Vinexin family (SORBS) proteins play different roles in stiffness-sensing and contractile force generation

Abstract

Vinexin, c-Cbl associated protein (CAP) and Arg-binding protein 2 (ArgBP2) constitute an adaptor protein family called the vinexin (SORBS) family that is targeted to focal adhesions (FAs). Although numerous studies have focused on each of the SORBS proteins and partially elucidated their involvement in mechanotransduction, a comparative analysis of their function has not been well addressed. Here, we established mouse embryonic fibroblasts that individually expressed SORBS proteins and analysed their functions in an identical cell context. Both vinexin-α and CAP co-localized with vinculin at FAs and promoted the appearance of vinculin-rich FAs, whereas ArgBP2 co-localized with α-actinin at the proximal end of FAs and punctate structures on actin stress fibers (SFs), and induced paxillin-rich FAs. Furthermore, both vinexin-α and CAP contributed to extracellular matrix stiffness-dependent vinculin behaviors, while ArgBP2 stabilized α-actinin on SFs and enhanced intracellular contractile forces. These results demonstrate the differential roles of SORBS proteins in mechanotransduction.

Keywords: Actin cytoskeleton; ArgBP2; CAP; Focal adhesion; Mechanotransduction; Vinexin.

Fig. 1.

Establishment of SORBS-re-expressing TKO MEFs. (A) Schematic diagram of the domain structure of SORBS proteins (vinexin-α, CAP and ArgBP2). SORBS proteins share one SoHo domain and three SH3 domains. (B) TKO MEFs re-expressing each SORBS protein were lysed and subjected to SDS-PAGE. Re-expression of SORBS proteins in TKO MEFs was examined by immunoblotting using the indicated antibodies. Vinculin was used as a loading control. The lysate of astrocytes served as a reference sample, which expressed all SORBS proteins. (C–E) SORBS-re-expressing cells were cultured on coverslips for 24 h. Cell membranes were stained with CellMask Orange and photographed (C). Cell area (D) and aspect ratio (E) were quantified from images of ninety cells from three separate experiments. N.S., not significant in Kruskal–Wallis ANOVA. Scale bars: 50 µm.

Fig. 2.

Localization of SORBS proteins in re-expressing cells. (A,B) SORBS-re-expressing cells cultured on coverslips for 24 h were immunostained using the indicated antibodies including anti-vinculin (A) or anti-α-actinin antibodies (B). Representative images are displayed from three independent experiments. The FA-containing areas indicated by yellow boxes are cropped and displayed at the bottom of the figure. ArgBP2 and α-actinin were co-localized at FAs (arrowhead) and on SFs (arrow). Scale bars: 20 µm. See also Figs S1C-E and S2.

Fig. 3.

Both vinexin-α and CAP induce vinculin-rich FAs, whereas ArgBP2 induces paxillin-rich FAs. SORBS-re-expressing cells cultured on coverslips were immunostained using antibodies indicated as follows: anti-vinculin (hVIN) (A,B), anti-paxillin (C–F), anti-talin (G,H) and anti-vinculin (ab73412) (E–H). Ninety (A–D) or thirty (E–H) individual cells from three separate experiments were photographed for each condition. Scale bars: 20 µm. The number of vinculin-positive FAs and paxillin-positive FAs per cell were quantified from the images in A and C, respectively (B,D). The intensity ratio of paxillin and talin to vinculin within vinculin-positive FAs was quantified from the images in E and G, respectively (F,H); P-value calculated by Mann–Whitney U-tests; n.s., not significant. See also Fig. S1F.

Fig. 4.

Both vinexin-α and CAP but not ArgBP2 contribute to CSK-resistant vinculin on rigid substrates. SORBS-re-expressing cells were cultured on polyacrylamide gel substrates with 2.2 kPa (soft) and 25 kPa (rigid) stiffness. Cells were treated without (A–C) or with (D–F) CSK, followed by immunostaining using an anti-vinculin antibody. Thirty individual cells from three separate experiments were photographed for each condition (A,D). Scale bars: 20 µm. The number (B,E) and integrated density (C,F) of total or CSK-resistant vinculin-positive FAs per cell were quantified from the images. Note that non-specific staining was observed in the nucleus after CSK treatment. n=30; P-value was calculated by Mann–Whitney U-test; n.s., not significant. See also Fig. S3C-E.

Fig. 5.

Both vinexin-α and CAP but not ArgBP2 immobilize vinculin at FAs and bind to vinculin. (A–C) SORBS-re-expressing cells stably expressing EGFP–vinculin were cultured on glass-based dishes and subjected to FRAP analysis. (A) More than 111 FAs containing GFP–vinculin from thirty individual cells in three independent experiments were photobleached for each condition, and the representative time-lapse montages are displayed. (B) The normalized fluorescence recovery of EGFP–vinculin at FAs in TKO MEF/mock (n=111), TKO MEF/vinexin-α (n=118), TKO MEF/CAP (n=116) or TKO MEF/ArgBP2 (n=115) was plotted as the mean±s.d. (C) The immobile fraction was calculated by curve-fitting the data. P-value was calculated by Mann–Whitney U-test; n.s., not significant. See also Fig. S3F and Movie 1. (D–F) Pulldown assay using GST–vinexin-αΔN, GST–CAPΔN and GST–ArgBP2ΔN. TKO MEFs were lysed and subjected to a pulldown assay. Input purified proteins were visualized by CBB staining (left panels). Co-precipitated proteins were visualized by immunoblotting using the indicated antibodies (right panels) (D). The amount of co-precipitated vinculin (E) or α-actinin (F) with GST-tagged proteins was quantified by Fiji software. The values represent the means±s.e.m. from three independent experiments.

