Figure S5.
Asymmetric composition of mitotic cell–cell junctions due to selective ability of α-catenin in neighbors of mitotic cells to recruit vinculin. (A) Coculture of MDCK cells expressing either GFP-vinculin or mScarlet-vinculin after 1 h incubation with 15 ng/ml HGF. A representative example (z-projection) with inset shows that vinculin is recruited to cell–cell adhesions upon HGF-induced cell scattering, in both of the two cells forming the focal adherens junction. (B) Left: Representative image (z-projection) with inset of a mitotic cell expressing GFP-vinculin with a neighbor expressing mScarlet-vinculin, displaying a similar asymmetry as demonstrated in Fig. 5 A. Right: Quantifications of the ratio of junctional versus cytosolic vinculin intensity of mitotic cells and their interphase neighbors (as in Fig. 5 B), separated by the vinculin tag in the mitotic and neighboring cells (GFP represented by green dots, mScarlet by magenta dots; n = 64 and n = 23 junctions, respectively). Gray bars show the mean ratio with SD. These data demonstrate that the asymmetry is not dependent on the fluorophore tagged to vinculin. Data were pooled from three independent experiments. ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (C) Coculture of MDCK cells expressing either α-catenin–GFP or E-cadherin–DsRed, with representative image of a mitotic cell expressing α-catenin–GFP that shows α-catenin remains present at the mitotic cortex. (D) x,z-representation of cocultures of parental MDCK cells and cells inducibly expressing either mCherry–α-cateninWT (left) or mCherry-α-cateninM319G, R326E (α-cateninCA; right), together with GFP-vinculin. Nuclei are visualized with Hoechst. (E) Quantification of cell perimeter of MDCK cells in metaphase expressing either WT- or CA α-catenin (n = 45 per condition). Gray bars show the mean cell perimeter with SD. Quantified data were pooled from three independent experiments. ****, P < 0.0001; Mann–Whitney. (F) Quantification of time from nuclear envelope breakdown (NEB) to full rounding in MDCK cells inducibly expressing either WT- (n = 81) or CA α-catenin (n = 93). Gray bars show the mean cell perimeter with SD. Quantified data were pooled from three independent experiments. n.s., not significant (P = 0.17); Mann–Whitney. All scale bars, 10 µm or 2 µm (insets, A and B). α-cat, α-catenin.

Asymmetric composition of mitotic cell–cell junctions due to selective ability of α-catenin in neighbors of mitotic cells to recruit vinculin. (A) Coculture of MDCK cells expressing either GFP-vinculin or mScarlet-vinculin after 1 h incubation with 15 ng/ml HGF. A representative example (z-projection) with inset shows that vinculin is recruited to cell–cell adhesions upon HGF-induced cell scattering, in both of the two cells forming the focal adherens junction. (B) Left: Representative image (z-projection) with inset of a mitotic cell expressing GFP-vinculin with a neighbor expressing mScarlet-vinculin, displaying a similar asymmetry as demonstrated in Fig. 5 A. Right: Quantifications of the ratio of junctional versus cytosolic vinculin intensity of mitotic cells and their interphase neighbors (as in Fig. 5 B), separated by the vinculin tag in the mitotic and neighboring cells (GFP represented by green dots, mScarlet by magenta dots; n = 64 and n = 23 junctions, respectively). Gray bars show the mean ratio with SD. These data demonstrate that the asymmetry is not dependent on the fluorophore tagged to vinculin. Data were pooled from three independent experiments. ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (C) Coculture of MDCK cells expressing either α-catenin–GFP or E-cadherin–DsRed, with representative image of a mitotic cell expressing α-catenin–GFP that shows α-catenin remains present at the mitotic cortex. (D) x,z-representation of cocultures of parental MDCK cells and cells inducibly expressing either mCherry–α-cateninWT (left) or mCherry-α-cateninM319G, R326E (α-cateninCA; right), together with GFP-vinculin. Nuclei are visualized with Hoechst. (E) Quantification of cell perimeter of MDCK cells in metaphase expressing either WT- or CA α-catenin (n = 45 per condition). Gray bars show the mean cell perimeter with SD. Quantified data were pooled from three independent experiments. ****, P < 0.0001; Mann–Whitney. (F) Quantification of time from nuclear envelope breakdown (NEB) to full rounding in MDCK cells inducibly expressing either WT- (n = 81) or CA α-catenin (n = 93). Gray bars show the mean cell perimeter with SD. Quantified data were pooled from three independent experiments. n.s., not significant (P = 0.17); Mann–Whitney. All scale bars, 10 µm or 2 µm (insets, A and B). α-cat, α-catenin.

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