Figure 5.
Figure 5. Metaphase plate thinning in preparation for anaphase is induced by a reduction in oscillation speed. (a) Histogram of average plate thicknesses in individual videos. (b) Scatter plot of oscillation extent versus plate thickness. Black line, one-line fit to the scatter plot; dark and light green lines, two-line fit to the scatter plot; dotted green line, transition point between the two scaling regimes. Triangles indicate data points deemed as outliers in the least-median square fit of the straight line. (inset) Line slopes matched by color; error bars indicate mean ± SD as estimated by the least-squares fit; p-values indicate comparison of slopes to 0 (see Materials and methods) to determine their significance. (c and d) Scatter plots of oscillation half-period (c) and center normal speed (d) versus plate thickness. (a–d) M, metaphase cells with no unaligned kinetochores; A, cells that enter anaphase during the course of a video. (e) Sum of square residuals from regressing plate thickness on oscillation half-period, center normal speed, or half-period and center normal speed combined. P-values indicate comparison of model fits using the F-test (Jaqaman and Danuser, 2006). (f) Scatter plot of center normal speed versus plate thickness, showing their variation within individual videos using the 25% of the videos that exhibited the largest decrease in plate thickness over the time course of the video. Gray circles are combined from late prometaphase cells with unaligned kinetochores near the metaphase plate (LPM near), late prometaphase cells with unaligned kinetochores far from the metaphase plate (LPM far) metaphase, and anaphase cells. (g and h) Scatter plots of center normal speed versus plate thickness after depolymerase depletion (g) and sister linkage perturbation (h). Outliers from the least-median square fit are not depicted. Crosses indicate cells that entered anaphase during a video, and circles indicate cells that did not. Green and red asterisks highlight values significantly larger and smaller than WT, respectively (P < 0.05). (i) Proposed model for the regulation of kinetochore breathing and oscillation and of metaphase plate thickness. Sister linkage at the centromere is set by a combination of chromatin stiffness and sister cohesion and determines both oscillation and breathing periods. Activity of MT depolymerases generates force at kinetochores, where net effect of antagonizing depolymerase activities at sister pairs sets baseline oscillation and breathing speeds. Plate thickness is primarily determined by oscillation speed with a minor contribution from oscillation period. Breathing speed and period affect distance between centromere and kinetochore and, thus, potentially the activity of kinetochore and centromere kinases and phosphatases on MT motors. Thick and thin arrows indicate strong and weak dependencies, respectively. The dashed arrow indicates the proposed feedback from breathing characteristics to depolymerase activity.

Metaphase plate thinning in preparation for anaphase is induced by a reduction in oscillation speed. (a) Histogram of average plate thicknesses in individual videos. (b) Scatter plot of oscillation extent versus plate thickness. Black line, one-line fit to the scatter plot; dark and light green lines, two-line fit to the scatter plot; dotted green line, transition point between the two scaling regimes. Triangles indicate data points deemed as outliers in the least-median square fit of the straight line. (inset) Line slopes matched by color; error bars indicate mean ± SD as estimated by the least-squares fit; p-values indicate comparison of slopes to 0 (see Materials and methods) to determine their significance. (c and d) Scatter plots of oscillation half-period (c) and center normal speed (d) versus plate thickness. (a–d) M, metaphase cells with no unaligned kinetochores; A, cells that enter anaphase during the course of a video. (e) Sum of square residuals from regressing plate thickness on oscillation half-period, center normal speed, or half-period and center normal speed combined. P-values indicate comparison of model fits using the F-test (Jaqaman and Danuser, 2006). (f) Scatter plot of center normal speed versus plate thickness, showing their variation within individual videos using the 25% of the videos that exhibited the largest decrease in plate thickness over the time course of the video. Gray circles are combined from late prometaphase cells with unaligned kinetochores near the metaphase plate (LPM near), late prometaphase cells with unaligned kinetochores far from the metaphase plate (LPM far) metaphase, and anaphase cells. (g and h) Scatter plots of center normal speed versus plate thickness after depolymerase depletion (g) and sister linkage perturbation (h). Outliers from the least-median square fit are not depicted. Crosses indicate cells that entered anaphase during a video, and circles indicate cells that did not. Green and red asterisks highlight values significantly larger and smaller than WT, respectively (P < 0.05). (i) Proposed model for the regulation of kinetochore breathing and oscillation and of metaphase plate thickness. Sister linkage at the centromere is set by a combination of chromatin stiffness and sister cohesion and determines both oscillation and breathing periods. Activity of MT depolymerases generates force at kinetochores, where net effect of antagonizing depolymerase activities at sister pairs sets baseline oscillation and breathing speeds. Plate thickness is primarily determined by oscillation speed with a minor contribution from oscillation period. Breathing speed and period affect distance between centromere and kinetochore and, thus, potentially the activity of kinetochore and centromere kinases and phosphatases on MT motors. Thick and thin arrows indicate strong and weak dependencies, respectively. The dashed arrow indicates the proposed feedback from breathing characteristics to depolymerase activity.

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