Fig. 1

Functional characterization of the SPLICS probes. a Cartoon showing the general approach used to design the SPLICS. The mitochondrial network, the ER network, and the ER–mitochondria contact sites are revealed by co-expression of the β₁₁-tagged cytosolic RFP (Kate) and the OMM-GFP_1–10 (left panel), of the ER_S/L-β₁₁ constructs and a cytosolic GFP_1–10 (middle panel) and of the SPLICS_S/L (right panel), respectively. b Experimental controls showing the correct targeting of the mitochondrial (OMM-GFP_1–10) and the ER (ER_S-β₁₁ and ER_L-β₁₁) targeted fragments verified by complementation with Kate-β₁₁ and GFP_1–10, respectively. Co-transfection of HeLa cells with OMM-GFP_1–10 and both ER_S-β₁₁ or ER_L-β₁₁ induces the appearance of a “dotted” fluorescence. c Quantification of ER–mitochondria contacts in HeLa cells. The SPLICS dots were quantified from the 3D rendering of a complete z-stack. Mean ± SEM: SPLICS_S 56 ± 4, n = 37 cells; SPLICS_L 229 ± 12, n = 25 cells. d Co-localization of SPLICS_S/L fluorescence with mitochondria (mtHSP60) and ER (CRT: calreticulin) markers. Representative traces e and statistical analysis f of mitochondrial Ca²⁺ uptake in HeLa cells transfected with SPLICS_S or SPLICS_L along with mtAeqmut. Mean ± SEM: Void Vector 75 ± 2, n = 65 wells; SPLICS_S 77 ± 1, n = 54 wells; SPLICS_L 71 ± 2, n = 54 wells. Scale bar 15 µm. Data shown are the result of 3–5 independent experiments.