Vinculin phosphorylation differentially regulates mechanotransduction at cell-cell and cell-matrix adhesions

Abstract

Cells experience mechanical forces throughout their lifetimes. Vinculin is critical for transmitting these forces, yet how it achieves its distinct functions at cell-cell and cell-matrix adhesions remains unanswered. Here, we show vinculin is phosphorylated at Y822 in cell-cell, but not cell-matrix, adhesions. Phosphorylation at Y822 was elevated when forces were applied to E-cadherin and was required for vinculin to integrate into the cadherin complex. The mutation Y822F ablated these activities and prevented cells from stiffening in response to forces on E-cadherin. In contrast, Y822 phosphorylation was not required for vinculin functions in cell-matrix adhesions, including integrin-induced cell stiffening. Finally, forces applied to E-cadherin activated Abelson (Abl) tyrosine kinase to phosphorylate vinculin; Abl inhibition mimicked the loss of vinculin phosphorylation. These data reveal an unexpected regulatory mechanism in which vinculin Y822 phosphorylation determines whether cadherins transmit force and provides a paradigm for how a shared component of adhesions can produce biologically distinct functions.

Figure 1.

Vinculin is specifically tyrosine phosphorylated at Y822 in cell–cell junctions. (A) Effect of cell density on vinculin Y822 phosphorylation. Subconfluent or confluent cultures of MCF10a vinculin knockdown cells rescued with GFP-vinculin were lysed and immunoblotted with phosphospecific Y822 antibodies and stripped and reprobed for total vinculin levels. The left panel shows representative immunoblots and the right panel shows the quantification of the average amount of phosphorylated vinculin normalized to total vinculin levels from three independent experiments. ##, P < 0.005. (B and C) The effect of inducing an epithelial-to-mesenchymal transition on vinculin Y822 phosphorylation. The levels of phosphorylated and total vinculin in MCF10a cells induced to scatter by application of HGF for 2 h (B) or in MDCK cells overexpressing Snail (C) were examined and presented as described in A. #, P < 0.05; *, P < 0.01. (D and E) Localization of phospho-Y822 in cells; MCF10a vinculin knockdown cells rescued with GFP-vinculin (E) or MCF10a parental cells (D) were examined by immunofluorescence with affinity-purified phosphospecific antibodies against Y822 (D and E) and E-cadherin or talin (D). The phospho-Y822 antibodies specifically stain adherens junctions but not focal adhesions. Bars, 20 µm. Images are representative of three independent experiments. Average Pearson correlation coefficient reported from three independent experiments ± SEM.

Figure 2.

Vinculin Y822 phosphorylation increases in response to engagement of and force on cadherins, but not integrins. (A) Examination of vinculin phosphorylation during the assembly of cell–cell junctions. Cell–cell junctions were manipulated using a calcium switch procedure as described in the Materials and methods. At the indicated times after calcium restoration, cells were lysed and phospho-vinculin levels were examined, quantitated, and presented as described in Fig. 1 A. “Short” denotes short exposure of the immunoblot; “Long” denotes long exposure of immunoblot. #, P < 0.05. (B) The effect that integrin- and cadherin-mediated adhesion have on phospho-Y822 levels. MCF10a cells were plated on tissue culture dishes coated with cadherin extracellular domains (CEC) or human fibronectin (FN). At the specified times, cells were lysed and assayed for the levels of phosphorylated vinculin as described above. (C and D) The effect that force on cadherins or integrins has on phosphorylated vinculin. MCF10a cells were incubated with beads coated with IgG or antibodies against E-cadherin (C) or with the integrin ligand, fibronectin, or poly-lysine as a control (D), and stimulated with tensional force using a permanent magnet. The levels of phosphorylated vinculin were assessed as described above. Y-27632 indicates cells that were incubated with a ROCK1/2 inhibitor before the application of force. Results represent the means from three independent experiments. ##, P < 0.005.

Figure 3.

