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Fig. 3 | BMC Biology

Fig. 3

From: A genetically encoded toolkit of functionalized nanobodies against fluorescent proteins for visualizing and manipulating intracellular signalling

Fig. 3

Targeting RNb-Ca2+ sensors to RFP-tagged proteins. a Schematic of RNb-GGECO fusion binding to RFP. bd HeLa cells expressing RNb-GGECO1.2 and TOM20-mCh, before and after addition of histamine (100 μM) and then ionomycin (5 μM). Cells were imaged in HBS using TIRFM. The TOM20-mCh image is shown after the histamine and ionomycin additions. The merged images are shown using images of RNb-GGECO1.2 after ionomycin (b, c) or histamine (d). The yellow and cyan-boxed regions in panel b are shown enlarged in panels c and d, respectively. Scale bars are 10 μm (b) or 1.25 μm (c, d). e Timecourse of the effects of histamine (100 μM) and ionomycin (5 μM) on the fluorescence of RNb-GGECO1.2 (F/F0, where F and F0 are fluorescence recorded at t and t = 0). The traces are from regions coinciding with a single mitochondrion or cytosol (regions identified in panel d), indicating changes in [Ca2+] at the OMM. f Enlarged region (70–180 s) of the graph is shown in e. Results are representative of cells from 13 independent experiments

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