Fig. 2

Internalized TSH/TSHRs meet the G_s-protein at the retromer-coated compartment (RCC) associated with the TGN. Primary mouse thyroid cells were cotransfected with different combinations of G_s-YFP and/or subcellular markers, followed by stimulation with fluorescent TSH (TSH-594 or TSH-647) for 10 min to visualize internalized TSH/TSHR complexes. Shown are images at the indicated time points from beginning of stimulation. a Co-localization between TSH-594 and transfected G_s-YFP at 10 min. The focus in the images shown in a was specifically set to also visualize TSH and the G_s-protein at the plasma membrane (arrowheads). b Co-localization between TSH-647, G_s-YFP and the early endosome marker Rab5-mCherry. Note the absence of triple co-localization after TSH stimulation. c Co-localization between TSH-594 and G_s-YFP. TSH is co-localizing with the G_s-protein on subdomains of tubulovesicular structures concentrated around the nucleus (arrowheads). d Quantification showing the percentage (mean ± s.e.m.; n = 10 cells) of TSH-containing structures co-localizing with G_s-YFP over time, calculated from images like those in c. e Enlarged view of a typical elongated membranous structure, showing presence of the G_s-protein on only one side and TSH on both. f Image sequences showing polarized fusion/splitting events among TSH- and G_s-protein-containing structures. Time is given relative to the first image. g Simultaneous visualization of TSH-647, G_s-YFP and Vps29-mCherry, used to localize the retromer complex. Structures analogous to those reported in c were observed (arrowheads). Bottom Enlarged view of a typical elongated membranous structure demonstrating the presence of retromer on the G_s-positive subdomain. h Quantification showing the percentage (mean ± s.e.m.; n = 4 cells) of structures containing both TSH-647 and G_s-YFP co-localizing with Vps29-mCherry over time, calculated from images like those in g. i Schematic view of the results. Where appropriate, cell edges and nucleus (N) are marked in white. Data in a–c and g are representative of 3, 3, 5 and 3 independent experiments, respectively