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
Related in: MedlinePlus
Mentions: First, the moderately pH-sensitive red fluorescent protein mApple was inserted into the extracellular domain of β2AR to make mApp-β2AR, which, in turn, allowed simultaneous imaging of TfR-phl (to detect single constitutive endocytic events) and mApp-β2AR (to detect ligand-triggered β2AR internalization) using the pulsed pH assay. The pKa of mApple (pKa 6.5; Shaner et al., 2008) is lower than that of sePhl (pKa 7.1; Sankaranarayanan and Ryan, 2000), and so a slightly more acidic wash of pH ∼5.0 was used to quench both red and green fluorescence. In an example HEK293 cell bathed in buffer at pH 7.4, TfR-phl was concentrated at CCSs and otherwise distributed evenly (Figure 4Ai, green). By contrast, mApp-β2AR fluorescence was uniform, with no concentration at CCSs (Figure 4Ai, magenta). Images were acquired over a period of 400 s in conjunction with alternating pH to detect single TfR-phl(+) scission events as described. Before the addition of isoproterenol, no mApp-β2AR signal was detected at TfR-phl(+) scission events (Figure 4Bi). At t = 402 s, isoproterenol was introduced into the rhythmically alternating perfusion streams to trigger mApp-β2AR endocytosis (Supplemental Figure S1 and Materials and Methods). After a further ∼60 s, mApp-β2AR coclustered with TfR-phl at CCS, and in an example TfR-phl(+) scission event, a robust step increase in mApp-β2AR fluorescence was detected (Figure 4Bii). This could occur only if TfR-phl and mApp-β2AR pinched off in the same clathrin-coated vesicle.
Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany Translational Lung Research Center, Department of Translational Pulmonology, University of Heidelberg, 69120 Heidelberg, Germany.