Fig. 1: Altered ER tether motion at ER–mitochondria contact sites.
From: Motion of VAPB molecules reveals ER–mitochondria contact site subdomains
a, Cartoon indicating ER–mitochondrial tethering. b, 3D FIB-SEM reconstruction of ER (cyan), mitochondria (blue) and ERMCSs (red). Inset shows representative EM slice overlaid with segmentation masks. c, 3D reconstruction of an ERMCS with aligned cristae. d, Single-molecule tracers used in e–f. e, sptPALM trajectories of either non-specific tail anchor (TA, top) or VAPB (bottom) overlaid on ER reference image (KDEL, white). Arrows indicate regions of spatially correlated motion. f, Spatial probability maps for TA (top) or VAPB (bottom) generated from trajectories in e. Hotspots indicate likely sites of VAPB tethering. e–f are representative examples of n = 13 (TA, regions i and ii) or n = 24 (VAPB, regions iii and iv) cells. g, 3D EM reconstruction of contact sites (CS) (red) between ER (cyan) and mitochondria (not shown), with simulated sptPALM localization densities from the volume projected below. Dashed lines indicate mitochondria borders. h, Simulated localization densities from g. i, Contact size measured from FIB-SEM simulation projections or from single-molecule localization density (n = 466 contact sites (sptPALM), 38 contact sites (FIB-SEM); P = 0.9724, Dunn’s multiple comparisons test, two-sided). Dotted line indicates confocal microscopy resolution limit. j, Micrograph of ensemble ER and mitochondria reporters (left), with corresponding TA or VAPB probability maps (centre) and magnified inset (right) in an example cell. Outlines indicate the area explored by each mitochondrion during the experiment. See Extended Data Fig. 2 for full dataset. k, Cartoon indicating VAPB single-molecule tracers used in l–m. ΔN-VAPB, deletion of the N terminus; ΔC-VAPB-TA; replacement of the C terminus with TA. l, Representative ERMCSs with associated localization (Loc.) densities for the tracer molecules shown in k. m, Number of mitochondria-associated regions of enhanced tethering per cell for the indicated VAPB reporters (n = 24 (VAPB), 12 (ΔN-VAPB), 8 (ΔC-VAPB-TA); P < 0.0001, P > 0.9999, Dunn’s multiple comparisons test, two-sided). Error bars show the median and 95% confidence interval. Scale bars, 100 nm (b), 5 µm (e,f,j), 1 µm (j, inset), 500 nm (g–h), 1 µm (l).All panels are representative of at least two independently performed experiments with similar results.
