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. 2024 Jan 24;626(7997):169–176. doi: 10.1038/s41586-023-06956-y

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 3 — a, Cartoon of VAPB-PTPIP51 tethering at an ERMCS. b, Representative trajectories and corresponding localization densities for VAPB at ERMCSs in control (left) or PTPIP51-overexpressing (right) cells. Dashed lines indicate contact site boundaries. c, Overlaid ERMCS footprints from b with axes indicated. d, VAPB-associated ERMCS size in control or PTPIP51-overexpressing cells (n = 160 ERMCSs (control), 64 ERMCSs (+PTPIP51); major P < 0.0001, minor P < 0.0001; Dunn’s multiple comparisons test, two-sided). Line indicates confocal microscopy resolution limit. e, Airyscan micrographs of ER (cyan) and mitochondria (magenta) in either control, VAPB-overexpressing, or PTPIP51-overexpressing cells. f, Number of total VAPB organelle contact sites in control or PTPIP51-overexpressing cells (n = 24 cells (control), 16 cells (+PTPIP51); P = 0.6570, P = 0.0005; Dunn’s multiple comparisons test, two-sided) and mitochondrial enrichment coefficient derived from the same cells (n = 22 cells (control), 14 cells (+PTPIP51); P = 0.0003; Dunnett’s T3 multiple comparisons test, two-sided). g, Airyscan micrograph of TA (cyan) and VAPB (red) in a COS7 overexpressing PTPIP51 (untagged). h, Representative VAPB localization density at ERMCSs in control (complete medium) or starved (HBSS 8 h) cells. Line profiles indicate axes used for i; dashed lines indicate contact site boundaries. i, Overlaid ERMCS footprints from h. j, Contact site size in control or starved cells (n = 160 ERMCSs (control), 96 ERMCSs (starved); major P < 0.0001, minor P < 0.0001; Dunn’s multiple comparisons test, two-sided); line indicates confocal microscopy resolution limit. k, Single-molecule steps associated with the contact sites in h, coloured by the mean 2D D_eff in the local neighbourhood. l, Effective 2D diffusion coefficient of VAPB from h, normalized to neighbouring ER regions (dashed line indicates contact site centre). m, Mean 2D D_eff within contact sites in either control or starved cells (n = 160 ERMCSs (control), 96 ERMCSs (starved); P = 0.0068; Dunn’s multiple comparisons test, two-sided). Error bars indicate 95% confidence interval of the median. Scale bars, 200 nm (b,h,k), 2.5 µm (e), 2.5 µm (g).

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