Figure 5. A vinculin-dependent... | Rockefeller University Press

Figure 5.

$A vinculin-dependent mechanoresponse occurs selectively in neighbors of mitotic cells.(A) Top: schematic representation of coculture of MDCK cells expressing either GFP- or mScarlet-vinculin. Bottom: Representative image (z-projection) of a mitotic cell expressing mScarlet-vinculin with a neighbor expressing GFP-vinculin (Video 10). The inset (yellow) shows that the mitotic cell–cell contact contains a strong cortical GFP-vinculin enrichment originating from the neighboring cell, while mScarlet-vinculin is absent from the cortex of the mitotic cell. Similar results were obtained when the mitotic cell expressed GFP-vinculin and was adjoined by a neighbor expressing mScarlet-vinculin (Fig. S5 B). (B) Quantification of the ratio of junctional versus cytosolic vinculin intensity of mitotic cells and their interphase neighbors (n = 87 junctions). Gray bars show the mean ratio with SD. Data were pooled from three independent experiments, and from both mScarlet-vinculin mitotic cells with GFP-vinculin neighbors and vice versa, as no differences between the tags could be observed (see Fig. S5 B). ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (C) Coculture of MDCK cells expressing either WT- or vinculin-binding deficient (ΔVBS) α-catenin, which are distinguished by expression of H2B-mCherry only in WT cells (all other nuclei are visualized with DAPI, shown in green). Left: Example of a mitotic cell expressing α-cateninWT and neighbor expressing α-cateninΔVBS. Right: Example of a mitotic cell expressing α-cateninΔVBS and neighbor expressing α-cateninWT. (D) Quantification of the fraction of mitotic cells with disrupted cell–cell contacts in cocultures of MDCK cells expressing WT and ΔVBS α-catenin. Data were pooled from three independent experiments, with at least 10 cells analyzed per condition in each experiment. Gray bars represent the mean and SD of the average fraction of disrupted cell–cell contacts in the independent experiments. **, P = 0.02; paired t test; n.s., not significant (P = 0.45). All scale bars, 10 µm or 2 µm (inset, A).$

A vinculin-dependent mechanoresponse occurs selectively in neighbors of mitotic cells. (A) Top: schematic representation of coculture of MDCK cells expressing either GFP- or mScarlet-vinculin. Bottom: Representative image (z-projection) of a mitotic cell expressing mScarlet-vinculin with a neighbor expressing GFP-vinculin (Video 10). The inset (yellow) shows that the mitotic cell–cell contact contains a strong cortical GFP-vinculin enrichment originating from the neighboring cell, while mScarlet-vinculin is absent from the cortex of the mitotic cell. Similar results were obtained when the mitotic cell expressed GFP-vinculin and was adjoined by a neighbor expressing mScarlet-vinculin (Fig. S5 B). (B) Quantification of the ratio of junctional versus cytosolic vinculin intensity of mitotic cells and their interphase neighbors (n = 87 junctions). Gray bars show the mean ratio with SD. Data were pooled from three independent experiments, and from both mScarlet-vinculin mitotic cells with GFP-vinculin neighbors and vice versa, as no differences between the tags could be observed (see Fig. S5 B). ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (C) Coculture of MDCK cells expressing either WT- or vinculin-binding deficient (ΔVBS) α-catenin, which are distinguished by expression of H2B-mCherry only in WT cells (all other nuclei are visualized with DAPI, shown in green). Left: Example of a mitotic cell expressing α-catenin^WT and neighbor expressing α-catenin^ΔVBS. Right: Example of a mitotic cell expressing α-catenin^ΔVBS and neighbor expressing α-catenin^WT. (D) Quantification of the fraction of mitotic cells with disrupted cell–cell contacts in cocultures of MDCK cells expressing WT and ΔVBS α-catenin. Data were pooled from three independent experiments, with at least 10 cells analyzed per condition in each experiment. Gray bars represent the mean and SD of the average fraction of disrupted cell–cell contacts in the independent experiments. **, P = 0.02; paired t test; n.s., not significant (P = 0.45). All scale bars, 10 µm or 2 µm (inset, A).