Figure 1. APC-m4 disrupts the molecular... | Rockefeller University Press

Figure 1.

$Figure 1. APC-m4 disrupts the molecular order of F-actin at FAs. All data are from U2OS cells. (A) Schematic of FA (gray) and emanating stress fiber (green), highlighting ROIs analyzed by polarization-resolved microscopy. ROIs overlapping with FAs are designated as “inside,” and ROIs just outside of FAs on the stress fiber are designated “outside.” (B) Schematic showing orientational order parameters ρ (mean orientation) and Ψ (mean order or angular aperture). In the cartoon, fluorophores that label actin filaments are depicted as gray ellipsoids. The color bar provides a scale quantifying Ψ for other panels. (C) Representative confocal images of cells grown on collagen micropatterns (H and Y shapes), depleted of endogenous APC and rescued with refractory APC constructs (APC-WT or APC-m4). Cells were fixed and stained with Alexa Fluor 488–phalloidin (F-actin, gray) and phospho-paxillin (P-Paxillin) antibodies (pink). Yellow boxes highlight areas analyzed in D and E. Scale bar, 20 µm. (D) Representative zoom images from boxed region of APC-WT cell in C showing both the inside (In) and outside (Out) ROIs analyzed. Left: Overlay of Alexa Fluor 488–phalloidin (gray) and phospho-paxillin (pink). Remaining panels (left to right) show the color-coded molecular order (Ψ) super-imposed on Alexa Fluor 488–phalloidin (F-actin, gray); intensity-thresholded image of FA (white) superimposed with stick representation of Ψ (encoded in stick color); and mean orientation (ρ) encoded in stick orientation with thresholded image of FAs (gray). Below the image panels are histograms, displaying the Ψ value distribution (in degrees) from the In and Out ROIs combined (<Ψ> = 133.8°), and separately from In (<Ψ> = 132.9°) and Out (<Ψ> = 131.1°). Scale bar, 2 µm. (E) Same as D except for APC-m4 cells. Histograms display the Ψ value distribution from the In and Out ROIs combined (<Ψ> = 143.0°), and separately from In (<Ψ> = 145.2°) and Out (<Ψ> = 140.8°). (F) Total F-actin intensity (from In and Out ROIs combined). (G) Bee swarm plot showing average molecular order (Ψ), separately for inside and outside ROIs. (H) Bee swarm plot showing the SDΨ, separately for inside and outside ROIs. (I) Bee swarm plot showing the SDρ, separately for inside and outside ROIs. Data in panels F–I averaged from three independent experiments. APC-WT (n = 25 ROIs) and APC-m4 (n = 31 ROIs) from n ≥ 15 cells per condition. Error bars, SD. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant; AU, arbitrary unit.$

APC-m4 disrupts the molecular order of F-actin at FAs. All data are from U2OS cells. (A) Schematic of FA (gray) and emanating stress fiber (green), highlighting ROIs analyzed by polarization-resolved microscopy. ROIs overlapping with FAs are designated as “inside,” and ROIs just outside of FAs on the stress fiber are designated “outside.” (B) Schematic showing orientational order parameters ρ (mean orientation) and Ψ (mean order or angular aperture). In the cartoon, fluorophores that label actin filaments are depicted as gray ellipsoids. The color bar provides a scale quantifying Ψ for other panels. (C) Representative confocal images of cells grown on collagen micropatterns (H and Y shapes), depleted of endogenous APC and rescued with refractory APC constructs (APC-WT or APC-m4). Cells were fixed and stained with Alexa Fluor 488–phalloidin (F-actin, gray) and phospho-paxillin (P-Paxillin) antibodies (pink). Yellow boxes highlight areas analyzed in D and E. Scale bar, 20 µm. (D) Representative zoom images from boxed region of APC-WT cell in C showing both the inside (In) and outside (Out) ROIs analyzed. Left: Overlay of Alexa Fluor 488–phalloidin (gray) and phospho-paxillin (pink). Remaining panels (left to right) show the color-coded molecular order (Ψ) super-imposed on Alexa Fluor 488–phalloidin (F-actin, gray); intensity-thresholded image of FA (white) superimposed with stick representation of Ψ (encoded in stick color); and mean orientation (ρ) encoded in stick orientation with thresholded image of FAs (gray). Below the image panels are histograms, displaying the Ψ value distribution (in degrees) from the In and Out ROIs combined (<Ψ> = 133.8°), and separately from In (<Ψ> = 132.9°) and Out (<Ψ> = 131.1°). Scale bar, 2 µm. (E) Same as D except for APC-m4 cells. Histograms display the Ψ value distribution from the In and Out ROIs combined (<Ψ> = 143.0°), and separately from In (<Ψ> = 145.2°) and Out (<Ψ> = 140.8°). (F) Total F-actin intensity (from In and Out ROIs combined). (G) Bee swarm plot showing average molecular order (Ψ), separately for inside and outside ROIs. (H) Bee swarm plot showing the SD_Ψ, separately for inside and outside ROIs. (I) Bee swarm plot showing the SD_ρ, separately for inside and outside ROIs. Data in panels F–I averaged from three independent experiments. APC-WT (n = 25 ROIs) and APC-m4 (n = 31 ROIs) from n ≥ 15 cells per condition. Error bars, SD. Statistical significance calculated by nonparametric Mann–Whitney two-tailed Student’s t test: ***, P < 0.001; **, P < 0.01; *, P < 0.05; n.s., not significant; AU, arbitrary unit.