Figure 8.
Figure 8. TfR-pHuji and SEP-β2AR are differentially sorted at the level of CCV formation. (A and B) Examples of a scission event visible with both TfR-pHuji and SEP-β2AR (A) or with TfR-pHuji only during isoproterenol application (B). Note the enrichment of β2AR at pH 7.4 but not in the CCV. (C) Fluorescence at the time of detection in the green and red channels of CCVs detected in a representative cell during isoproterenol application. Yellow dots, CCVs detected in both channels (11 CCVs); green dots, CCVs detected in the SEP channel only (14 CCVs); red dots, clusters detected in the pHuji channel only (76 CCVs). Black dashed line shows linear regression of all TfR-pHuji clusters (R = 0.33). Green and red lines show the lower limits of SEP and pHuji CCV detection, respectively. (D, a) Proportion for n = 7 cells of vesicles detected in both channels (yellow) or with either TfR-pHuji (red) or SEP-β2AR (green). (b) TfR-pHuji CCVs enriched in SEP-β2AR (yellow) or not (red) according to the top-right quadrant defined by the SEP and pHuji lower detection limits determined as in B and vice-versa (yellow and green). (E) Mean TfR-pHuji and SEP-β2AR fluorescence at pH 7.4 (top) and 5.0 (bottom) of scission events detected in the red channel (570 events in seven cells) aligned to the time of CCV detection. The black lines indicate 95% confidence intervals for significant enrichment. (F) Same as E with 324 scission events detected in the green channel. (G) Working model in which TfR (red lollipops) is constitutively clustered at all clathrin-coated structures (brown lines) via AP-2 binding (orange squares) and internalized in vesicles via the action of dynamin (blue rings) regardless of isoproterenol application. On the opposite, SEP-β2AR (green lollipops) in basal conditions is homogenously distributed at the cell surface (black line) and occasionally internalized (a), whereas during application of isoproterenol it is actively targeted to CCPs via recruitment of cytosolic β-arrestin (green squares; b). This relocalization of ligand-bound receptors to all CCPs leads to potential surface-localized intracellular signaling platforms (yellow bolts), whereas ligand-induced internalization of β2AR only occurs in a subpopulation of CCVs. Error bars represent SEM.

TfR-pHuji and SEP-β2AR are differentially sorted at the level of CCV formation. (A and B) Examples of a scission event visible with both TfR-pHuji and SEP-β2AR (A) or with TfR-pHuji only during isoproterenol application (B). Note the enrichment of β2AR at pH 7.4 but not in the CCV. (C) Fluorescence at the time of detection in the green and red channels of CCVs detected in a representative cell during isoproterenol application. Yellow dots, CCVs detected in both channels (11 CCVs); green dots, CCVs detected in the SEP channel only (14 CCVs); red dots, clusters detected in the pHuji channel only (76 CCVs). Black dashed line shows linear regression of all TfR-pHuji clusters (R = 0.33). Green and red lines show the lower limits of SEP and pHuji CCV detection, respectively. (D, a) Proportion for n = 7 cells of vesicles detected in both channels (yellow) or with either TfR-pHuji (red) or SEP-β2AR (green). (b) TfR-pHuji CCVs enriched in SEP-β2AR (yellow) or not (red) according to the top-right quadrant defined by the SEP and pHuji lower detection limits determined as in B and vice-versa (yellow and green). (E) Mean TfR-pHuji and SEP-β2AR fluorescence at pH 7.4 (top) and 5.0 (bottom) of scission events detected in the red channel (570 events in seven cells) aligned to the time of CCV detection. The black lines indicate 95% confidence intervals for significant enrichment. (F) Same as E with 324 scission events detected in the green channel. (G) Working model in which TfR (red lollipops) is constitutively clustered at all clathrin-coated structures (brown lines) via AP-2 binding (orange squares) and internalized in vesicles via the action of dynamin (blue rings) regardless of isoproterenol application. On the opposite, SEP-β2AR (green lollipops) in basal conditions is homogenously distributed at the cell surface (black line) and occasionally internalized (a), whereas during application of isoproterenol it is actively targeted to CCPs via recruitment of cytosolic β-arrestin (green squares; b). This relocalization of ligand-bound receptors to all CCPs leads to potential surface-localized intracellular signaling platforms (yellow bolts), whereas ligand-induced internalization of β2AR only occurs in a subpopulation of CCVs. Error bars represent SEM.

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