Limits...
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.

Show MeSH

Related in: MedlinePlus

Lifetimes analysis of clathrin spots. (A) Example fluorescence traces for Mu2-mCherry (magenta) and phl-β2AR (green) for CCSs that hosted one, two, and seven scission events, respectively (arrows). (B) Histogram of the number of scission events detected per punctate CCS. A significant proportion (∼20%) of punctate CCSs hosted more than one scission event. (C) Histogram of the time between CCS nucleation and the first detected scission event for β2AR (median lifetime, 119.6 s). (D) The time between CCS nucleation and the first, second, or third detected scission event for TfR(+) scission events (black circles), β2AR(+) scission events (open circles), or MOR (gray circles) revealed that CCSs matured more slowly when loaded with β2AR or MOR. (E) Lifetimes histograms of punctate CCSs in cells expressing β2AR before challenge (Ei), during challenge (Eii), and after washout (Eiii) of isoproterenol. Eiii insert shows increase in median lifetime of CCS lifetime on isoproteronol challenge for four cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230595&req=5

Figure 3: Lifetimes analysis of clathrin spots. (A) Example fluorescence traces for Mu2-mCherry (magenta) and phl-β2AR (green) for CCSs that hosted one, two, and seven scission events, respectively (arrows). (B) Histogram of the number of scission events detected per punctate CCS. A significant proportion (∼20%) of punctate CCSs hosted more than one scission event. (C) Histogram of the time between CCS nucleation and the first detected scission event for β2AR (median lifetime, 119.6 s). (D) The time between CCS nucleation and the first, second, or third detected scission event for TfR(+) scission events (black circles), β2AR(+) scission events (open circles), or MOR (gray circles) revealed that CCSs matured more slowly when loaded with β2AR or MOR. (E) Lifetimes histograms of punctate CCSs in cells expressing β2AR before challenge (Ei), during challenge (Eii), and after washout (Eiii) of isoproterenol. Eiii insert shows increase in median lifetime of CCS lifetime on isoproteronol challenge for four cells.

Mentions: We next focused on a more detailed analysis of spot-like CCS lifetimes, since the dynamics of these structures are most readily analyzed, and the lifetime of spot-like coated pits was used previously as a readout of clathrin-coated pit maturation (Ehrlich et al., 2004; Puthenveedu and von Zastrow, 2006; Loerke et al., 2009; Henry et al., 2012). To determine the maturation time of productive clathrin-coated buds, we tracked Mu2-mCherry labeled CCSs and mapped them to bona fide scission events (Merrifield et al., 2005). As shown previously for TfR in fibroblasts (Merrifield et al., 2005; Taylor et al., 2011, 2012), spot-like CCSs most commonly hosted only one scission event but could potentially host multiple scission events (Figure 3, A and B). Strikingly, many scission events coincided with partial dimming but not complete disappearance of the host CCS, and repeated trains of partial dimming resulted in characteristic sawtooth-type profiles of Mu2-mCherry fluorescence (Figure 3A, right pair of traces). As described previously for NIH fibroblasts (Merrifield et al., 2005; Taylor et al., 2011), “nonterminal” events, for which the host CCS did not completely disappear after scission, comprised a significant proportion of the total number of scission events (∼50%). By definition, terminal events coincided with the final disappearance of the host spot-like CCS (Figure 3A).


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)

Lifetimes analysis of clathrin spots. (A) Example fluorescence traces for Mu2-mCherry (magenta) and phl-β2AR (green) for CCSs that hosted one, two, and seven scission events, respectively (arrows). (B) Histogram of the number of scission events detected per punctate CCS. A significant proportion (∼20%) of punctate CCSs hosted more than one scission event. (C) Histogram of the time between CCS nucleation and the first detected scission event for β2AR (median lifetime, 119.6 s). (D) The time between CCS nucleation and the first, second, or third detected scission event for TfR(+) scission events (black circles), β2AR(+) scission events (open circles), or MOR (gray circles) revealed that CCSs matured more slowly when loaded with β2AR or MOR. (E) Lifetimes histograms of punctate CCSs in cells expressing β2AR before challenge (Ei), during challenge (Eii), and after washout (Eiii) of isoproterenol. Eiii insert shows increase in median lifetime of CCS lifetime on isoproteronol challenge for four cells.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230595&req=5

Figure 3: Lifetimes analysis of clathrin spots. (A) Example fluorescence traces for Mu2-mCherry (magenta) and phl-β2AR (green) for CCSs that hosted one, two, and seven scission events, respectively (arrows). (B) Histogram of the number of scission events detected per punctate CCS. A significant proportion (∼20%) of punctate CCSs hosted more than one scission event. (C) Histogram of the time between CCS nucleation and the first detected scission event for β2AR (median lifetime, 119.6 s). (D) The time between CCS nucleation and the first, second, or third detected scission event for TfR(+) scission events (black circles), β2AR(+) scission events (open circles), or MOR (gray circles) revealed that CCSs matured more slowly when loaded with β2AR or MOR. (E) Lifetimes histograms of punctate CCSs in cells expressing β2AR before challenge (Ei), during challenge (Eii), and after washout (Eiii) of isoproterenol. Eiii insert shows increase in median lifetime of CCS lifetime on isoproteronol challenge for four cells.
Mentions: We next focused on a more detailed analysis of spot-like CCS lifetimes, since the dynamics of these structures are most readily analyzed, and the lifetime of spot-like coated pits was used previously as a readout of clathrin-coated pit maturation (Ehrlich et al., 2004; Puthenveedu and von Zastrow, 2006; Loerke et al., 2009; Henry et al., 2012). To determine the maturation time of productive clathrin-coated buds, we tracked Mu2-mCherry labeled CCSs and mapped them to bona fide scission events (Merrifield et al., 2005). As shown previously for TfR in fibroblasts (Merrifield et al., 2005; Taylor et al., 2011, 2012), spot-like CCSs most commonly hosted only one scission event but could potentially host multiple scission events (Figure 3, A and B). Strikingly, many scission events coincided with partial dimming but not complete disappearance of the host CCS, and repeated trains of partial dimming resulted in characteristic sawtooth-type profiles of Mu2-mCherry fluorescence (Figure 3A, right pair of traces). As described previously for NIH fibroblasts (Merrifield et al., 2005; Taylor et al., 2011), “nonterminal” events, for which the host CCS did not completely disappear after scission, comprised a significant proportion of the total number of scission events (∼50%). By definition, terminal events coincided with the final disappearance of the host spot-like CCS (Figure 3A).

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