For spindle width measurements, the two lateral edges of the mitotic spindle were identified by tubulin fluorescence and the distance between them was measured using ImageJ.
For pole to pole distance and spindle width measurements, at least 3 independent experiments were performed with at least 20 cells per condition measured in each experiment.
While siRNA-mediated CRMP4 knockdown had no effect on spindle width (Fig. 10C), we did observe a significant decrease in the pole to pole distance compared to control treated cells (Fig. 10A and 10B).
We performed a morphometric analysis of the mitotic spindle by measuring spindle length (pole to pole distance) and spindle width 9 hours following release from a double thymidine block (Fig. 10A 10C), as described previously with GSK3 inhibitors [33].
Overall, our data suggest that spindle width and length are coupled anisotropically.
Taken together, this suggests a universal behavior for the relationship between spindle width and spindle length.
However, in contrast to spindle length, the increase of spindle width in Clasp-depleted cells was monophasic.
Unlike Nuf2 depletions, spindle width also increased to 10.2 ± 0.8 µm following Kif18a depletion (Fig. 3D; supplementary material Fig. S2H).
We found that spindle width decreased in absence of kinetochore-fibers (8.7 ± 1.2 µm and 9.0 ± 1.0 µm for Nuf2/Clasp2 and Nuf2-depleted cells; Fig. 3A,C).
Spindle width rapidly reached a steady-state equilibrium around 40 minutes after the initiation of elongation, regardless of the specific perturbation analyzed (Fig. 1E, Fig. 2D, Fig. 3E).
These Clasp1/Clasp2 co-depleted spindles lost their diamond-like shape, with the spindle width nearing spindle length at steady-state equilibrium.
