Fig. 2: The RspA-splitFAST system to investigate ER-mit, ER-PM, PM-mit MCSs in vitro and in vivo.

a Representative confocal images of HeLa cells expressing short ER-mit RspA-splitFAST and stained with MitoTracker Deep Red. The box plot shows the percentage of mitochondrial surface co-localized with ER-mit RspA-splitFAST. b Representative confocal image (top) of a region of a HeLa cell expressing short ER-mit RspA-splitFAST and stained with MitoTracker Red. The fluorescent splitFAST dots (two representative dots were marked “1”/“2”) correspond to sites of close ER-mit membrane juxtaposition, as revealed by the EM slice of the very same cell region (bottom; see also Supplementary Movie 2). The fraction of mitochondrial perimeter in close contact with ER, analysed by CLEM, is similar in untransfected and ER-mit RspA-splitFAST-expressing (transfected) cells (box plots). c Representative confocal images of HeLa cells expressing ER-PM RspA-splitFAST, in which PM was labelled by either WGA staining or MyrPalm-D1cpv expression. ER-PM MCSs were visualized by addition of respectively HMBR (left) or HBR-3,5DOM (right). d Representative confocal images of PM-mit RspA-splitFAST-expressing HeLa cells, stained with MitoTracker and WGA (PM). The box plots represent the percentage of mitochondrial surface in contact with either ER (same as in panel (a)) or PM. e Representative confocal images of HeLa cells co-expressing ER-PM fr-splitFAST and ER-mit RspA-splitFAST. The far-red signal is exclusively emitted by ER-PM fr-splitFAST (marking ER-PM MCSs); the green signal can be emitted by both ER-mit RspA-splitFAST and ER-PM fr-splitFAST (as fr-splitFAST can also incorporate the green and red fluorogens). As the lifetimes of the ER-mit RspA-splitFAST and ER-PM fr-splitFAST green signals are different (see FLIM Phasor Plot, Supplementary Fig. 1i and Methods), ER-mit and ER-PM MCSs were distinguished in the very same cells by FLIM. f Representative confocal images of a HeLa cell, co-expressing ER-long-RspA-NFAST, ER-short-frNFAST and OMM-short-RspA-CFAST to simultaneously visualize short and long ER-mit MCSs. The signals were separated by FLIM (see e). Arrows indicate few long MCSs that do not colocalize with short ones (see box plots on the right). g Tomographic 3D reconstruction of a portion of HeLa cell, showing mitochondrial surface (red) and the regions of juxtaposition with ER, either close (<12 nm, cyan areas, corresponding to short ER-mit MCSs) or wider (12–24 nm range, yellow areas, corresponding to long ER-mit MCSs). Most short ER-mit MCSs are continuous with long ones (white arrows, front view box). The box plot represents the surface of either the few long contacts not containing short ones (Long, white arrowheads in the top view) or those continuous with short MCSs. See also Supplementary Movie 3. h Representative confocal images of the Caenorhabditis elegans strain pHX6743 (expressing ER-mit RspA-splitFAST under the myo3 promoter) or N2 (not expressing RspA-splitFAST). The fluorescent signals of ER-mit RspA-splitFAST (complemented with HBR-3,5DOM) and of background were separated by FLIM (See Phasor Plot). Scale bar: 10 µm (a, c–f, h); 2 µm (b); 500 nm (g).