Semi-synthetic approach for optical interrogation of the nervous system
Biofisika - Basque Centre for Biophysics
Optical monitoring of the membrane potential is a powerful approach to understand the cellular and molecular logic of the nervous system, especially when applied to an ensemble of cells or neurons otherwise not accessible by microelectrodes. Voltage sensitive fluorescent indicators provide a direct measurement of membrane potential changes through variation of fluorescence. Organic voltage-sensitive dyes possess excellent photophysical properties but lack spatial specificity. Recently developed fluorescent voltage-sensitive proteins have a benefit of genetic targeting but pose challenges because of difficulties in expression in vivo and low brightness.
In this talk, I will present a hybrid semi-synthetic approach to combine the advantages of organic indicators with genetic targeting to specific neuronal types. We use the genetically encoded expression of small protein tags, which can be enzymatically labelled with respective fluorescent ligands to label the neuronal membrane. Previously we used a membrane-bound self-labeling enzyme SNAP-tag as an in vivo reporter for Cre-dependent recombination, confirming the possibility of cell-specific targeting of synthetic fluorophores in mice (1). Therefore, we used a SNAP-tag tethered fluorophore and intramembrane voltage-sensitive molecule to create a FRET-based hybrid indicator for membrane potential. Moreover, we identified a new voltage sensitive dye and derivatized it to allow tethering to several membrane protein tags (2). The dye retained its voltage sensitivity as tested by simultaneous fluorescence microscopy and patch clamp electrophysiology. Transgenic mouse technology or viral expression of small protein tags would open the possibility for the implementation of these semi-synthetic approaches in vivo.
1) Yang G. et al., Nat Methods, 12, 137–9 (2015).
2) Sundukova et al., Angew Chem 8 (8), 2341-2344 (2019).
Host: Maria Kukley