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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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mApple-β2AR clusters and internalizes with TfR-phl. (A) Example HEK293 cell expressing TfR-phl (green, left) and mApp-β2AR (magenta, right) at pH 7.4, 50 s before isoproterenol addition (top) and 550 s after isoproterenol challenge (bottom). After isoproterenol challenge, mApp-β2AR clustered with TfR-phl at CCSs. Images are averages of 50 frames, acquired at 0.5 Hz, centered on the relative time point. (B) Example TfR-phl scission events before (Bi) and after (Bii) isoproterenol addition. After isoproterenol addition, mApp-β2AR signal coincided with TfR-phl(+) scission events, indicating that the two receptors cointernalized in the same vesicles. (Ci) Density plot of aligned fluorescence changes (horizontal axis) associated with bona fide TfR-phl(+) scission events plotted against global time (vertical axis). The flux of mApp-β2AR receptor through the constitutive CME pathway appeared as a transient pulse of fluorescence signal (right). (Cii) Quantified average fluorescence traces of TfR-phl and mApp-β2AR over the time windows indicated in Ci.
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Figure 4: mApple-β2AR clusters and internalizes with TfR-phl. (A) Example HEK293 cell expressing TfR-phl (green, left) and mApp-β2AR (magenta, right) at pH 7.4, 50 s before isoproterenol addition (top) and 550 s after isoproterenol challenge (bottom). After isoproterenol challenge, mApp-β2AR clustered with TfR-phl at CCSs. Images are averages of 50 frames, acquired at 0.5 Hz, centered on the relative time point. (B) Example TfR-phl scission events before (Bi) and after (Bii) isoproterenol addition. After isoproterenol addition, mApp-β2AR signal coincided with TfR-phl(+) scission events, indicating that the two receptors cointernalized in the same vesicles. (Ci) Density plot of aligned fluorescence changes (horizontal axis) associated with bona fide TfR-phl(+) scission events plotted against global time (vertical axis). The flux of mApp-β2AR receptor through the constitutive CME pathway appeared as a transient pulse of fluorescence signal (right). (Cii) Quantified average fluorescence traces of TfR-phl and mApp-β2AR over the time windows indicated in Ci.

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.


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)

mApple-β2AR clusters and internalizes with TfR-phl. (A) Example HEK293 cell expressing TfR-phl (green, left) and mApp-β2AR (magenta, right) at pH 7.4, 50 s before isoproterenol addition (top) and 550 s after isoproterenol challenge (bottom). After isoproterenol challenge, mApp-β2AR clustered with TfR-phl at CCSs. Images are averages of 50 frames, acquired at 0.5 Hz, centered on the relative time point. (B) Example TfR-phl scission events before (Bi) and after (Bii) isoproterenol addition. After isoproterenol addition, mApp-β2AR signal coincided with TfR-phl(+) scission events, indicating that the two receptors cointernalized in the same vesicles. (Ci) Density plot of aligned fluorescence changes (horizontal axis) associated with bona fide TfR-phl(+) scission events plotted against global time (vertical axis). The flux of mApp-β2AR receptor through the constitutive CME pathway appeared as a transient pulse of fluorescence signal (right). (Cii) Quantified average fluorescence traces of TfR-phl and mApp-β2AR over the time windows indicated in Ci.
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Figure 4: mApple-β2AR clusters and internalizes with TfR-phl. (A) Example HEK293 cell expressing TfR-phl (green, left) and mApp-β2AR (magenta, right) at pH 7.4, 50 s before isoproterenol addition (top) and 550 s after isoproterenol challenge (bottom). After isoproterenol challenge, mApp-β2AR clustered with TfR-phl at CCSs. Images are averages of 50 frames, acquired at 0.5 Hz, centered on the relative time point. (B) Example TfR-phl scission events before (Bi) and after (Bii) isoproterenol addition. After isoproterenol addition, mApp-β2AR signal coincided with TfR-phl(+) scission events, indicating that the two receptors cointernalized in the same vesicles. (Ci) Density plot of aligned fluorescence changes (horizontal axis) associated with bona fide TfR-phl(+) scission events plotted against global time (vertical axis). The flux of mApp-β2AR receptor through the constitutive CME pathway appeared as a transient pulse of fluorescence signal (right). (Cii) Quantified average fluorescence traces of TfR-phl and mApp-β2AR over the time windows indicated in Ci.
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.

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