Mathes, B. | bioRxiv Science

bioRxiv2021

Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization

Sulfonated rhodamines that endow xanthene dyes with cellular impermeability are presented. We fuse charged sulfonates to red and far-red dyes to obtain Sulfo549 and Sulfo646, respectively, and further link these to SNAP- and Halo-tag substrates for protein self-labelling. Cellular impermeability is validated in live cell imaging experiments in transfected HEK cells and neurons derived from induced pluripotent stem cells (iPSCs). Lastly, we show that Sulfo646 is amenable to STED nanoscopy by recording membranes of SNAP/Halo-surface-labelled human iPSC-derived neuronal axons. We therefore provide an avenue for rendering dyes impermeable for exclusive extracellular visualization via self-labelling protein tags.

biochemistry↗

bioRxiv2020

Interrogating surface versus intracellular transmembrane receptor populations using cell-impermeable SNAP-tag substrates

Employing self-labelling protein tags for the attachment of fluorescent dyes has become a routine and powerful technique in optical microscopy to visualize and track fused proteins. However, membrane permeability of the dyes and the associated background signals can interfere with the analysis of extracellular labeling sites. Here we describe a novel approach to improve extracellular labeling by functionalizing the SNAP-tag substrate benzyl guanine ("BG") with a charged sulfonate ("SBG"). This chemical manipulation improves solubility, reduces non-specific staining and renders the bioconjugation handle impermeable while leaving its cargo untouched. We report SBG-conjugated fluorophores across the visible spectrum, which cleanly label SNAP-fused proteins in the plasma membrane of living cells. We demonstrate the utility of SBG-conjugated fluorophores to interrogate class A, B and C G protein-coupled receptors (GPCRs) using a range of imaging approaches including nanoscopic super-resolution imaging, analysis of GPCR trafficking from intra- and extracellular pools, in vivo labelling in mouse brain and analysis of receptor stoichiometry using single molecule pull down.

physiology↗