Fig. 1: Inducing ER–mitochondrial associations in neuronal HT22 cells increases [Ca²⁺]_m uptake.

a, b Representative fluorescent images of an individual HT22 cell expressing (a) ER–mitochondrial linkers or (b) ER-Flipper-GFP and TOM70-FKBP12-RFP prior to and 10 min following rapamycin (100 nM) addition. Fluorescent traces are shown individually (left and middle panels) and as an overlay of both fluorescent channels (right panels). Cyan: ER-CFP-9xFRB, green: ER-Flipper-GFP, red: TOM70-FKBP12-RFP. c Transfection efficiency of HEK293T cells transfected with mtGA^wt or mtGA^mut. Data are presented as mean ± SD, n = 8–9, Student’s t-test, ***p < 0.0001. d Representative measurement of [Ca²⁺]_m uptake in HEK293T cells transfected with mtGA^wt stimulated with buffer (CTR) or 50 mM CaCl₂ in the presence or absence of 100 nM rapamycin. Data are presented as mean ± SD, n = 3–4. e, f Quantification of [Ca²⁺]_m in HEK293T cells expressing (e) mtGA^wt or (f) mtGA^mut following stimulation with buffer or 50 mM CaCl₂ with/without 100 nM rapamycin. Luminescence in mitoGA^wt stimulated cells was normalized to the total luminescence (L_{t = 5–30}/L_total). Data are presented as mean ± SD, Student’s t-test, n = 3-4, ***p < 0.0001 compared to untreated control, ^#p < 0.05 compared to 50 mM CaCl₂ without rapamycin