Figure 2.
Tension-sensitive recruitment of vinculin to adherens junctions during mitosis.(A) Representative image (z-projection) of MDCK cells expressing GFP-vinculin in interphase and following entry in mitosis (Video 3). (B) Quantification of the ratio of junctional versus cytosolic GFP-vinculin in MDCK cells in interphase and following entry in mitosis (n = 27). Data were pooled from three independent experiments. ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (C) Representative x,z-projection of MDCK cell expressing GFP-vinculin in interphase and following entry in mitosis. (D) Average cell perimeter ± SD (blue line) and the ratio of junctional versus cytosolic GFP-vinculin intensity ± SD (green line) during progression of cells through mitosis. Time-lapse images were aligned at the moment of nuclear envelope breakdown (NEB). Perimeter analyses were performed until the initiation of cytokinesis (dotted line represents average perimeter after measurement of first cell was completed). Note that the decrease in cell perimeter initiates shortly before NEB, in accordance with previous analyses of mitotic rounding in nonepithelial cells (Matthews et al., 2012). Data were pooled from three independent experiments (n = 9). (E) Representative image (z-projection) of mitotic MDCK cells coexpressing GFP-vinculin and unconjugated mScarlet. (F) Western blot of lysates from parental, α-catenin knockout (KO), and α-catenin KO with addback of WT- or vinculin-binding deficient (ΔVBS) α-catenin, MDCK cells probed for α-catenin and α-tubulin. (G) Immunostaining of MDCK cells (z-projection) expressing WT- or vinculin-binding deficient (ΔVBS) α-catenin for endogenous vinculin, together with DAPI. (H) Representative image of a mitotic MDCK cell expressing GFP-vinculin before and after laser ablation of the cortex of a cell–cell contact perpendicular to the mitotic cell (indicated with orange scissors; Video 6). The inset shows the tricellular junction associated with the ablated cell–cell contact over time. Note that the gradual decrease indicates that the disappearance of the GFP-vinculin signal is not due to bleaching during laser ablation of the junction, and that the tricellular junction itself remains intact as visualized by CellMask. (I) Quantification of the ratio of junctional versus cytosolic GFP-vinculin in mitotic MDCK cells before and after laser cuts (n = 12). Data were pooled from three independent experiments. ****, P < 0.0001; paired t test. All scale bars, 10 µm or 2 µm (inset, H). IB, immunoblot; t, time; Vin, vinculin.

Tension-sensitive recruitment of vinculin to adherens junctions during mitosis. (A) Representative image (z-projection) of MDCK cells expressing GFP-vinculin in interphase and following entry in mitosis (Video 3). (B) Quantification of the ratio of junctional versus cytosolic GFP-vinculin in MDCK cells in interphase and following entry in mitosis (n = 27). Data were pooled from three independent experiments. ****, P < 0.0001; Wilcoxon matched-pairs signed-rank test. (C) Representative x,z-projection of MDCK cell expressing GFP-vinculin in interphase and following entry in mitosis. (D) Average cell perimeter ± SD (blue line) and the ratio of junctional versus cytosolic GFP-vinculin intensity ± SD (green line) during progression of cells through mitosis. Time-lapse images were aligned at the moment of nuclear envelope breakdown (NEB). Perimeter analyses were performed until the initiation of cytokinesis (dotted line represents average perimeter after measurement of first cell was completed). Note that the decrease in cell perimeter initiates shortly before NEB, in accordance with previous analyses of mitotic rounding in nonepithelial cells (Matthews et al., 2012). Data were pooled from three independent experiments (n = 9). (E) Representative image (z-projection) of mitotic MDCK cells coexpressing GFP-vinculin and unconjugated mScarlet. (F) Western blot of lysates from parental, α-catenin knockout (KO), and α-catenin KO with addback of WT- or vinculin-binding deficient (ΔVBS) α-catenin, MDCK cells probed for α-catenin and α-tubulin. (G) Immunostaining of MDCK cells (z-projection) expressing WT- or vinculin-binding deficient (ΔVBS) α-catenin for endogenous vinculin, together with DAPI. (H) Representative image of a mitotic MDCK cell expressing GFP-vinculin before and after laser ablation of the cortex of a cell–cell contact perpendicular to the mitotic cell (indicated with orange scissors; Video 6). The inset shows the tricellular junction associated with the ablated cell–cell contact over time. Note that the gradual decrease indicates that the disappearance of the GFP-vinculin signal is not due to bleaching during laser ablation of the junction, and that the tricellular junction itself remains intact as visualized by CellMask. (I) Quantification of the ratio of junctional versus cytosolic GFP-vinculin in mitotic MDCK cells before and after laser cuts (n = 12). Data were pooled from three independent experiments. ****, P < 0.0001; paired t test. All scale bars, 10 µm or 2 µm (inset, H). IB, immunoblot; t, time; Vin, vinculin.

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