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Semisynthetic fluorescent pH sensors for imaging exocytosis and endocytosis - November 2017

Magalie Martineau, Agila Somasundaram, Jonathan B. Grimm, Todd D. Gruber, Daniel Choquet, Justin W. Taraska, Luke D. Lavis, and David Perrais
Nature Communications Nov 10;8(1):1423 doi: 10.1038/s41467-017-01752-5.

The GFP-based superecliptic pHluorin (SEP) enables the detection of exocytosis and endocytosis, revealing the change in pH experienced by vesicular proteins from acidic intraluminal pH (5.5) to neutral extracellular pH (7.4). Red fluorescent proteins such as pHuji, which we have developed previously (Chen et al. J Cell Biol 2014) approach, but do not match, the sensitivity of SEP to pH changes. Here we describe ‘semisynthetic’ pH-sensitive protein conjugates with organic fluorophores, carbofluorescein and Virginia Orange, which match the properties of SEP (Figure 1a). Conjugation to genetically encoded self-labeling tags or antibodies allows visualization of both exocytosis and endocytosis of synaptic vesicles (Figure 1b,c), constituting new bright sensors for these key steps of synaptic transmission.

Figure 1: Antibodies labelled with pH dependent Virginia Orange (VO), a new tool to visualize the exocytosis and endocytosis of endogenous vesicular proteins. a, Structure and pH dependency of fluorescein, carbofluorescein (CFl) and VO. b, protocol to label endogenous synaptotagmin 1 (Syt1), a vesicular protein. c, images of axons of neurons transfected with Syt1-SEP and incubated with anti-Syt1-VO before (0s) and at the end of stimulation (10 s). Note the large increase in fluorescence in axonal boutons of transfected (green arrow) and untransfected (white arrow) neurons. d, Quantification of fluorescence increases with the two sensors.

Contact: David Perrais