Figure 1: The role of Kin-1 in regulating spindle assembly is dependent on integrin binding and activation.

(a) Kin-1 colocalizes with activated FAK (tyrosine 397 phosphorylated, pFAK) at focal adhesions, in non-mitotic cells (white arrowheads; scale bar, 8 μm). (b) Kin-1 is enriched at multiple sites along the adhesion plane of Cell 1 (left panels, white arrowheads) and at the spindle poles in Cell 2 in the mitotic plane (right panels, white arrowheads; scale bar, 8 μm). For visualization purposes, the outline of the mitotic cell (Cell 2, dashed white oval) has been shown. (c) siRNA-mediated Kin-1 depletion leads to abnormal spindle formation (Kin-1 siRNA, right column of images, three cells are shown). A non-targeting siRNA (Scram siRNA)-treated cell is shown as control (left panel of images; scale bar, 4 μm). (d) Schematic of a bipolar wild-type spindle and the breakdown of the four abnormal spindle phenotypes seen upon Kin-1 depletion (Misorientation, Monopolar-I, Monopolar-II and Multi-phenotype). The grey line represents the substrate the cells are adhered to, and misorientated spindles were defined as spindles that were not parallel to the substrate. (e) Quantification of the incidence of abnormal spindles in Scram siRNA, Kin-1^UTR1+2 siRNA and Kin-1^UTR1+2 siRNA plus Kin-1:GFP-expressing cells (n=8, ±s.e.m., ≥250 spindles scored for each condition). The dashed black line corresponds to the average percentage of abnormal spindle incidence in untreated control cells (5.3±1.9, n=3, ±s.e.m, ≥250 spindles scored). (f) Kin-1:GFP and Kin-1^W/A(612):GFP both localize to the spindle poles of mitotic cells (white arrowheads) as well as to the plasma membrane (yellow arrowhead; scale bar, 8 μm). (g) Quantification of the incidence of abnormal spindles in Scram siRNA, Kin-1^UTR1+2 siRNA, Kin-1^UTR1+2 siRNA plus Kin-1:GFP and Kin-1^UTR1+2 siRNA plus Kin-1^W/A(612):GFP-expressing cells (n=4, ±s.e.m., ≥250 spindles scored for each condition). (h) Kin-1 and talin-1 siRNA treatment both resulted in a similar increase in mutant spindles formation, whereas FAK siRNA had no effect. There was no co-operative increase in mutant spindles when both Kin-1 and talin were depleted (n=4, ±s.e.m., ≥250 spindles scored for each condition). Student’s t-tests were performed where indicated.