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: This is, to our knowledge, the first time that ligand-triggered endocytosis has been imaged at the level of single-scission events. The parallels with a similar analysis of constitutive TfR(+) scission events in NIH3T3 fibroblasts are striking (Merrifield et al., 2005; Taylor et al., 2011, 2012). As for constitutive TfR endocytosis, the ligand-triggered endocytosis of β2AR proceeds via quantized scission events hosted by an apparently diverse array of clathrin spots and plaques. A simple model to explain the observed diversity of CCS dynamics is that CCSs may occur as discrete clathrin-coated buds at the plasma membrane (spot-like CCSs) or in association with a large, flat or gently curved patch of clathrin at the plasma membrane (plaque-like CCSs; Figure 7). As noted previously, the latter types of structure have been observed by electron microscopy (Heuser, 1980; Maupin and Pollard, 1983). To fully understand the CCS dynamics observed by microscopy, the dynamics of both the bud and, if present, the associated clathrin patch must be considered. If the patch of clathrin at the membrane is absent, the coated bud will disappear once scission occurs in a terminal event (Figure 7). If a small patch of clathrin is left after scission, a nonterminal event will occur if a fresh bud regrows at the same site (Figure 7). If the associated patch is sufficiently large and perhaps stabilized by adhesion (Batchelder and Yarar, 2010), it might sufficiently bright to obscure the loss of clathrin coat during budding events at or close to its edges, which would thus evade detection by conventional fluorescence microscopy, and it would appear as a plaque (Figure 7). Therefore CCSs are superficially heterogeneous even though they represent a common pathway of receptor internalization.
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany Translational Lung Research Center, Department of Translational Pulmonology, University of Heidelberg, 69120 Heidelberg, Germany.