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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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Single-event analysis of β2AR internalization. (Ai) Example HEK293 cell expressing Clc-mCh (left) and phl-β2AR at pH7.4 (middle) and 2 s later at pH 5.5 (right); first image taken ∼428 s before addition of isoproterenol. At pH 5.5, cell-surface phl-β2AR quenched and no pH-resistant, phl-β2AR(+)endosomes were visible close to the plasma membrane. (Aii) Same cell 240 s after addition of isoproterenol. Phl-β2AR became concentrated at coated pits at pH 7.4 (middle). When cell-surface phl-β2AR was quenched 2 s later at pH 5.5, acid-resistant endosomes containing phl-β2AR were clearly visible close to the plasma membrane. (Aiii) Expanded region indicated in Aii, showing Clc-mCh (magenta, top left), phl-β2AR at p H7.4 (green, middle left), and overlay (lower left). Three acid-resistant, phl-β2AR(+) endosomes were visible in the field (green, middle right) colocalized with CCS (yellow circles). (B) Time-resolved images of a single-scission event engulfing phl-β2AR. The fluorescence was quantified and bona fide scission events defined primarily on the basis of S/N (lower graph, scale: vertical 2.5 a.u., horizontal 33 s; see Materials and Methods for details). (C) Time course of detected scission events before, during, and after addition of isoproterenol. Summarized data from four cells showing the incidence of scission events over a 20-s time window (gray histogram) and the cumulative number of scission events (blue line).
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Figure 1: Single-event analysis of β2AR internalization. (Ai) Example HEK293 cell expressing Clc-mCh (left) and phl-β2AR at pH7.4 (middle) and 2 s later at pH 5.5 (right); first image taken ∼428 s before addition of isoproterenol. At pH 5.5, cell-surface phl-β2AR quenched and no pH-resistant, phl-β2AR(+)endosomes were visible close to the plasma membrane. (Aii) Same cell 240 s after addition of isoproterenol. Phl-β2AR became concentrated at coated pits at pH 7.4 (middle). When cell-surface phl-β2AR was quenched 2 s later at pH 5.5, acid-resistant endosomes containing phl-β2AR were clearly visible close to the plasma membrane. (Aiii) Expanded region indicated in Aii, showing Clc-mCh (magenta, top left), phl-β2AR at p H7.4 (green, middle left), and overlay (lower left). Three acid-resistant, phl-β2AR(+) endosomes were visible in the field (green, middle right) colocalized with CCS (yellow circles). (B) Time-resolved images of a single-scission event engulfing phl-β2AR. The fluorescence was quantified and bona fide scission events defined primarily on the basis of S/N (lower graph, scale: vertical 2.5 a.u., horizontal 33 s; see Materials and Methods for details). (C) Time course of detected scission events before, during, and after addition of isoproterenol. Summarized data from four cells showing the incidence of scission events over a 20-s time window (gray histogram) and the cumulative number of scission events (blue line).

Mentions: In an example HEK293 cell coexpressing green phl-β2AR and Clc-mCherry, phl-β2AR fluorescence was homogeneous across the plasma membrane and did not colocalize with punctate CCSs (Figure 1Ai). The cell was imaged with pH cycling for 250 s, and in an example image acquired at pH 5.5, no pH-resistant phl-β2AR was detected close to the plasma membrane (Figure 1Ai). At 250 s, the perfusion stream was switched to buffers containing 20 μM isoproterenol, which triggered rapid recruitment of phl-β2AR into CCSs (Figure 1Ai). In an example image acquired at pH 5.5, a population of brightly fluorescent punctae was revealed colocalized with Clc-mCherry labeled CCSs and with patches of phl-β2AR at pH 7.4 (Figure 1Aii, yellow circles). Not all clathrin patches colocalized with a correlated spot of phl-β2AR at pH 7.4 (Figure 1Aii, white arrowhead). These rare CCSs could represent coated pits that entirely lacked phl-β2AR (Cao et al., 1998), or they could be CCSs that pinched of at pH 5.5 or otherwise naturally acidified after scission; in either of the latter cases, the phl-β2AR cargo would be quenched and therefore invisible. The pH-resistant spots of phl-β2AR fluorescence in images acquired at pH 5.5 represented newly formed endocytic vesicles containing phl-β2AR that pinched off of the plasma membrane as the cell was perfused with a pulse of buffer at pH 7.4 in the preceding image (Figure 1Aii, yellow circles).


