kif18B-2 siRNAs were treated with DMSO or cytochalasin D for 30 min before being fixed and stained for MTs (green), actin (red), and DNA (blue). Images were collected on a GE Healthcare OMX 3D-SIM microscope. (B) Cells treated as in A, except that they were treated with monastrol for 3 h before cytochalasin D treatment and fixation. (C) Monopolar spindle area from 45 spindles in three independent experiments. Dots represent individual measurements, and bar and whiskers represent mean ± SD. ***p < 0.001. Scale bar, 10 μm.
FIGURE 6:
FIGURE 6:
Kif18B knockdown causes an increase in the lifetime of astral MTs and a decrease in MT growth rate in general. (A, B) First frame of EB1-GFP image series of spindles in PtK1 cells transfected with (A) control or (B) Kif18B-2 siRNA. Scale bar, 10 μm. (C, D) The plusTipTracker software was used to detect and link EB1 comets from time-lapse images of spindles in A and B. Tracked comets were divided into three subspindle regions and color coded accordingly: astral MTs to the left and right of spindle poles (aster_lr) are in magenta, astral MTs in the top and bottom region of the spindle body (aster_tb) are in green, and MTs in the central region of the spindle body are in cyan (Spindle_midzone). (E) Lifetime of EB1 tracks in the three subspindle regions was fit with a one-phase exponential decay to determine the rate constant K (s−1), and the average lifetime (τ) was determined from K−1. Data represent mean ± 95% confidence interval from the fit (see also Supplemental Figure S2) for 11 spindles for the control with 1045 tracks in aster_lr region, 1063 tracks in aster_tb region, and 4030 tracks in spindle_midzone region and 16 Kif18B RNAi spindles with 3605 tracks in aster_lr region, 2295 tracks in aster_tb region, and 7828 tracks in spindle_midzone region. (F) Histograms of EB1 track velocity in the entire spindle of control and Kif18B knockdowns graphed with the fitting curve of a Gaussian distribution. The mean and SD derived from each fit is indicated. Control RNAi, 11 spindles with 7782 tracks; Kif18B RNAi, 16 spindles with 16414 tracks.

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