Fig. 2: Impact of feeding and fasting on the endoplasmic reticulum (ER) 3D architecture and ER-mitochondria interactions in liver tissue.

A, B Reconstruction of ER network in fed (A) and fasted (B) state (1000 × 1000 × 400 voxels, 8 × 8 × 3.2 µm³). Scale bars: 500 nm. Images show SEM (left) and 3D rendering (right) of FIB-SEM data, rendered in Houdini software. In B, lower panel shows 3D rendering of ER and lipid droplet from FIB-SEM data using Houdini software. C Quantification of length of parallel organized ER stacks per total ER from TEM (n = 5 images per condition). D Ratio of ER tubules per total ER volume quantified from FIB-SEM data (n = 5 cells volumes for fed and n = 6 cell volumes for fasted). E Right: TEM image of mitochondria surrounded by rough ER. Scale bar: 100 nm. Left: quantification of distance between rough ER sheets-mitochondria membranes in TEM images. Number of mitochondria quantified n = 248 fed; n = 300 fasted. F Right: TEM image of mitochondria surrounded by smooth ER. Scale bar: 100 nm. Left: Quantification of distance between smooth ER-mitochondria membranes in TEM images. Number of mitochondria quantified n = 246 fed, n = 183 fasted. G 3D reconstruction of mitochondria volumes marked by areas of interactions with the ER at 0–24 nm distance (red) and 25–56 nm distance (purple) in fed (left, n = 14,855 mitochondria) and fasted (right, n = 6678 mitochondria) conditions. Scale bars: 1 µm. H, I Quantification of the mitochondria surface covered by ER at 24–56 nm distance in fed (n = 14,855 mitochondria) and fasted (n = 6678 mitochondria) conditions. Inset: 95% of confidence interval for data in H. J, K Quantification of the mitochondria surface covered by ER at 0–24 nm distance in fed (n = 14,855 mitochondria) and fasted (n = 6678 mitochondria) conditions. Inset: 95% of confidence interval for data in K. L Quantification of % of mitochondria surface covered by ER sheets at 0-56 nm in a whole hepatocyte volume (n = 2786 mitochondria for fed and n = 1275 mitochondria for fasted). ER sheets were segmented using the previously described algorithm¹⁰. M 3D rendering of a mitochondrion from fasted cell covered by a single ER sheet (purple). Scale bar: 500 nm. For box-and-whisker plots in C and D, the line inside the box shows the median value. The bounds of the box represent the 25th–75th percentiles, with whiskers at minimum and maximum values, two-tailed unpaired t-test, ***p = 0.0002. For H and J, two-tailed unpaired t-test ****p < 0.0001 and permutation test ****p < 0.0001.