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. 2019 Nov 18;51(4):460–475.e10. doi: 10.1016/j.devcel.2019.09.006

Figure 2.

Figure 2

Low Membrane Tension Promotes Caveolae Formation at the Rear of Fast-Moving Cells in 3D and Durotactic Gradients

(A) A2780 cells expressing EGFP-cavin-1 and mCherry-caveolin-1 were imaged as in Figure 1A; pairwise Pearson’s correlation coefficient for co-localization of EGFP-cavin-1 and mCherry-caveolin-1 in rear compared to peri-nuclear regions (N=28 cells, mean value above).

(B) A2780 cells expressing Lifeact-EGFP and mCherry-caveolin-1 imaged as (A), maximum intensity projection (MIP) shown for whole cell (left), individual Z-slices for rear regions (right).

(C) Left: A2780 cells expressing mCherry-caveolin-1 seeded onto 2D durotactic gradients and imaged as in (A), yellow line indicates outline of the cell; right: orientation of caveolae with respect to the gradient shown by rose plot (N=58 cells, 3 repeats).

(D and E) A2780 (D) or N15A (E) cell spheroids within collagen and fibronectin hydrogels stained for F-actin (phalloidin) and Cavin-1. Spheroid edge denoted by yellow dotted line; inset: higher-resolution image of the highlighted individual invading cells. MIPs of Z-stacks shown.

(F) Pairwise normalized rear and nuclear Cavin-1 intensity in individual invading A2780 and N15 cells (N>8 cells analyzed, 3 repeats).

(G) A2780 cell seeded in 3D CDM and fixed and stained for F-actin, cavin-1, and EHD2; whole cell and separately captured zoomed rear MIPs of confocal stacks shown.

(H) Serial transmitted electron microscopy (sTEM, 100-nm sections) of A2780 cell expressing mCherry-Caveolin-1 in CDM. Manual segmentation of the nucleus, the plasma membrane and caveolae are displayed as a 3D model over the entire cell. Both caveolae connected to the plasma membrane (yellow) and caveolae-associated vesicles (pink) were segmented (segm. + sTEM image). Both structures were merged in yellow in the overall model. An individual sTEM image is presented. Insets show higher-resolution images of multilobed (arrows) or membrane-attached (arrowhead) caveolae (different z-section of the same cells). Scale bar, 500 nm (inset: 100 nm). ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. See also Figure S2 and Videos S3 and S4.