Fig. 4.

Targeted GNb-Ca²⁺ sensors detect changes in [Ca²⁺] at the surface of mitochondria. a Schematic of GNb-RGECO fusion binding to GFP. b, c Representative HeLa cells co-expressing TOM20-GFP and GNb-RGECO1.2 imaged in HBS using TIRFM before and after addition of histamine (100 μM) and then ionomycin (5 μM). The TOM20-GFP images are shown after the histamine and ionomycin additions. Histamine and ionomycin evoked changes in fluorescence of GNb-RGECO1.2 at the OMM. The yellow boxed region in panel B is shown enlarged in panel c. d–f Similar analyses of HeLa cells co-expressing TOM20-GFP and GNb-LAR-GECO1.2 (GNb-LARG1.2). Histamine (100 μM) evoked changes in fluorescence of GNb-LARG1.2 at the OMM of mitochondria in the perinuclear region (region of interest 1 (ROI 1) in e), but not in a peripheral region (ROI 2 in f). All mitochondria responded to ionomycin (5 μM), indicating that histamine evoked local changes in [Ca²⁺] at the OMM. The cyan and yellow boxed regions in d are shown enlarged in e and f, respectively. Scale bars 10 μm (b, d) or 2.5 μm (c, e and f). g Timecourse of the changes in fluorescence of GNb-RGECO1.2 at the OMM evoked by histamine and ionomycin for the entire cell shown in B. h Fluorescence changes recorded from ROI 1 and ROI 2 in panels e and f. Results are representative of cells from 4 independent experiments