Fig. 5.
Astrin-SKAP complex mediates a late step of the end-on conversion process. a Experimental regime: Cells treated with Monastrol and MG132 for 2 h and ZM447439 for 45 min were immunostained using antibodies against Tubulin and SKAP and CREST antisera. DNA was stained using DAPI. b Immunofluorescence images of HeLa cells transfected with control, SKAP or Astrin siRNA, as indicated, and treated as in a. Magnified images in the right-most panels correspond to boxed area. Scale bar: 5 μm. c Single-plane time-lapse images of Z-stacks show the fate of lateral kinetochores (red) attached to MTs (green) in Control or SKAP siRNA treated HeLaYFP-Tub; CenpB-Red cells in Monastrol. White arrowheads mark the KT tracked. Yellow arrows mark shrinking lateral K-fibre and white arrows highlight synchronous movement of the KT and MT-end. Scale bar: 2 μm. d Graph shows percentage of lateral kinetochores that remained lateral (L) or transitioned into other attachment states (D–detached; E–end-on) in time-lapse movies as in c. Error bars represent SEM from four experiments. e Graph of time spent by lateral kinetochores on MT-walls, before changing into another attachment state, in control or SKAP siRNA treated cells. Red and black bars mark mean-time and SD values, respectively, across kinetochores from four independent experiments. # indicates statistically insignificant difference (unpaired Student’s t-test). f Immunoblots of lysates of cells treated with control or SKAP siRNA harvested after time-lapse microscopy. Antibodies against SKAP and γ-Tubulin (loading control) were used. g Images of bipolar spindles with congressed chromosomes in control or SKAP siRNA transfected cells treated with MG132 and ZM447439 and immunostained as in a. Scale bar: 5 μm in uncropped and 2 μm in cropped images. Boxed areas correspond to cropped images. h Cartoon illustrates SKAP’s role in the end-on conversion process: (i) SKAP is selectively recruited to end-on (mature) but not lateral (immature) kinetochores. (ii) SKAP is crucial for the lateral to end-on conversion event but not required for tethering kinetochores onto microtubule walls. Finally, reducing Aurora-B allows end-on attachments in a SKAP dependent manner