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Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis.

Lampe M, Pierre F, Al-Sabah S, Krasel C, Merrifield CJ - Mol. Biol. Cell (2014)

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.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany Translational Lung Research Center, Department of Translational Pulmonology, University of Heidelberg, 69120 Heidelberg, Germany.

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The flux of TfR-phl(+) scission events through the constitutive endocytic pathway is only mildly affected by ligand-triggered β2AR internalization. (A) A group of 3 HEK293 cells before, during, and after challenge with isoproterenol, showing surface TfR-phl fluorescence (left) and mApple-β2AR fluorescence (right) at pH 7.4. (B) Quantification of fluorescence changes in cell indicated in A by asterisk. TfR-phl fluorescence (green) showed a moderate decrease during the course of the experiment. (C) By contrast, mApple-β2AR (magenta) showed a robust decrease in fluorescence, followed by a moderate increase upon isoproterenol washout (indicated by arrow). (D) The incidence rate of TfR-phl(+) scission events, expressed as events μm−2 s−1, before, during, and after isoproterenol challenge.
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Figure 5: The flux of TfR-phl(+) scission events through the constitutive endocytic pathway is only mildly affected by ligand-triggered β2AR internalization. (A) A group of 3 HEK293 cells before, during, and after challenge with isoproterenol, showing surface TfR-phl fluorescence (left) and mApple-β2AR fluorescence (right) at pH 7.4. (B) Quantification of fluorescence changes in cell indicated in A by asterisk. TfR-phl fluorescence (green) showed a moderate decrease during the course of the experiment. (C) By contrast, mApple-β2AR (magenta) showed a robust decrease in fluorescence, followed by a moderate increase upon isoproterenol washout (indicated by arrow). (D) The incidence rate of TfR-phl(+) scission events, expressed as events μm−2 s−1, before, during, and after isoproterenol challenge.

Mentions: We established, in agreement with published results (Puthen­veedu and von Zastrow, 2006), that an influx of β2AR into the clathrin-mediated endocytic machinery extended the lifetime of spot-like CCS. However, did this correspond to a general slowing of the constitutive endocytic rate? To answer this question, we measured the incidence rate of TfR-phl(+) scission events before, during, and after challenge with isoproterenol (Figure 5). In an example cluster of three cells, TfR-phl fluorescence at pH 7.4 showed a moderate decrease through the experiment, which was most likely due to bleaching (Figure 5, A and B). By contrast, mApp-β2AR fluorescence at pH 7.4 dramatically decreased with first-order kinetics upon isoproterenol addition, followed by a recovery upon washout as the mApp-β2AR was internalized and subsequently recycled (Puthenveedu and von Zastrow, 2006; Figure 5, A and C). For each cell, we defined a polygon of constant area that was contained within the footprint of the cell before, during, and after isoproterenol addition and measured the number of TfR-phl(+) scission events that occurred within the polygon over time. On addition of isoproterenol, there was a very modest, although insignificant, increase in the incidence rate of TfR-phl scission events (Figure 5D). We therefore conclude that ligand-triggered endocytosis of β2AR does not significantly change the overall incidence rate of constitutive endocytic scission events.


Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis.

Lampe M, Pierre F, Al-Sabah S, Krasel C, Merrifield CJ - Mol. Biol. Cell (2014)

The flux of TfR-phl(+) scission events through the constitutive endocytic pathway is only mildly affected by ligand-triggered β2AR internalization. (A) A group of 3 HEK293 cells before, during, and after challenge with isoproterenol, showing surface TfR-phl fluorescence (left) and mApple-β2AR fluorescence (right) at pH 7.4. (B) Quantification of fluorescence changes in cell indicated in A by asterisk. TfR-phl fluorescence (green) showed a moderate decrease during the course of the experiment. (C) By contrast, mApple-β2AR (magenta) showed a robust decrease in fluorescence, followed by a moderate increase upon isoproterenol washout (indicated by arrow). (D) The incidence rate of TfR-phl(+) scission events, expressed as events μm−2 s−1, before, during, and after isoproterenol challenge.
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Related In: Results  -  Collection

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Figure 5: The flux of TfR-phl(+) scission events through the constitutive endocytic pathway is only mildly affected by ligand-triggered β2AR internalization. (A) A group of 3 HEK293 cells before, during, and after challenge with isoproterenol, showing surface TfR-phl fluorescence (left) and mApple-β2AR fluorescence (right) at pH 7.4. (B) Quantification of fluorescence changes in cell indicated in A by asterisk. TfR-phl fluorescence (green) showed a moderate decrease during the course of the experiment. (C) By contrast, mApple-β2AR (magenta) showed a robust decrease in fluorescence, followed by a moderate increase upon isoproterenol washout (indicated by arrow). (D) The incidence rate of TfR-phl(+) scission events, expressed as events μm−2 s−1, before, during, and after isoproterenol challenge.
Mentions: We established, in agreement with published results (Puthen­veedu and von Zastrow, 2006), that an influx of β2AR into the clathrin-mediated endocytic machinery extended the lifetime of spot-like CCS. However, did this correspond to a general slowing of the constitutive endocytic rate? To answer this question, we measured the incidence rate of TfR-phl(+) scission events before, during, and after challenge with isoproterenol (Figure 5). In an example cluster of three cells, TfR-phl fluorescence at pH 7.4 showed a moderate decrease through the experiment, which was most likely due to bleaching (Figure 5, A and B). By contrast, mApp-β2AR fluorescence at pH 7.4 dramatically decreased with first-order kinetics upon isoproterenol addition, followed by a recovery upon washout as the mApp-β2AR was internalized and subsequently recycled (Puthenveedu and von Zastrow, 2006; Figure 5, A and C). For each cell, we defined a polygon of constant area that was contained within the footprint of the cell before, during, and after isoproterenol addition and measured the number of TfR-phl(+) scission events that occurred within the polygon over time. On addition of isoproterenol, there was a very modest, although insignificant, increase in the incidence rate of TfR-phl scission events (Figure 5D). We therefore conclude that ligand-triggered endocytosis of β2AR does not significantly change the overall incidence rate of constitutive endocytic scission events.

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.

View Article: PubMed Central - PubMed

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