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: To define the dynamic characteristics of CCSs that hosted β2AR(+) scission events, we transfected HEK293 cells with phl-β2AR and Mu2-mCherry to label CCSs and detected and analyzed scission events as described. Similar to NIH3T3 fibroblasts, both spot-like CCSs and larger and longer-lived clathrin plaques could exist at the adherent surface of HEK293 cells (Figure 2A). Persistent clathrin plaques occurred in a significant proportion (∼10%) of cells growing on fibronectin-coated coverslips and were most likely triggered by tight adhesion to the substrate (Batchelder and Yarar, 2010). On challenge with isoproterenol, phl-β2AR rapidly clustered at preexisting spot-like CCSs (Puthenveedu and von Zastrow, 2006) and large clathrin plaques (Figure 2, A and Bi). As shown earlier, not all punctate CCSs showed clear colocalization with phl-β2AR at pH 7.4 (Figure 1Aii), and fine, finger-like projections at the lower surface of some HEK293 cells obscured things further (Figure 2Ai). These background features were quenched at pH 5.5 and so did not interfere with the detection of CCSs or scission events. Strikingly, when β2AR(+) scission events were mapped to their host CCS, both clathrin punctae and clathrin plaques could host spot-like scission events (Figure 2, Bi and Bii). To quantify the distribution of scission events among spot-like CCSs and clathrin plaques for five cells, we segmented images (see Materials and Methods) and quantified the pixel area of the phl-β2AR(+)-marking scission and the corresponding “host” CCS, labeled with Mu2-mCherry, and plotted the result as a density scatterplot (Figure 2C). All scission events were the size of diffraction-limited spots, but a significant proportion (∼10%) of clathrin objects hosting scission events were significantly larger blobs and so classified as “plaques.” Therefore (and as shown previously for TfR-phl; Taylor et al., 2011), both clathrin spots and plaques could host discrete, quantized scission events of phl-β2AR, and the relative sizes of clathrin spots and plaques are poor predictors of the endocytic potential of any given CCS. In previous studies, it was likely that the endocytic activity at the edges of clathrin plaques was masked by the bright fluorescence of the plaque itself and so remained undetected. This was verified when Mu2-mCherry fluorescence traces were classified according to whether they occurred at spots or plaques: the average fluorescence traces in each case showed a discrete dip after scission, presumably as the clathrin bud separated from the host structure and was uncoated (Figure 2D).
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany Translational Lung Research Center, Department of Translational Pulmonology, University of Heidelberg, 69120 Heidelberg, Germany.