Fig. 3: Molecular basis for Astrin’s dual role in maintaining and recognising mono-oriented end-on attachments.

a Representative images of immunostained cells show the localisation of YFP tagged Astrin fragments (694–1193 or 851–1193, as indicated) following 5 h of STLC treatment to increase mono-oriented kinetochores and exposed to CDK1 inhibitor (RO3306) or Aurora-B inhibitor (ZM447439) for 15 min prior to fixation. Cells were immunostained with antibodies against GFP, Tubulin and CREST (as kinetochore marker). Only instances where YFP crescent signals are sandwiched between Tubulin and CREST signals are scored as positive for YFP-Astrin recruitment at the outer-kinetochore. White boxes mark the area of cropped images. Scale bar as indicated. b Graph shows the percentage of YFP-Astrin positive kinetochores in cells expressing YFP-Astrin WT or mutant and treated with CDK1i or Aurora-B inhibitors as indicated in a. c Representative super-resolution microscopy images of cells treated as in a. Measurements in crops indicate intra-kinetochore distances between peak intensities of CREST and YFP signals. d Graph shows the average intra-kinetochore distance between CREST and YFP signals in kinetochores of cells expressing Astrin full-length protein or a 694–1193 a.a fragment. Colours in super-plots b and d represent two (DMSO) or three (CDK1i or CDK1i and Aurora-Bi) independent experimental repeats (b) or cells (d), respectively. Horizontal black bars and whiskers mark average value and standard deviation, respectively, across two (WT) or three (fragment) independent experiments. ‘*’ and ‘ns’ indicate significant and insignificant statistical differences, respectively, as determined by the Non-parametric two-sided Mann-Whitney test. Exact p-values can be found in Source data. e Schematic of Astrin fragments used to dissect Astrin’s dual role in maintaining and sensing end-on attachments. Kinetochore binding C-terminus and microtubule-binding N- terminus of Astrin contribute to the sensing and maintenance of end-on attachments, respectively. The C-terminal region of Astrin that is responsible for sensing end-on kinetochores, bears at least two domains that are differently regulated by Aurora-B and CDK1 pathways.