ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells (2022)
Figure 7

Figure 7. Cortical actin tension modulates plasma membrane protein composition.

(A) Diagram showing spatial regions associated with plasma membrane actin-associated projections including the basal domain, the annulus and the protrusion.

(B) Line graph of the excess chemical potential required to recruit negative curvature sensing domain proteins to the basal, annulus, and protrusion region of the plasma membrane as a function of excess membrane area (A/Ap), which is the variable conjugate to the cortical actin tension.

(C) (Top) Model of how exocyst components (including Exo70) tether vesicles during exocytosis. Representative fluorescence microscopy images of MECs stably co-expressing EGFP-Exo70 (Exo70) and the plasma membrane reporter farnesylated mCherry (PM) ligated to rBM in 2D, 3D, and treated with blebbistatin (2D+Bleb). Fluorescent images of whole cells are shown (left) and cell-edge protrusions are magnified within inset (right). Colocalization of Exo70 and membrane protrusions are highlighted with yellow arrows. Scale bar (whole cell), 5 μm; Scale bar (magnified inset), 1 μm.

(D) Bar graph of the relative level of EGFP-Exo70 at the plasma membrane versus cytoplasm in MECs ligated to rBM in 2D or 3D in the absence and presence of blebbistatin (Bleb). Membrane localization of EGFP-Exo70 was quantified as a ratio of plasma membrane to cytoplasmic fluorescence (mean ± SEM; 2D, n= 59; 3D, n=60; 2D + Bleb, n=57; 3D + Bleb, n=54 cells from three independent experiments). Statistical analysis by one-way ANOVA followed by Uncorrected Fisher's LSD. ****P < 0.0001.

(E) 30-nm thick slices through a cellular tomogram focusing on subplasmalemmal regions enriched with membrane-encased compartments of vitrified MECs cultured on rBM treated with (2D, right panel) or without (2D+Bleb, left panel) blebbistatin. The position of the cell body is towards the upper right-hand corners (marked with white asterisk). Scale bars, 200 nm.

(F) Quantification of the number of membrane-enclosed compartments (vesicles) proximal to the plasma membrane that were classified as either 'filled' (black) or 'empty' (white), based on the absence or presence of encapsulated macromolecules. Membrane-enclosed compartments were independently classified by three cryo-EM experts using 28 tomograms per condition. The resulting standard deviation for assigning 'empty' versus 'filled' was 10.3% (n=3 independent expert classifications). Statistical significance of differences between the distributions was assessed using Mann-Whitney rank tests. ****P < 0.0001.

(G) Schematic of the proximity-based biotinylation assay used to label proteins localized to the plasma membrane under different ECM ligation conditions. Expression of a recombinant APEX2-CAAX probe within cells facilitated biotin labelling of proteins in close proximity to the plasma membrane upon addition of biotin-phenol and H2O2.

(H) Volcano plot showing differentially associated proteins in MECs ligated to rBM in 2D treated with vehicle (DMSO; blue; log 2> 0.5; high cortical actin tension) versus blebbistatin (Bleb; red; log 2> 0.5; low cortical actin tension). Differentially expressed genes with an adjusted p-value<0.1 and log2 (fold change) > 0.5 are shown (n=2 biological replicates).

(I) List of proteins enriched at the plasma membrane in cells ligated to rBM in 2D and treated with blebbistatin (low cortical actin tension).

(J) Representative immunofluorescence microscopy images of MECs ligated to rBM in 2D, 3D, or 2D treated with blebbistatin (2D+ Bleb) and immunostained with antibodies targeting ANXA2 or S100A14 (green). Cellular F-actin was counterstained using phalloidin (red) and actin-rich protrusions are highlighted with yellow arrows. Fluorescent images of whole cells (left) and actin-rich protrusions containing ANXA2 or S100A14 are magnified in the inset (right). The edge of the actin cortex in MECs ligated to rBM in 2D was marked with a yellow dashed line. Scale bar (whole cell), 5 μm; Scale bar (magnified insets), 1 μm.