Supplementary Figure 8: Enlarged kinetochores in metaphase have immature attachments. Related to Figure 8.
(a,b) Representative images (a) and quantification of kinetochore levels (b) of Astrin in metaphase cells transfected with siRNA to Spindly and expressing the indicated GFP-Spindly variants. The graph shows the mean kinetochore intensity normalized to SpindlyFL. Each dot represents one cell: FL (n=55 cells), ΔN (n=48 cells) pooled from two independent experiments. Source data can be found in Supplementary Table 3. (c) Representative images of metaphase HeLa cells transfected with siRNA to Spindly, expressing GFP-SpindlyΔN and mCherry-Tubulin, fixed at 37°C or after cold treatment, and stained for indicated antigens. (‘b’, bridging fibre; ‘k’, k-fibres). The asterisk indicates the region of the enlarged kinetochore that normally mediates the lateral interaction with microtubules at 37°C. The experiment was repeated at least three times with similar results. (d) Live-cell imaging of HeLa cells transfected with siRNA to Spindly and expressing GFP-SpindlyΔN and mCherry-Tubulin. (‘Gr’, Growing microtubules; ‘Sh’, Shrinking microtubules; ‘e’ end-on attachment; ‘l’, lateral attachment). Maximum projections of several z-planes are shown. See also Supplementary Movies 5. The experiment was repeated at least three times with similar results. (e) Model of the mechanism of kinetochore expansion and compaction performed by the axis RZZ-Spindly-dynein and MPS1. See discussion for details.
