The kinetics of expansion. (A) Examples of kinetochore staining at time points shortly after nuclear envelope breakdown (∼10 min after addition of fresh CSF extract to induce M phase entry). (left) Images of two nuclei at each time point to illustrate the types of figures seen at that time point. Bars, 1 µm. (right) Higher magnification images of a group of kinetochores from each nucleus. Note that after 8 min, small amounts of BubR1 have loaded only in nuclei that have lost their circularity and have presumably completed nuclear envelope breakdown (NEBD). In contrast, after 12 min, much more BubR1 staining can be seen, particularly in kinetochores in locations where the spindle is more disorganized (inset 3 and 4; bars, 0.2 µm). All images are maximum intensity projections of 3D-SIM datasets. (B and C) Quantification of individual kinetochore signals for the indicated protein over a time course during spindle assembly. Significance relative to the 45-min value. Mann–Whitney test: ***, P < 0.001; **, P < 0.01. n > 100 kinetochores from 5–8 nuclei. (D and E) Quantification of total signal from chromosome clusters for the indicated kinetochore component over a time course to indicate the rate of kinetochore expansion. Mean and standard deviation are plotted in black. A.U., arbitrary unit.