Figure 1.
Persistent coupling of α-cat to F-actin enhances binucleation rate. (A) Schematic of α-cat domains based on crystal structure. α-cat-H0-ABD+ replaces four amino acids (RAIM>GSGS) in the first kinked helix of α-cat’s five-helical bundle actin-binding domain (whitening of red domain), compromising normal force-gated binding of α-cat to F-actin. This generates an α-cat that binds F-actin more strongly (+++) than wild-type α-cat (+) via in vitro pelleting assays (Ishiyama et al., 2018). (B) Immunoblot of MDCK KO2.2 cells transfected with GFP-tagged versions of WT α-cat or an F-actin-binding-domain (ABD) unfolding mutant (α-catH0-ABD+). GAPDH was used as loading control. Asterisk (*) marks very low levels of an α-cat fragment generated by our CRISPR-targeting strategy via a previously suggested alternative translational start site (Bullions et al., 1997). As this α-cat isoform lacks the β-cat binding region, fails to robustly localize to cell-cell contacts (Quinn et al., 2024) and is not consistently present in our MDCK cultures, it does not likely contribute to the cytokinesis interference pathway in this study. (C) Representative immunofluorescence images of MDCK α-cat KO cells restored with GFP-α-cat-WT (Top) or GFP-α-cat-H0-ABD+ (Bottom). GFP-α-cat (green), nuclei (magenta), binucleated cells (outlined, yellow dashed line). Scale bar, 30 μm. Rectangular regions framed in white-dashed boxes are enlarged to show detail (right, i). Scale bar, 15 μm. Yellow arrows denote binucleated cells chosen for orthogonal views (right, ii). Yellow arrowhead shows plane of x-z slice. Scale bar, 5 μm. (D) Graph shows percentage of wound distal versus wound proximal cells displaying binucleation, P < 0.05 (*) and P < 0.001 (***) by unpaired t test. Symbols denote measurements from distinct fields of view; different colors reflect biological replicates (n = 3) mean ± SD. (E) Leading edge of MDCK α-cat KO cells expressing GFP-α-cat-WT (Top) or GFP-α-cat-H0-ABD+ (Bottom) 40-h post scratch wound. GFP- α-cat (green), nuclei (magenta). Yellow arrows denote binucleated cells. Scale bar, 40 μm. The rectangular regions framed in yellow are enlarged to show detail (right, i). Scale bar, 40 μm. Source data are available for this figure: SourceData F1. Refer to the image caption for details.

Persistent coupling of α-cat to F-actin enhances binucleation rate. (A) Schematic of α-cat domains based on crystal structure. α-cat-H0-ABD+ replaces four amino acids (RAIM>GSGS) in the first kinked helix of α-cat’s five-helical bundle actin-binding domain (whitening of red domain), compromising normal force-gated binding of α-cat to F-actin. This generates an α-cat that binds F-actin more strongly (+++) than wild-type α-cat (+) via in vitro pelleting assays (Ishiyama et al., 2018). (B) Immunoblot of MDCK KO2.2 cells transfected with GFP-tagged versions of WT α-cat or an F-actin-binding-domain (ABD) unfolding mutant (α-catH0-ABD+). GAPDH was used as loading control. Asterisk (*) marks very low levels of an α-cat fragment generated by our CRISPR-targeting strategy via a previously suggested alternative translational start site (Bullions et al., 1997). As this α-cat isoform lacks the β-cat binding region, fails to robustly localize to cell-cell contacts (Quinn et al., 2024) and is not consistently present in our MDCK cultures, it does not likely contribute to the cytokinesis interference pathway in this study. (C) Representative immunofluorescence images of MDCK α-cat KO cells restored with GFP-α-cat-WT (Top) or GFP-α-cat-H0-ABD+ (Bottom). GFP-α-cat (green), nuclei (magenta), binucleated cells (outlined, yellow dashed line). Scale bar, 30 μm. Rectangular regions framed in white-dashed boxes are enlarged to show detail (right, i). Scale bar, 15 μm. Yellow arrows denote binucleated cells chosen for orthogonal views (right, ii). Yellow arrowhead shows plane of x-z slice. Scale bar, 5 μm. (D) Graph shows percentage of wound distal versus wound proximal cells displaying binucleation, P < 0.05 (*) and P < 0.001 (***) by unpaired t test. Symbols denote measurements from distinct fields of view; different colors reflect biological replicates (n = 3) mean ± SD. (E) Leading edge of MDCK α-cat KO cells expressing GFP-α-cat-WT (Top) or GFP-α-cat-H0-ABD+ (Bottom) 40-h post scratch wound. GFP- α-cat (green), nuclei (magenta). Yellow arrows denote binucleated cells. Scale bar, 40 μm. The rectangular regions framed in yellow are enlarged to show detail (right, i). Scale bar, 40 μm. Source data are available for this figure: SourceData F1.

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