Spatial relationships between vinculin tension and actin at cell adhesions define cell colony positioning and identity

Figures

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  Figure 1. Knockin of a tension-sensitive module to the endogenous vinculin locus in iPSCs. (A) Schematic of heterozygous knock-in of a FRET tension-sensitive module in iPSCs at the endogenous vinculin locus. (B) Schematic of force sensor at cell adhesion complexes, where strain across vinculin reduces FRET. (C) Western blot using C-terminal antibody for vinculin shows full-length wild-type (WT) vinculin expression in both WT cells and edited clones, but only vinculin knockin sensor expression in edited cells. (D) Normalized protein abundance of total full-length vinculin (combined WT and knockin bands) in WT and edited cells. N = 6 bands from 3 cell passages per line. Lines are mean ± SEM, unpaired t-tests, ∗∗p < 0.01. (E) Pie graph representing the average relative proportion of the full-length vinculin knockin protein abundance to that of the unedited WT allele in VinTS knock-in cells. (F) VinTS-iPSC adhesions showing sensor fluorophores colocalized with vinculin. Scale bar, 25 μm. (G) VinTS-iPSC colony expressing sensor and pluripotency marker Oct4. Scale bar, 25 μm. (H) FRET analysis pipeline: Clover channel marks adhesions, force calculated per pixel, segmented adhesions, and vinculin…

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  Figure 2. Engineered VinTS iPS cells have differential adhesion and cytoskeletal properties in edge and middle iPSCs. (A) Representation of middle versus edge cell classification in iPSC colonies. (B) Top view of 3D rendering of VinTS-iPSC colony stained for phalloidin (gray) and Dapi (blue). (C–F) Quantification of average (C) adhesion number, (D) adhesion z-position, (E) phalloidin signal of segmented adhesions, and (F) average vinculin tension force at adhesions per cell in iPSC colonies, n = 816 cells from 14 colonies. Lines are mean ± SEM, unpaired t-tests, ∗∗∗∗p < 0.001. (G) Representative VinTS-iPSC colony images at substrate (left) and apical (right) planes. Scale bar, 20 μm. (H) Example colony z-projection showing classified “adherens junction” or “focal” adhesions. (I) Average vinculin tension forces of focal and adherens junction adhesion forces per cell. Unpaired t-test, lines are mean ± SEM. (J) Comparison of average vinculin tension in focal and adherens junction adhesions of middle versus edge cells. Two-way ANOVA with uncorrected Fisher’s LSD, interaction p < 0.0001. Multiple comparisons test, ∗∗∗p < 0.005, ∗∗∗∗p < 0.001.

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  Figure 3. Edge cells have stronger response to latrunculin treatment than middle cells. (A) Representative images of VinTS-iPSCs stained for actin in (left) untreated and (right) latrunculin-treated colonies on top, and corresponding vinculin tension force maps of average z-projections on bottom. Scale bars, 25 μm. A.U., arbitrary units of fluorescence. (B and C) Average adhesion (B) volume and (C) vinculin tension force of untreated versus latrunculin-treated cells, untreated n = 816 cells from 14 colonies, latrunculin n = 729 cells from 15 colonies. Lines are mean ± SEM, unpaired t-tests, ∗∗∗∗p < 0.001. (D) Average vinculin tension force at adhesions in untreated versus latrunculin-treated middle and edge cells. Two-way ANOVA, lines are mean ± SEM, p < 0.05 for row, column, and interaction factors. Multiple comparisons test, ∗∗∗∗p < 0.001. (E) Linear regression of average adhesion vinculin tension force versus actin colocalization to adhesions: middle untreated slope = 6.027, R2 = 0.0435; edge untreated slope = 11.98, R2 = 0.0889; middle latrunculin slope = 6.122, R2 = 0.0407; edge latrunculin slope = −5.285, R2 = 0.0227. (F) Average adhesion vinculin tension force of classified…

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  Figure 4. 3D Averaged morphed cell representations reveal differential spatial positioning of adhesion vinculin tension force in middle and edge cells. (A) Example of 3D rendered edge (top) and middle (bottom), untreated, and latrunculin-treated cells. Left images per set are adhesion vinculin tension force calculated from FRET, right column is adhesion vinculin tension force merged with phalloidin (gray). (B) Averaged morphed cell shape showing (left) average cell boundary and (right) adhesion vinculin tension force distribution in untreated middle cells. (C) Single-slice views of average adhesion vinculin tension force in x, y, and z cross sections, with colony center to the left. (D and E) Quantification of average adhesion vinculin tension force through morphed cells of each slice from (D) bottom to top and (E) from colony middle to edge. (F) Schematic of radial density binning. (G and H) Average radial distribution of (G) adhesion vinculin tension force and (H) actin from cell center to periphery. (I) Schematic of depth density binning. (J and K) Average depth distribution of (J) adhesion vinculin tension force and (K) actin from cell center to periphery.

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  Figure 5. Adhesion state space predicts cell colony positioning. (A–D) K-means clustering of adhesion properties visualized in PCA plots, n = 1545 cells. Cells colored by (A) cluster identity, (B) treatment, (C) edge state, and (D) average adhesion vinculin tension force. (E and F) Distribution plots with individual measurements versus fraction of cells, colored by k-means cluster for (E) cell geometry and (F) adhesion properties. Dotted lines are average measurement per cluster. (G) Heatmap of Pearson’s correlation value between scaled cell geometry factors and adhesion properties within four largest clusters, with only values of p < 0.05 labeled. Dashed outline highlights cluster-specific correlations.

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  Figure 6. Adhesion state space predicts cell fate progression down differentiated lineages. (A and B) Schematic of cardiomyocyte (A) and neuron (B) differentiations, with boxes marking imaging time points. (C and D) Representative max-intensity z-stacks of (top) nuclei and actin at time points during (C) cardiomyocyte and (D) neural differentiation, with corresponding (bottom) adhesion vinculin tension force maps of segmented adhesions. (E) PCA performed on all cardiac and neural cells, plotted along the first three principal-component dimensions, showing from left to right: all cells colored by k-means cluster, all cells colored by differentiation type, cardiac cells colored by differentiation day, and neural cells colored by differentiation day. (F and G) Cells plotted by PCA1 versus (F) first or (G) second pseudotime lineage, from left to right colored: all cells by k-means cluster, cardiac cells by differentiation day, and neural cells by differentiation day. (H) Heatmap of Pearson’s correlation value between scaled cell geometry factors and adhesion properties to pseudotime lineage value, with values of p < 0.05 labeled.