Vinculin and metavinculin exhibit distinct effects on focal adhesion properties, cell migration, and mechanotransduction

Figures

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  Fig 1. Sequence and structural differences between Vcn and MVcn. (A) Diagram comparing domain architecture of full-length Vcn and MVcn. (B) Crystal structures of Vcn (PDB: 1TR2) and MVcn [37]. Regions that lack electron density are represented by a dotted line. (C) Diagram comparing sequence differences in the Vcn and MVcn tail domain. MVcn tail contains an insert of 68 amino acid between residue 915 and 916 (H1’ in red, residues 916–983). (D) Structural schematic depicts sequence differences that lead to a helix replacement of H1 with H1’ (red) in MVcn tail (Vh: Vcn head domain; Vt: Vcn tail domain; MVt: MVcn tail domain (PDB:3MYI); H: helix; NT: N-terminus; CT: C-terminus; and P: proline-rich region).

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  Fig 2. MVcn-expressing cells have larger but fewer FAs compared to Vcn-expressing cells. (A) Western blot shows the expression level of either Vcn or MVcn in Vcn null MEF background. Both expression levels are equivalent to endogenous Vcn expression in WT MEFs. (B) Fluorescent images of WT MEF, Vcn-null parent MEF cell line, exogenous Vcn-expressing, and MVcn-expressing Vcn-null MEFs stained for paxillin and showing expression of either fluorescently-tagged Vcn or MVcn. Scale bar = 10 μm. FA number (C) and overall FA area per cell (D) were quantified. Graphs represent data pooled from 4 independent experiments (n ≥ 100 cells; ***, p<0.001).

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  Fig 3. MVcn rescues decreased cell area in Vcn-null MEFs. (A) Stable fluorescently-tagged Vcn or MVcn expression in Vcn-null MEFs, co-stained with phalloidin, to allow cell area quantification. WT MEFs and Vcn-null MEFs stained with phalloidin and Vcn antibody are shown for comparison. (B) Cell area quantification. (n ≥ 150 cells per cell type, data pooled from 3 independent experiments; ***, p<0.001). (C) Representative real-time impedance traces from the RTCA xCELLigence system, measuring impedance every 15 s for the first 4 hours, and then every 3 min for the next 6 hours. Cell Index (CI) represents electrical impedance. First 8 hours are shown. CI is higher in cells expressing Vcn and MVcn compared to Vcn-null MEFs, indicating greater cell spreading. (D) Quantification of CI at 2 hours after seeding cells. Data pooled from 4 independent experiments (n ≥ 3500 cells per cell type; ***, p<0.001).

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  Fig 4. Random cell migration analysis shows enhanced migration velocity and higher persistence of migration for cells expressing MVcn compared to Vcn-expressing cells. (A) Representative cell tracks plotted for n>50 cells. (B) Quantification of random migration velocity. (C) Quantification of directional persistence. Data pooled from 3 independent experiments (n ≥ 140 cells; ***, p<0.001).

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  Fig 5. Focal adhesion assembly and disassembly rates are higher for Vcn-expressing cells compared to MVcn-expressing cells. (A) Representative time-lapse image sequences of Vcn-null MEFs stably expressing either mEmerald-Vcn or mRFP-MVcn migrating on 10 μg/ml FN. Images (shown in grayscale) are taken every 15 sec. Green outlines (generated by focal adhesion analysis program) show individual FA. Scale bar = 10 μm. Graph of average rate constants of FA assembly (B) and disassembly (C) from FAs in each cell type. Data pooled from 3 independent experiments (n ≥ 13 cells (and at least 400 adhesions); ***, p<0.001).

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  Fig 6. 3D-Force microscopy (3DFM) shows reduced ability of MVcn-expressing cells to rescue cell stiffening response compared to Vcn-expressing cells. (A) 3D-force microscopy (3DFM) setup used to measure cell stiffening response. Constant force was applied for 5 sec intervals followed by 10 sec relaxation; this pattern was repeated for a total of 5 pulses. (B) Cell stiffening response is measured by quantifying the decrease in bead displacement after each subsequent pulse of magnetic force in Vcn-null, Vcn-, and MVcn-expressing cells. Cell stiffening response is lost in MVcn-expressing cells. Data pooled from 3 independent experiments (n ≥19 cells each cell type; ***, p<0.001).