α-cat-H0-ABD ⁺ -induced cytokinesis failure requires the mechanosensitive M-domain. (A) Schematic of α-cat mutants used for analysis. More (+++) or less (+) F-actin binding is predicted from previous in-solution actin co-sedimentation assays carried out on wild-type α-cat actin-binding domain (amino acids 663–696) or H0-mutant α-cat ABD, which disrupts helicity of the H0 portion of the first α-helix of the five-helical ABD and increases F-actin binding affinity (Ishiyama et al., 2018). Since the extended αH0-αH1 region gates force-dependent binding to F-actin in context of a reconstituted E-cad cytodomain/β-cat/α-cat complex (Wang et al., 2022), and this has never been formally tested for an α-cat lacking its middle domain, α-catΔM and α-catΔM-H0-ABD⁺ constructs binding to F-actin is predicted (+++ and + in gray), rather than formally tested (black). (B) Live imaging analysis of MDCK α-cat KO cells restored with α-cat-WT, α-cat-H0-ABD⁺, α-catΔM and α-catΔM-H0-ABD⁺ during mitosis phases (gray). Time stamp in minutes (‘, white); yellow arrowheads show dividing cells; GFP-α-cat (green), nuclei (magenta). Scale bar, 50 μm. (C) Graph quantification of mitosis phase lengths as minutes from prophase. ** and ****P < 0.01 and 0.001 by ANOVA. Symbols in blue or with a centered “x” show time-point where cytokinesis failed. (D) Graph quantification of cytokinesis failure events. Graphs indicate mean ± SD.