Fig. 2: Kinetochore-autonomous regulation of Astrin dynamics at end-on attachments.

a Representative time-lapse images of Fluorescence Recovery After Photobleaching (FRAP) of HeLa cells expressing YFP-Astrin following one hour of MG132 treatment prior to photobleaching. Cropped images show the recovery of YFP intensities after bleaching one spindle area (green circle) or one kinetochore (red circle). Cartoons on the left display a cross-sectional view of the spindle showing the plane of focus (orange bar) for microtubules (top) or kinetochores (middle) of the mitotic spindle (shown as a grey ellipse). Scale bar as indicated. b and c, Graphs show curves of normalised YFP fluorescence intensities on spindle (b) or kinetochores (c), respectively, versus time from FRAP experiments as in a. Lines and whiskers mark the average and standard deviation, respectively, from two independent experiments (kinetochore data) and three independent experiments (microtubules data). The calculated half-life time of recovery is indicated in each graph. d Representative time-lapse images of Fluorescence Recovery After Photobleaching (FRAP) of HeLa cells expressing YFP-Astrin, acquired once every 15 s, following one hour of STLC treatment prior to photobleaching in the presence of CDK1 inhibitor (CDK1i, RO3306) or DMSO (control) as indicated. Cropped images outlined in yellow and blue indicate bleached and unbleached (control) kinetochore, corresponding to yellow and blue arrowheads, respectively. Scale bar as indicated. e Kinetochore-bound Astrin recovery times with or without CDK1 inhibitor obtained using movies as in d. Recovery time was visually scored to indicate the first time point (30 s bin) where Astrin signal at the kinetochore was seen following the bleaching event. The values shown are from two independent experimental repeats.