Y822F vinculin does not support cadherin-mediated adhesion or force transmission. MCF10a cells were infected with GFP, GFP-tagged chick vinculin (GFP-WT), or a mutant version of vinculin with a Y822F substitution (GFP-822F), and then infected a second time with either an empty vector (Cont) or an shRNA vector targeting human vinculin (KD). (A) Examination of E-cadherin localization by immunofluorescence. The Y822F mutant vinculin does not rescue E-cadherin localization to adherens junctions in cells with low levels of vinculin. Bar, 10 µm. (B) Examination of the adhesion of the indicated cells to cadherin extracellular (EC) domains. For assessing homophilic ligation, cells were plated on dishes coated with human E-cadherin extracellular domains fused to Fc, and were washed. The percentages of cells that adhered (±SEM from three independent experiments) are shown. #, P ≤ 0.05. (C) Pulses of force were applied to magnetic beads coated with Fc-tagged E-cadherin that have been incubated on MCF10a knockdown cells rescued with either GFP-WT or GFP-Y822F vinculin. Experiments are the relative means ± SEM and have been normalized to the first pulse. WT vinculin + E-cadherin beads (n = 30); Y822F vinculin + E-cadherin beads (n = 30). Statistical significance was determined by two-tailed Student’s t test. #, P ≤ 0.05. (D) The effect of Y822F substitution on E-cadherin induced vinculin Y822 phosphorylation in response to force. Force was exerted on magnetic beads as described in the legend of Fig. 2 C and vinculin phosphorylation was measured and depicted as described in Fig. 1 A. Results are representative of three independent experiments. (E) The levels of β-catenin that coimmunoprecipitated with GFP, GFP-WT vinculin, or GFP-Y822F vinculin were assessed using immunoblotting. The left panel shows representative immunoblots and the right panel is a quantification of the average amount of bound protein normalized for the amount of GFP protein recovered in three independent experiments. β-Catenin bound GFP-Y822F at reduced levels compared with WT vinculin. *, P ≤ 0.01.

Figure 4.

Blocking vinculin phosphorylation at Y822 does not inhibit integrin adhesion, force transmission, or talin recruitment. (A–D) Examination of the ability of Y822F to support integrin-mediated adhesion and force transmission in epithelial cells. (A) Subconfluent cultures of vinculin knockdown MCF10a cells rescued with GFP-vinculin (KD-GFP-WT) or Y822F vinculin (KD/GFP-822F) or control MCF10a cells expressing GFP (Cont/GFP) were examined by phase-contrast microscopy. No differences were observed in cell morphology. Bar, 100 µm. (B) Examination of Y822F localization in focal adhesions. The cells were stained with antibodies against paxillin. GFP-WT and GFP-Y822F vinculin localized to focal adhesions to similar extents. Bars, 20 µm. (C) The levels of talin that coimmunoprecipitated with GFP, GFP-WT vinculin, or GFP-Y822F vinculin were assessed using immunoblotting. Unlike β-catenin, talin bound GFP-Y822F to wild-type levels. (D) Summary of the ability of Y822F vinculin to support integrin-mediated events. Matrix adhesion indicates the percentage of cells that adhered to 10 µg/ml fibronectin. Cell spreading indicates the area of cells (as measured using ImageJ) that were allowed to spread on 10 µg/ml of fibronectin for 4 h. Cell migration indicates the cell speed of individual cells randomly migrating on fibronectin. The average cell speed (±SEM) was calculated from three independent experiments. (E and F) An examination of the ability of Y822F to support integrin-mediated events in mouse embryo fibroblasts isolated from the vinculin-null mouse. (E) Colocalization of GFP-WT or GFP-Y822F vinculin with paxillin as examined by immunofluorescence. Bars, 20 µm. (F) The ability of the WT and Y822F cells to spread on fibronectin-coated surfaces was examined as described in D; the average number of focal adhesions per cell were counted and expressed as the average ± SD. Cells expressing Y822F spread slightly better and had slightly more focal adhesions than those expressing WT vinculin. (G) Pulses of force were applied to fibronectin-coated magnetic beads that were incubated on MCF10a knockdown cells rescued with either GFP-WT or GFP-Y822F vinculin. Experiments are the relative means ± SEM and have been normalized to the first pulse. WT vinculin + fibronectin beads (n = 19); Y822F vinculin + fibronectin beads (n = 20). Statistical significance was determined by two-tailed Student’s t test; *, P ≤ 0.01.