Fig. 6.

The central region of vinexin-α and CAP play critical roles in regulating vinculin. (A) Schematic diagram of the domain structure of chimeric proteins between SORBS proteins. Each SORBS protein was divided into three parts: the N-terminal part containing one SoHo domain, the C-terminal part containing three SH3 domains and the central part not containing any domain structure. (B) HA-tagged chimeric protein-expressing cells cultured on coverslips were immunostained using anti-vinculin and anti-HA antibodies. The FA-containing areas indicated by yellow boxes are cropped and displayed at the bottom of the figure. Scale bars: 20 µm. (C) Cells cultured on collagen-coated dishes were treated with or without CSK and then lysed with 1% SDS, followed by immunoblotting using the indicated antibodies (lower panels). The ratio of CSK-resistant vinculin to total vinculin was quantified by Fiji software. The values represent the means±s.e.m. from three independent experiments. *P<0.05 was compared with HA-expressing samples using an unpaired Student's t-test. (D) GFP–depletion assay using purified GST–vinculin. 293T cells expressing GFP–chimeric proteins were subjected to a pulldown assay. Intensity of the unbound fraction was measured by spectrofluorimeter and the bound fraction was calculated (upper panel). Co-precipitated chimeric proteins were visualized by immunoblotting using the indicated antibodies (lower panels). The values represent the means±s.e.m. from three independent experiments. *P<0.05 was compared with GFP-expressing samples using an unpaired Student's t-test.

Fig. 7.

ArgBP2 but neither vinexin-α nor CAP immobilizes α-actinin on SFs. SORBS-re-expressing cells stably expressing EGFP–α-actinin were cultured on glass-based dishes and subjected to FRAP analysis. (A–C) More than 78 FAs containing GFP–α-actinin from twenty individual cells in three independent experiments were photobleached for each condition, and the representative time-lapse montages are displayed (A). The normalized fluorescence recovery after photobleaching of EGFP–α-actinin at FAs in TKO MEF/mock (n=79), TKO MEF/vinexin-α (n=78), TKO MEF/CAP (n=79) or TKO MEF/ArgBP2 (n=79) was plotted as the mean±s.d. (B) The immobile fraction was calculated by curve-fitting the data (C); N.S., non-significant in Kruskal–Wallis ANOVA. See also Fig. S3H and Movie 2. (D–F) More than 25 cells were selected in three independent experiments, and one circular region along SFs containing GFP–α-actinin in each cell were photobleached for each condition (D). The normalized fluorescence recovery after photobleaching of EGFP–α-actinin at SFs in TKO MEF/mock (n=26), TKO MEF/vinexin-α (n=26), TKO MEF/CAP (n=25) or TKO MEF/ArgBP2 (n=25) was plotted as the mean±s.d. (E). The immobile fraction was calculated by curve-fitting the data (F). P-value was calculated by Tukey's post hoc test; n.s., not significant. See also Fig. S3I and Movie 3.

Fig. 8.

ArgBP2 generates the greatest contractile force in the wrinkle formation assay. (A–F) SORBS-re-expressing cells were cultured on type I collagen-coated silicone substrates and subjected to the cell contraction assay. Left panels: more than twenty individual cells were observed for 2 h and four independent experiments were performed for each combination. Representative images of cells that showed maximum wrinkle length during the 2 h observation in each combination are displayed. Scale bars: 50 µm. Right panels: the total length of wrinkles per cell at the time of maximum was quantified from images in the representative experiment with a custom-made program written in Fiji software. See also Fig. S4B. The number of cells is as follows: (A) n=42 (TKO MEF/mock) and n=30 (TKO MEF/vinexin-α), (B) n=27 (TKO MEF/mock) and n=22 (TKO MEF/CAP), (C) n=42 (TKO MEF/vinexin-α) and n=28 (TKO MEF/CAP), (D) n=47 (TKO MEF/mock) and n=39 (TKO MEF/ArgBP2), (E) n=40 (TKO MEF/vinexin-α) and n=32 (TKO MEF/ArgBP2), (F) n=24 (TKO MEF/CAP) and n=24 (TKO MEF/ArgBP2). P-value was calculated by Mann–Whitney U-test. (G,H) SORBS-re-expressing cells cultured on coverslips for 24 h were immunostained using anti-diphosphorylated (T18/S19) MRLC antibody with phalloidin staining (G). Scale bars: 20 µm. The integrated density of diphosphorylated MRLC per cell was quantified from images of ninety individual cells from three separate experiments (H). n=90; P-value calculated by Mann–Whitney U-tests; n.s., not significant.