Fig. 3: CLEM reveals cloud-like meshwork structure of Apaf1 foci.

See Supplementary Fig. 4 for workflow details. a–e CLEM of resin-embedded HeLa cells transiently expressing Apaf1-GFP, treated with ABT-737 and QVD. Representative of a total of 13 electron tomograms. a Fluorescence image of a section through a resin-embedded cell; Apaf1-GFP (green) and MitoTracker DeepRed (magenta). White squares indicate areas imaged by electron tomography and shown in b and d. b Virtual slice from electron tomogram acquired at area indicated in a. White square indicates area that contains an Apaf1-GFP localisation, shown in c. Magenta arrow indicates mitochondrion, white arrow indicates endoplasmic reticulum. c Zoomed-in area from the electron tomogram shown in b. Here shown virtual slice corresponds to a different z-position than the virtual slice shown in b. Yellow arrows indicate parts of the cloud-like meshwork structure, white arrows indicate putative ribosomes. d Virtual slice from electron tomogram acquired at area indicated in a. White square indicates area that contains an Apaf1-GFP localisation, shown in e. White arrows indicate Golgi cisternae. e Zoomed-in area from the electron tomogram shown in d. Here shown virtual slice corresponds to a different z-position than the virtual slice shown in d. Yellow arrows indicate parts of the cloud-like meshwork structure, white arrows indicate putative ribosomes. f–i Pre-FIB milling cryo-CLEM of vitrified HeLa cells stably expressing Apaf1-GFP, treated with ABT-737 and QVD. Representative of a total of 3 cryo-electron tomograms. f Cryo-fluorescence image of a cell grown on an EM grid, imaged before cryo-FIB milling; Apaf1-GFP (green) and MitoTracker DeepRed (magenta). The white dashed circle indicates the target Apaf1-GFP focus. g Virtual slice from cryo-electron tomogram acquired in the target area indicated in f. White square indicates area shown in h and i. h Zoomed-in area from the cryo-electron tomogram shown in g. Here shown virtual slice corresponds to a different z-position than the virtual slice shown in g. White arrows indicate putative ribosomes. i Segmentation model indicating area shown in h that contains meshwork structure. j–n Pre- and post-FIB milling cryo-CLEM of vitrified HeLa cells transiently expressing Apaf1-SNAP labelled with SNAP-Cell 647-SiR (Apaf1-SNAP647), treated with ABT-737 and QVD. Representative of a total of 8 cryo-electron tomograms. j Cryo-fluorescence image of a cell grown on an EM grid, imaged before cryo-FIB milling; Apaf1-SNAP647 (magenta) and MitoSpy Green (green). White square indicates area targeted by cryo-FIB milling. k Cryo-fluorescence image of the cell area indicated in j, imaged after cryo-FIB milling. The white dashed circle indicates the Apaf1-SNAP647 (magenta) focus that was imaged by cryo-ET in l. l Virtual slice from cryo-electron tomogram acquired in the area indicated in k. White square indicates area shown in m and n. White arrow indicates mitochondrion. m Zoomed-in area from the cryo-electron tomogram shown in l. Here shown virtual slice corresponds to a different z-position than the virtual slice shown in l. n Segmentation model indicating area shown in m that contains meshwork structure. Scale bars in a: 2 μm. In b, d, g and l: 100 nm. In c, e, h and m: 50 nm. In f and k: 3 μm. In j: 10 μm.