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

Abstract

<p>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...</p>

Ligand-Specific Factors Influencing GLP-1 Receptor Post-Endocytic Trafficking and Degradation in Pancreatic Beta Cells

“…To examine the consequences of these apparent differences in post-endocytic sorting on GLP-1R degradation, experiments were performed in INS-1-SNAP-GLP-1R cells treated with cycloheximide to arrest protein translation. After continuous exposure to 10 nM of either ligand, applied in a time series in reverse order, a cell permeating SNAP-tag probe, BG-conjugated Oregon Green (BG-OG) [25], was used to label total residual intact SNAP-GLP-1R. Over an 8 h time-course, a steady reduction of BG-OG labelling was apparent with both ligands, suggesting destruction of the SNAP moiety (and by inference, the GLP-1R itself), but this was faster and more pronounced with exendin-4 treatment ( Figure 1D).…”

Deuterated rhodamines for protein labelling in nanoscopy

“…fluoresceins, rhodols, SNARFs, and quenchers like QSYs), and other dye scaffolds (e.g. coumarins, cyanines, BODIPYs, EDANS, NBDs, Hoechst) including quenchers at any C-H bond positions for improving and fine-tuning spectroscopic properties; ii) to be further explored with other isotopes, such as 13 C, 15 N or even radioactive 3 H; iii) to be used in different labelling approaches, such as the attachment to sulfonated BG (SBG) scaffolds allowing the separation of SNAP-tagged receptor pools [19] , to biomolecule targeting probes [20][21][22] , to "click chemistry" reagents (e.g. cyclopropenes, cyclooctenes) [23] or to photoswitchable ligands [24,25] including black hole quenchers (BHQs), and iv) to serve as multimodal dyes for confocal fluorescence and Raman microscopy [26] .…”

Sulfonated rhodamines as impermeable labelling substrates for cell surface protein visualization

“…Accordingly, we synthesized Sulfo549 and Sulfo646 based on JF 549 and JF 646 , each bearing two sulfonate groups, and further linked them to SNAP- and Halo-tag substrates BG and CA, respectively. Our probes add favorably to a previous study, where we reported on a strategy to limit any dye to cell surface exposed SNAP tags by altering the BG substrate to a sulfonate itself (termed SBG) [16] . With a simple chloride anion being the leaving group for the Halo-tag, introducing a charged sulfonate is not tolerated.…”

“…[15] Moreover, this might be the case for cell surface receptor localization ( Figure 1A), and as such it remains difficult to isolate surface and intracellular pools of receptors not only for nanoscopy. [16] To restrict a priori fluorogenic dyes to the cell surface, we set out to synthesize rhodamines endowed with a cell impermeable sulfonate moiety. To achieve this, the rhodamine scaffold of fluorogenic and bright JaneliaFluor (JF) dyes [8] was extended with a short sulfonate-containing linker to provide red and far-red colors (Sulfo549 and Sulfo646), which can be targeted to cell surface tags and receptors ( Figure 1B).…”