Fig. 4

Fig. 4

a Schematic representation of the constructs SPLICS_S/L P2A^ER–MT–PM able to detect short (8–10 nm) and long (40–50 nm) range contact sites between the ER–mitochondria and the ER–PM simultaneously in different spectral variants. The cartoon shows the approach used to design the SPLICS reporters and indicates that the complementation of either short- and long-range ER-β_{11 fragments} with OMM-GFP_1–10 and PM-YFP_1–10 will reveal a florescence signal at the level of the contact sites. b, d Cartoon showing the short- and long-range contact sites measured by the indicated reporters. Confocal images of HeLa cells transfected with SPLICS_S P2A^ER–MT–PMc and SPLICS_L P2A^ER-MT-PMe showing the appearance of fluorescent “dots” upon excitation at 488 and 514 nm wavelengths confirming the complementation with GFP_1–10 (in green) and YFP_1–10 (in pseudocolour, red) at the ER–MT and the ER–PM contact sites, respectively. The co-localization of the SPLICS_S/L P2A^ER–MT–PM reporters with PM-cherry (in cyan) as plasma membrane marker and with an endogenous marker of ER (KDEL, in magenta) (upper panels) and with mitochondria (TOM20, in cyan)/ER (KDEL, in magenta)/(lower panels) is shown. Scale bar 25 µm. Cytosolic f and mitochondrial g Ca²⁺ transients in cells overexpressing SPLICS_S-P2A^ER–MT–PM and SPLICS_L-P2A^ER–MT–PM probes, mean ± SEM. To induce ER Ca²⁺ depletion cells were incubated for 5 min in KRB supplemented with 100 nM thapsigargin, 100 μM histamine, 100 μM EGTA and then exposed, where indicated, to KRB containing 2 mM CaCl₂. The traces are obtained from at least three independent transfections, values. h Quantification of the ER–mitochondria and the ER–PM (short and long) contacts in HeLa cells. The SPLICS dots were quantified from the 3D rendering of a complete Z-stack, mean ± SEM. The data were obtained from three independent transfections. (*P = 0.013, **P = 0.0052 one-way ANOVA). YFP emitted fluorescence is shown as pseudocolour in red to better appreciate in yellow the co-localization with the green signal of GFP. Source data are provided as a source data file.