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Fig. 18 | BMC Biology

Fig. 18

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

Fig. 18

Nanobody-mediated targeting of low-affinity Ca2+ sensors allows measurement of changes in [Ca2+] in an ER sub-compartment at ER-PM MCS. a Schematic of ssRNb-Ca2+ sensor bound to RFP. b Schematic of ssGNb-Ca2+ sensor bound to GFP. cf HeLa cells co-expressing the indicated combinations of mCh-MAPPER, GFP-MAPPER, ssRNb-GCEPIA (ssRNb-GC), ssRNb-GEMCEPIA (ssRNb-GEM; the image is shown for the 525-nm emission channel), ssGNb-LAR-GECO1 (ssGNb-LGECO) or ssGNb-RCEPIA were imaged in Ca2+-free HBS using TIRFM. Yellow boxes indicate regions enlarged in subsequent images. Scale bars 10 μm (main images) and 2.5 μm (enlargements). gj Timecourses of fluorescence changes recorded from cells co-expressing mCh-MAPPER and ssRNb-GCEPIA (g), mCh-MAPPER and ssRNb-GEMCEPIA (h), GFP-MAPPER and ssGNb-LAR-GECO1 (ssGNb-LARG1) (i) and GFP-MAPPER and ssGNb-RCEPIA (j) in response to emptying of intracellular Ca2+ stores with ionomycin (5 μM). k Summary results (with mean ± SD, n = 4 cells) show fractional decreases (ΔF) in either fluorescence or emission ratio (for ssRNb-GEM) recorded 90 s after addition of ionomycin

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