Fig. 3: ER-mit MCS dynamics. | Nature Communications

Fig. 3: ER-mit MCS dynamics.

From: Simultaneous detection of membrane contact dynamics and associated Ca2+ signals by reversible chemogenetic reporters

Fig. 3

a Representative Airyscan 3D image of a COS-7 cell co-expressing ER-mit RspA-splitFAST (surface rendering, yellow), the ER-marker ER-StayGold and stained with MitoTracker Deep Red. Distribution graphs represent the Kernel density (orange) and the histogram (green) of volume, area and sphericity values of ER-mit RspA-splitFAST dots (see “Methods”). Dispersion plots show a negative correlation between volume and sphericity (left) and a positive correlation between volume and ellipticity (right). b Representative lattice light-sheet microscopy 3D image of a HeLa cell clone expressing ER-mit RspA-splitFAST (surface rendering, yellow) and stained with MitoTracker Deep Red. The time-lapse shows the maintenance of an ER-mit MCS during an extensive mitochondrial movement, followed by branching at the MCSs (arrows). See also the corresponding Supplementary Movie 4. c Representative Airyscan image of a COS-7 cell as in panel (a). The time-lapse shows co-sliding of mitochondria, ER and ER-mit RspA-splitFAST dots (arrow), as well as the disappearance of an ER-mit MCS (circle, first frame) upon mitochondria detachment from ER tubules. See also the corresponding Supplementary Movie 5. d Representative Airyscan 3D image (surface rendering) of a COS-7 cell co-expressing ER-mit RspA-splitFAST, mCherry-Drp1 and stained with MitoTracker Deep Red. The time-lapse shows mCherry-Drp1 localization at a site of mitochondria and ER-mit MCS fission (arrow). See also the corresponding Supplementary Movie 6. e Representative image as for panel (b). The time-lapse shows an ER-mit MCS movement (arrow) at a site of mitochondria “Kiss-and-run” fusion (40–70 s) followed by organelle fission (100–140 s). See also the corresponding Supplementary Movie 7. f Representative 3D image of a HeLa cell expressing ER-mit RspA-splitFAST (surface rendering). ER-mit RspA-splitFAST dots (tracked using IMARIS, see Methods) move in space and interact each other. Tracks (coloured continuous lines) report MCS movements and interactions. ER-mit MCSs undergoing fusion/fission are considered part of the same track. Enlarged regions show the time-lapse of a MCS undergoing fission (309–329 s), and two MCSs fusing together (448–488 s). Charts represent the percentage of interacting ER-mit MCS subgroups (tracks) for each range of fission/fusion events, as indicated. See also the corresponding Supplementary Movie 8. g Representative Airyscan 3D image of a COS-7 cell co-expressing ER-mit RspA-splitFAST, ER-StayGold and stained with MitoTracker Deep Red. Enlarged regions show the time-lapse of the appearance (38 s) of an ER-mit MCS (arrow), upon ER and mitochondria membrane docking, and its following disappearance (153 s), upon organelle distancing (arrow). See also the corresponding Supplementary Movie 9. h Representative 3D image of a COS-7 cell co-expressing ER-mit RspA-splitFAST, ER-StayGold and stained with MitoTracker Deep Red. ER-StayGold signal has been segmented (see “Methods”) to separate ER tubules from sheets. Manders’ co-localization M2 coefficient of ER-mit MCSs with ER sheets/tubules is shown (box plots). Scale bar: 5 µm (a–h).

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