Figure 2.
Figure 2. Automated image analysis and quantitative analysis of sister kinetochore dynamics. (a) The four steps of image analysis to measure sister kinetochore dynamics. White lines show boundaries of the 3D dataset, and the black rectangle indicates the magnified region shown in b. Bar, 2 µm. (b) Basic parameters of kinetochore dynamics: sister separation d and center normal position x. Bar, 0.5 µm. (c) Experimental time courses of sister center normal position x and sister separation d. (top) Green and orange indicate movements in the positive and negative directions along the normal. (bottom) Green and orange indicate phases of increase and decrease in sister separation. In both, black indicates unclassified intervals. Error bars indicate SD of the measured coordinates. (d) Illustration of sister (ΔprL and ΔprR) and center (ΔprC) displacement projections onto the vector connecting sister pairs (black). (e) Sister center normal and projection speeds and sister breathing speed in WT and fixed cells. Error bars indicate mean ± SD, representing cell to cell variation. Red double asterisks highlight values significantly smaller than WT (P < 0.001 by one-tailed t test). (f) Distribution of oscillation half-periods τx in WT. (inset) Illustration of the oscillation half-period τx; green and orange as in c (top). (g) Distribution of breathing half-periods τd in WT. (inset) Illustration of breathing half-period τd; green and orange as in c (bottom). (h) Three models with increasing width of the distribution of sister oscillation half-period. (i) Sister displacement cross-correlation (left) and sister center autocorrelation (right) resulting from the models in h and from a series of random sister positions. The blue and cyan curves were generated by the same half-period distribution but with more or less coupling, respectively, between the two sisters. (j) Sister displacement cross-correlation and sister center autocorrelation in WT and fixed cells calculated by combining all aligned sister kinetochores in each condition. (i–k) Line thickness represents the 95% confidence interval of the correlations about their calculated values (see Materials and methods). (k) Sister breathing autocorrelation in WT and fixed cells. Details as in j. Arrows, mean oscillation half-period; arrowheads, mean oscillation full period; carets, cross-correlation at lag ΔT = 0; stars, −0.5 autocorrelation at lag ΔT = 7.5 s (one frame), which is indicative of a series of random positions.

Automated image analysis and quantitative analysis of sister kinetochore dynamics. (a) The four steps of image analysis to measure sister kinetochore dynamics. White lines show boundaries of the 3D dataset, and the black rectangle indicates the magnified region shown in b. Bar, 2 µm. (b) Basic parameters of kinetochore dynamics: sister separation d and center normal position x. Bar, 0.5 µm. (c) Experimental time courses of sister center normal position x and sister separation d. (top) Green and orange indicate movements in the positive and negative directions along the normal. (bottom) Green and orange indicate phases of increase and decrease in sister separation. In both, black indicates unclassified intervals. Error bars indicate SD of the measured coordinates. (d) Illustration of sister (ΔprL and ΔprR) and center (ΔprC) displacement projections onto the vector connecting sister pairs (black). (e) Sister center normal and projection speeds and sister breathing speed in WT and fixed cells. Error bars indicate mean ± SD, representing cell to cell variation. Red double asterisks highlight values significantly smaller than WT (P < 0.001 by one-tailed t test). (f) Distribution of oscillation half-periods τx in WT. (inset) Illustration of the oscillation half-period τx; green and orange as in c (top). (g) Distribution of breathing half-periods τd in WT. (inset) Illustration of breathing half-period τd; green and orange as in c (bottom). (h) Three models with increasing width of the distribution of sister oscillation half-period. (i) Sister displacement cross-correlation (left) and sister center autocorrelation (right) resulting from the models in h and from a series of random sister positions. The blue and cyan curves were generated by the same half-period distribution but with more or less coupling, respectively, between the two sisters. (j) Sister displacement cross-correlation and sister center autocorrelation in WT and fixed cells calculated by combining all aligned sister kinetochores in each condition. (i–k) Line thickness represents the 95% confidence interval of the correlations about their calculated values (see Materials and methods). (k) Sister breathing autocorrelation in WT and fixed cells. Details as in j. Arrows, mean oscillation half-period; arrowheads, mean oscillation full period; carets, cross-correlation at lag ΔT = 0; stars, −0.5 autocorrelation at lag ΔT = 7.5 s (one frame), which is indicative of a series of random positions.

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