Fig. 2: Nanopillar-induced membrane invaginations promote the local formation of ER–PM contacts in cardiomyocytes.
From: Plasma membrane curvature regulates the formation of contacts with the endoplasmic reticulum
a, Schematic of using FIB-SEM to examine the cell–nanopillar interface. b, Left: A FIB-SEM image of the interface between an iPSC-CM and a nanopillar. This image is black–white inverted for clarity. Scale bar, 1 µm. Middle: An enlarged view of the region shown in the red box. Scale bar, 100 nm. Right: As for the middle image, but with the PM (green) and ER (magenta) highlighted in pseudo colours. c, Left: Three different FIB-SEM sections of an HL-1 cardiomyocyte on the same nanopillar. The red arrows indicate ER–PM contact sites. Scale bars, 1 µm. Middle and right: Enlarged views as in b. Scale bars, 100 nm. d, ExM imaging of an iPSC-CM cultured on nanopillars. GFP-CAAX is shown in green and anti-JPH2 is shown in magenta. Scale bars, 10 µm (in each dimension). e, An x–y image focused on the middle height of nanopillars using an expansion microscope. Scale bar, 10 µm. f, The x–z view along the yellow dashed line shown in e. The z dimension was scaled by the ratio between the z-step size and x–y pixel size to exhibit x and z at the same dimensional scale. Scale bar, 10 µm. g, An averaged x–z image of the ten pillars shown in the yellow box in e. The intensities were normalized by the inter-pillar intensities on flat membranes for both CAAX and JPH2 channels and then displayed at the same scale. Right: The ratiometric image is the ratio between JPH2 and CAAX channels. Scale bars, 5 µm. h, Quantifications of the nanopillar-to-flat intensity ratios for GFP-CAAX and JPH2, normalized by the average intensity ratio for GFP-CAAX. Each dot represents the averaged ratio for a region of nanopillars inside a cell (typically five or six pillars); n = 52 regions from 15 cells for each probe. The data are presented as means ± s.d. ****P < 0.0001. The experiments in b and c were replicated independently twice with similar results and the other experiments were replicated independently three times with similar results. Statistical significance in h was determined by two-tailed Mann–Whitney test. Source numerical data are available Source data.