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)

Single-event analysis of β2AR internalization. (Ai) Example HEK293 cell expressing Clc-mCh (left) and phl-β2AR at pH7.4 (middle) and 2 s later at pH 5.5 (right); first image taken ∼428 s before addition of isoproterenol. At pH 5.5, cell-surface phl-β2AR quenched and no pH-resistant, phl-β2AR(+)endosomes were visible close to the plasma membrane. (Aii) Same cell 240 s after addition of isoproterenol. Phl-β2AR became concentrated at coated pits at pH 7.4 (middle). When cell-surface phl-β2AR was quenched 2 s later at pH 5.5, acid-resistant endosomes containing phl-β2AR were clearly visible close to the plasma membrane. (Aiii) Expanded region indicated in Aii, showing Clc-mCh (magenta, top left), phl-β2AR at p H7.4 (green, middle left), and overlay (lower left). Three acid-resistant, phl-β2AR(+) endosomes were visible in the field (green, middle right) colocalized with CCS (yellow circles). (B) Time-resolved images of a single-scission event engulfing phl-β2AR. The fluorescence was quantified and bona fide scission events defined primarily on the basis of S/N (lower graph, scale: vertical 2.5 a.u., horizontal 33 s; see Materials and Methods for details). (C) Time course of detected scission events before, during, and after addition of isoproterenol. Summarized data from four cells showing the incidence of scission events over a 20-s time window (gray histogram) and the cumulative number of scission events (blue line).
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Related In: Results  -  Collection

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Figure 1: Single-event analysis of β2AR internalization. (Ai) Example HEK293 cell expressing Clc-mCh (left) and phl-β2AR at pH7.4 (middle) and 2 s later at pH 5.5 (right); first image taken ∼428 s before addition of isoproterenol. At pH 5.5, cell-surface phl-β2AR quenched and no pH-resistant, phl-β2AR(+)endosomes were visible close to the plasma membrane. (Aii) Same cell 240 s after addition of isoproterenol. Phl-β2AR became concentrated at coated pits at pH 7.4 (middle). When cell-surface phl-β2AR was quenched 2 s later at pH 5.5, acid-resistant endosomes containing phl-β2AR were clearly visible close to the plasma membrane. (Aiii) Expanded region indicated in Aii, showing Clc-mCh (magenta, top left), phl-β2AR at p H7.4 (green, middle left), and overlay (lower left). Three acid-resistant, phl-β2AR(+) endosomes were visible in the field (green, middle right) colocalized with CCS (yellow circles). (B) Time-resolved images of a single-scission event engulfing phl-β2AR. The fluorescence was quantified and bona fide scission events defined primarily on the basis of S/N (lower graph, scale: vertical 2.5 a.u., horizontal 33 s; see Materials and Methods for details). (C) Time course of detected scission events before, during, and after addition of isoproterenol. Summarized data from four cells showing the incidence of scission events over a 20-s time window (gray histogram) and the cumulative number of scission events (blue line).
Mentions: In an example HEK293 cell coexpressing green phl-β2AR and Clc-mCherry, phl-β2AR fluorescence was homogeneous across the plasma membrane and did not colocalize with punctate CCSs (Figure 1Ai). The cell was imaged with pH cycling for 250 s, and in an example image acquired at pH 5.5, no pH-resistant phl-β2AR was detected close to the plasma membrane (Figure 1Ai). At 250 s, the perfusion stream was switched to buffers containing 20 μM isoproterenol, which triggered rapid recruitment of phl-β2AR into CCSs (Figure 1Ai). In an example image acquired at pH 5.5, a population of brightly fluorescent punctae was revealed colocalized with Clc-mCherry labeled CCSs and with patches of phl-β2AR at pH 7.4 (Figure 1Aii, yellow circles). Not all clathrin patches colocalized with a correlated spot of phl-β2AR at pH 7.4 (Figure 1Aii, white arrowhead). These rare CCSs could represent coated pits that entirely lacked phl-β2AR (Cao et al., 1998), or they could be CCSs that pinched of at pH 5.5 or otherwise naturally acidified after scission; in either of the latter cases, the phl-β2AR cargo would be quenched and therefore invisible. The pH-resistant spots of phl-β2AR fluorescence in images acquired at pH 5.5 represented newly formed endocytic vesicles containing phl-β2AR that pinched off of the plasma membrane as the cell was perfused with a pulse of buffer at pH 7.4 in the preceding image (Figure 1Aii, yellow circles).

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