Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis.
Bottom Line: Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles.Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak.These data support a simple model in which different cargoes internalize through common CCSs.
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany Translational Lung Research Center, Department of Translational Pulmonology, University of Heidelberg, 69120 Heidelberg, Germany.Show MeSH
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Mentions: These analysis depended on using the robust TfR-phl scission signal as a reference signal to measure constitutive scission events, and so it could not detect potential mApp-β2AR(+)/TfR-phl(–) scission events. These could correspond to scission of vesicles from “specialized” CCSs that internalized β2AR but excluded TfR. Moreover, using TfR-phl(+) scission events as a reference most likely underestimated the proportion of TfR-phl(+)/mApp-β2AR(+) scission events, as the red mApple scission signal was dimmer than the green phl signal and prone to contamination by internalized (but incompletely quenched) mApple-β2AR fluorescence. The automated scission detection algorithm was adapted to detect the very faint candidate mApp-β2AR scission events (see Materials and Methods). Briefly, spot fluorescence was measured using a circle-minus-annulus measurement as before, but segmented spots were excluded from the annulus measurement, giving a less noisy estimate of local background fluorescence. Candidate mApp-β2AR(+) scission events were classified as bona fide if they coincided with a cluster of mApp-β2AR at pH 7.4 and showed a stepwise fluorescence increase with S/N ≥ 10 (see Materials and Methods). This eliminated false-positive events, which most likely represent endocytic vesicles briefly visiting the plasma membrane. The mApp-β2AR(+) scission events identified were classified as TfR-phl(+) if there was a correlated stepwise increase in TfR-phl fluorescence with S/N ≥ 5. Of the mApp-β2AR scission events identified, 95% coincided with a correlated TfR-phl(+) scission event (Figure 6, A–C). From this we conclude that, upon isoproterenol challenge, there is only one population of constitutive endocytic vesicles in HEK293 cells that internalize both mApp-β2AR and TfR-phl.
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany Translational Lung Research Center, Department of Translational Pulmonology, University of Heidelberg, 69120 Heidelberg, Germany.