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Fig. 10 | BMC Biology

Fig. 10

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

Fig. 10

RNb-FKBP inducibly recruits ER transmembrane proteins to mitochondria. a Schematic of RNb-FKBP bound to RFP. b Schematic of GNb-FKBP bound to GFP. c, d HeLa cells co-expressing RNb-FKBP, mitochondrial TOM70-GFP-FRB and mCh-Sec61β were imaged using TIRFM. A representative cell (n = 7) is shown before (c) and after (d) treatment with rapamycin (100 nM, 10 min). The boxed region is enlarged in subsequent images. Scale bars 10 μm (main images) and 2.5 μm (enlargements). e Timecourse of mCh-Sec61β fluorescence changes (F/F0) evoked by rapamycin recorded at a representative mitochondrion and in nearby reticular ER. Results show ~ 80% loss of fluorescence from the ER devoid of mitochondrial contacts. f, g HeLa cells co-expressing endogenously tagged GFP-IP3R1, GNb-FKBP and mitochondrial TOM70-mCh-FRB were imaged using TIRFM. A representative cell (n = 6) is shown before (f) and after (g) treatment with rapamycin (100 nM, 10 min). The boxed region is enlarged in subsequent images. Scale bars 10 μm (main images) and 2.5 μm (enlargements). h HeLa cells co-expressing GFP-calmodulin (GFP-CaM), GNb-FKBP and TOM20-mCh-FRB were imaged using epifluorescence microscopy. A representative cell (n = 3) is shown before and after treatment with rapamycin (100 nM, 10 min). The image for TOM-mCh-FRB is shown in the presence of rapamycin. Scale bar 10 μm

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