Figure 3. The vinculin mechanoresponse... | Rockefeller University Press

Figure 3.

$The vinculin mechanoresponse is a direct consequence of mitotic rounding.(A) Representative examples (z-projections) of MDCK cells expressing GFP-vinculin in interphase and following entry in mitosis in the presence of 10 µM para-nitro-blebbistatin, 2 µg/ml cytochalasin D, or DMSO control (Video 8). (B) Quantification of the fraction of mitotic cells with enrichment of junctional vinculin upon entry in mitosis in the presence of DMSO, para-nitro-blebbistatin, or cytochalasin D. Note that the small number of cells (8 out of 92) that still showed mitotic vinculin enrichment in the presence of blebbistatin also showed moderate mitotic rounding (not shown). Data were pooled from at least three independent experiments. DMSO versus blebbistatin: P < 0.0001; DMSO versus cytochalasin D: P = 0.0028; paired t test. (C) Top: Schematic diagram showing method of cellular confinement, in which MDCK cells are confined in height by a polydimethylsiloxane (PDMS)-coated glass slide during interphase to prevent rounding when cells enter mitosis. Bottom: Representative still images (z-projections) of MDCK cells expressing GFP-vinculin and H2B-mCherry upon induction and release of confinement (Video 9). (D) Representative x,z-projection of a mitotic MDCK cell expressing GFP-vinculin and H2B-mCherry under confinement and following release of confinement. The locations of the cell–cell contacts are indicated with gray arrowheads, and the height of PDMS-coated glass slide (6 µm) is indicated by the purple dashed line. (E) Quantification of cell perimeter of cells upon induction and release of confinement (n = 16 cells). Gray bars show the mean cell perimeter with SD. Data were pooled from two independent experiments. ****, P < 0.0001; paired t test. (F) Quantification of the fraction of mitotic cells with enrichment of junctional vinculin following induction and release of confinement (n = 16). Data were pooled from two independent experiments. All scale bars, 10 µm.$

The vinculin mechanoresponse is a direct consequence of mitotic rounding. (A) Representative examples (z-projections) of MDCK cells expressing GFP-vinculin in interphase and following entry in mitosis in the presence of 10 µM para-nitro-blebbistatin, 2 µg/ml cytochalasin D, or DMSO control (Video 8). (B) Quantification of the fraction of mitotic cells with enrichment of junctional vinculin upon entry in mitosis in the presence of DMSO, para-nitro-blebbistatin, or cytochalasin D. Note that the small number of cells (8 out of 92) that still showed mitotic vinculin enrichment in the presence of blebbistatin also showed moderate mitotic rounding (not shown). Data were pooled from at least three independent experiments. DMSO versus blebbistatin: P < 0.0001; DMSO versus cytochalasin D: P = 0.0028; paired t test. (C) Top: Schematic diagram showing method of cellular confinement, in which MDCK cells are confined in height by a polydimethylsiloxane (PDMS)-coated glass slide during interphase to prevent rounding when cells enter mitosis. Bottom: Representative still images (z-projections) of MDCK cells expressing GFP-vinculin and H2B-mCherry upon induction and release of confinement (Video 9). (D) Representative x,z-projection of a mitotic MDCK cell expressing GFP-vinculin and H2B-mCherry under confinement and following release of confinement. The locations of the cell–cell contacts are indicated with gray arrowheads, and the height of PDMS-coated glass slide (6 µm) is indicated by the purple dashed line. (E) Quantification of cell perimeter of cells upon induction and release of confinement (n = 16 cells). Gray bars show the mean cell perimeter with SD. Data were pooled from two independent experiments. ****, P < 0.0001; paired t test. (F) Quantification of the fraction of mitotic cells with enrichment of junctional vinculin following induction and release of confinement (n = 16). Data were pooled from two independent experiments. All scale bars, 10 µm.