Figure 5.

Abl is the tyrosine kinase that phosphorylates vinculin Y822. (A and B) Examination of vinculin phosphorylation on Y822 as described in Fig. 1 A. (A) NIH3T3 cells, NIH3T3 cells transformed with the Rous sarcoma virus (v-Src), or BALB/c fibroblasts transformed with Abelson mouse leukemia virus (v-Abl) were left resting (−) or treated with pervanadate (+) before examination of pY822 phosphorylation. (B) MCF10a parental cells were left resting or treated with the phosphatase inhibitor pervanadate; Gleevec indicates cells pretreated with this Abl inhibitor. (C) In vitro kinase assay using Abl. Purified GST or GST vinculin 811–881 proteins were incubated with recombinant Abl in the presence or absence of 5 µM Gleevec. The samples were fractionated by SDS-PAGE and the bottom portion of the gel was stained to show the amounts of protein used (bottom); the top of the gel was immunoblotted with a phospho-Y822 specific antibody. All results are representative of three independent experiments. (D and E) Effect that application of force on E-cadherin or integrins has Abl tyrosine kinase activity. Force was exerted on magnetic beads coated with antibodies against E-cadherin (D) or fibronectin (E) as described in the legend of Fig. 2 C and phosphorylation of CrkL, an Abl substrate, was examined by immunoblotting with antibodies that recognize Y207 (the Abl-specific site) or total CrkL levels (left). Quantification of the results of three independent experiments is shown in the graph in the right-hand panel. IgG and poly-l-lysine indicate the controls. *, P < 0.01.

Figure 6.

Inhibition of Abl mimics loss of vinculin phosphorylation at Y822. (A and B) β-catenin recruitment to and colocalization with vinculin when Abl is inhibited. MCF10a cells were left resting (−) or treated with Gleevec. (A) Vinculin was immunoprecipitated from parental cells and the levels of bound β-catenin were examined by immunoblotting. The blot was stripped and reprobed with antibodies against vinculin to reveal the amounts of proteins recovered. (B) MCF10a vinculin knockdown cells expressing GFP-vinculin were left untreated or treated with Gleevec and then analyzed by immunofluorescence with antibodies against β-catenin. Average Pearson correlation coefficient is reported below images (±SD). Bars, 10 µm. (C) Effects of Gleevec on E-cadherin–induced cellular stiffening. MCF10a vinculin knockdown cells re-expressing WT or Y822F vinculin were incubated with magnetic beads coated with Fc-tagged E-cadherin and were treated with or without Gleevec. 3DFM was used to measure the bead displacement on individual cells. Relative bead displacement is shown. WT vinculin (n = 30); Y822F vinculin (n = 30); WT vinculin + Gleevec (n = 30); Y822F vinculin + Gleevec (n = 30). (D) MCF10a cells were incubated with magnetic beads coated with antibodies against E-cadherin or IgG in the presence (+) or absence (−) of Gleevec. Tensional force was generated on the beads using a permanent magnet and phospho-Y822 or total vinculin levels were examined as described in Fig. 1 A. All results are representative of at least three independent experiments. ##, P < 0.005. (E) Effects of Gleevec on cell–matrix events. The ability of the GFP-WT cells to adhere to or spread on fibronectin-coated surfaces was examined as described in Fig. 4 D after 2 h of treatment with 20 µM Gleevec and expressed as averages ± SEM.