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. 2023 Dec 6;9(49):eadi8076. doi: 10.1126/sciadv.adi8076

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Fig. 6. — (A) Schematic of labeling scheme used to visualize only internalized receptors. Surface receptors are labeled with a cleavable fluorophore (CA-SS–Alexa-488) and internalized by agonist incubation. MESNA treatment (100 mM) is then used to cleave the remaining surface fluorophores. (B) Fluorescence cell images of HA-Halo-mGluR3 labeled with CA-SS–Alexa-488 followed by 1 mM glutamate treatment before and after MESNA application. (C) Representative SiMPull images showing HA-Halo-mGluR2 (left) and HA-Halo-mGluR3 (right) after MESNA treatment in control (i.e., antagonist) versus Glu conditions. Single-molecule spots are circled in green. A representative fluorescence intensity trace is plotted with arrows marking photobleaching steps. (D) Quantification of total spots from control or Glu-treated conditions. mGluR3 and mGluR8, but not mGluR2, show glutamate-driven internalized receptors. (E) Photobleaching step distribution for immobilized spots from either untreated or cells treated with glutamate and MESNA. A similar distribution is seen for surface and internalized mGluR3 or mGluR8. (F) Two-color SiMPull images of HA-Halo-mGluR2 coexpressed with either SNAP-mGluR3 or SNAP-mGluR8 labeled with CA-SS–Alexa-488 and SBG-JF646, followed by 1 mM Glu incubation and MESNA treatment. A representative fluorescence intensity trace is plotted in each color for a colocalized spot with arrows marking photobleaching steps. (G) Quantification from two-color SiMPull experiments showing total number of HA-Halo-mGluR2 spots when coexpressed with SNAP-mGluR2, mGluR3, or mGluR8 treated with either antagonist or Glu. (H) Photobleaching step distribution for colocalized spots, indicating strict 1:1 heterodimerization. (I) Two-color SiMPull images of HA-Halo-mGluR8 coexpressed with SNAP-mGluR3 labeled with CA-SS–Alexa-488 and SBG-JF646, followed by 1 mM Glu incubation and MESNA treatment. A fluorescence intensity trace is plotted for a colocalized spot with arrows marking photobleaching steps. (J) Photobleaching step distribution for colocalized HA-Halo-mGluR8/SNAP-mGluR3 spots, indicating strict 1:1 heterodimerization. One-way ANOVA test, ***P < 0.001. Data represented as means ± SEM.