Figure 5.
Figure 5. Defects in microtubule bundle assembly increase the rate of anaphase spindle elongation. (A–D) Live-cell imaging of centromere and spindle pole position by spinning disk confocal microscopy. Example hTERT-RPE1 cells expressing GFP-CENP-A to label the kinetochores and GFP-Centrin to label the centrosomes are shown. Select images from time series are shown. A control cell (A and B) and a cell expressing shRNA to PRC1 (shPRC1; C and D) are shown. T = 0 was assigned to the frame immediately prior to that with detectable sister kinetochore separation. (A and C) Maximum-intensity projections (MIPs) show select time points from a cell in anaphase. Time-lapse recordings are provided in Videos 6 and 7. (B and D) Single image planes show consecutive images (15-s intervals) from the cells shown in A (B) or C (D). Selected kinetochore pairs are highlighted (orange and yellow circles). (E and F) Kymograph generated from the time-lapse videos of the cell shown in A (E) or C (F). Horizontal scale bar, 3 µm. Vertical scale bar, 2 min. (G and H) Analysis of normalized kinetochore-to-pole (k-to-p) distance show individual kinetochores (thin lines) and average (bold line) traces from an example control cell (G) and an example shPRC1 cell (H). Error bars are SD. (I) Box-and-whisker plots of kinetochore-to-pole velocity. Each point represents the velocity of a single kinetochore. Velocity was determined by linear fit to kinetochore-to-pole distance versus time in the T = 0.5–3-min time window. Negative sign indicates direction of slope. Data were pooled data from all experiments (n = 59 and 111 kinetochores for control and shPRC1 cells, respectively) and fit to a Gaussian. (J) Plots of pole-to-pole distance versus time for control (n = 12 cells; black traces) and shPRC1 (n = 21 cells; red traces) cells. (K) Plot of relative pole separation distance (ΔLength), defined as the pole-to-pole distance at time t (Lt) minus the pole-to-pole distance at T = 0 (L0). (L) Pole separation velocity for control and shPRC1 cells (***, P < 0.003). Mean ± SD.

Defects in microtubule bundle assembly increase the rate of anaphase spindle elongation. (A–D) Live-cell imaging of centromere and spindle pole position by spinning disk confocal microscopy. Example hTERT-RPE1 cells expressing GFP-CENP-A to label the kinetochores and GFP-Centrin to label the centrosomes are shown. Select images from time series are shown. A control cell (A and B) and a cell expressing shRNA to PRC1 (shPRC1; C and D) are shown. T = 0 was assigned to the frame immediately prior to that with detectable sister kinetochore separation. (A and C) Maximum-intensity projections (MIPs) show select time points from a cell in anaphase. Time-lapse recordings are provided in Videos 6 and 7. (B and D) Single image planes show consecutive images (15-s intervals) from the cells shown in A (B) or C (D). Selected kinetochore pairs are highlighted (orange and yellow circles). (E and F) Kymograph generated from the time-lapse videos of the cell shown in A (E) or C (F). Horizontal scale bar, 3 µm. Vertical scale bar, 2 min. (G and H) Analysis of normalized kinetochore-to-pole (k-to-p) distance show individual kinetochores (thin lines) and average (bold line) traces from an example control cell (G) and an example shPRC1 cell (H). Error bars are SD. (I) Box-and-whisker plots of kinetochore-to-pole velocity. Each point represents the velocity of a single kinetochore. Velocity was determined by linear fit to kinetochore-to-pole distance versus time in the T = 0.5–3-min time window. Negative sign indicates direction of slope. Data were pooled data from all experiments (n = 59 and 111 kinetochores for control and shPRC1 cells, respectively) and fit to a Gaussian. (J) Plots of pole-to-pole distance versus time for control (n = 12 cells; black traces) and shPRC1 (n = 21 cells; red traces) cells. (K) Plot of relative pole separation distance (ΔLength), defined as the pole-to-pole distance at time t (Lt) minus the pole-to-pole distance at T = 0 (L0). (L) Pole separation velocity for control and shPRC1 cells (***, P < 0.003). Mean ± SD.

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