Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis - PubMed (original) (raw)

Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis

Marcel Mettlen et al. J Cell Biol. 2010.

Abstract

Clathrin-mediated endocytosis of surface receptors and their bound ligands (i.e., cargo) is highly regulated, including by the cargo itself. One of the possible sources of the observed heterogeneous dynamics of clathrin-coated pits (CCPs) might be the different cargo content. Consistent with this, we show that CCP size and dynamic behavior varies with low density lipoprotein receptor (LDLR) expression levels in a manner dependent on the LDLR-specific adaptors, Dab2 and ARH. In Dab2-mCherry-expressing cells, varying LDLR expression leads to a progressive increase in CCP size and to the appearance of nonterminal endocytic events. In LDLR and ARH-mCherry-expressing cells in addition to an increase in CCP size, turnover of abortive CCPs increases, and the rate of CCP maturation decreases. Altogether, our results underscore the highly dynamic and cargo-responsive nature of CCP assembly and suggest that the observed heterogeneity is, in part, related to compositional differences (e.g., cargo and adaptors) between CCPs.

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Figures

Figure 1.

Figure 1.

Dab2/LDLRs regulate CCP size. BSC1 cells stably expressing LCa-EGFP were transfected with Dab2-mCherry and infected with adenoviruses coding for a tet-repressible LDLR expression system. Cells were cultured in the presence of decreasing concentrations of tet, fixed/permeabilized, and observed by epifluorescence or electron microscopy. [tet]: (A–D) 1,000 ng/ml, no CD8/LDLR; (E–H): 25 ng/ml, low CD8/LDLR; arrowheads: normal-sized CCPs; arrows: enlarged CCPs (I–L): 0 ng/ml, high CD8/LDLR. Bars, 10 µm. (D, H, and L) Encircled areas indicate clathrin lattices at the ventral membrane of BSC-1 cells. Insets show normal-sized CCPs in each condition.

Figure 2.

Figure 2.

Requirements for Dab2/LDLR-induced GCCS formation. BSC1-wt cells were transfected with either myristoylated Dab2-mCherry (A) or Dab2-p67-mCherry (B and C) and were infected with adenoviruses coding for CD8/LDLR as indicated. Cells were fixed, permeabilized, immunolabeled in green for clathrin heavy chain (CHC; B and C), and observed by epifluorescence. Bars, 10 µm.

Figure 3.

Figure 3.

GCCSs are dynamic and, at moderate CD8/LDLR levels, generate nonterminal endocytic events. BSC1 cells were cultured as described in Fig. 1 and observed by dual-color TIRF-FM (A, Bb, C), electron microscopy (Ba), or spinning disk confocal microscopy (D). (A) Montage shows lifetime of a CCP. Arrowhead indicates nonterminal event. (B) In the presence of mild CD8/LDLR expression, CCPs tend to cluster and undergo nonterminal endocytic events. (C) GCCSs are dynamic and change morphology. (D) A small area in GCCSs has been bleached (indicated by square) and FRAP was recorded and quantified. Bars: (A) 400 nm; (Ba) 200 nm; (Bb) 600 nm; (C and D) 2 µm.

Figure 4.

Figure 4.

ARH localization is cargo dependent. BSC1 cells stably expressing ARH-mCherry were infected with adenoviruses coding for CD8/LDLR and cultured in the presence (A and B) or absence (C and D) of tet. Cells were then fixed, permeabilized, immunolabeled as indicated, and observed by epifluorescence. Insets show enlarged areas. (E) BSC1 cells coexpressing Dab2-mCherry, ARH-EGFP, and CD8/LDLR were fixed and visualized by spinning disk confocal microscopy. Bars, 10 µm.

Figure 5.

Figure 5.

Requirements for ARH targeting to CCPs. BSC1 cells stably expressing ARH F259A-mCherry (A and B) or ARH S117Y;F165A-mCherry (C) were left untreated (A) or infected with adenoviruses coding for CD8/LDLR (B and C). Cells were then fixed, permeabilized, immunolabeled as indicated, and observed by epifluorescence. Bars, 10 µm. (D) Indicated cell lines were cultured as previously described and analyzed by Western blot after subcellular fractionation. S: cytosolic fraction (diluted 4x); P: membrane fraction. Actin served as loading control. The cell fractionation experiment has been repeated at least three times and a typical experiment is shown.

Figure 6.

Figure 6.

ARH partially rescues saturated LDLR uptake. (A) BSC1-wt cells infected with adenovirus coding for CD8/LDLR were cultured in the presence of the indicated concentrations of tet and the rate of anti-CD8 antibody internalization relative to surface bound ligand was measured at 37°C, as described in Materials and methods. (B) Time course of single round internalization of prebound anti-CD8 antibody in BSC1 cells expressing EGFP-CLCa (○, •) or mCherry-ARH (□, ■). Shown are means (±SD) of at least three independent experiments. (C) BSC-wt cells or cells stably expressing LCa-EGFP, σ2-EGFP, ARH-wt-, ARH-F259A-, or ARH-252A255-mCherry were treated (+ µ2 k.d.) or not with µ2 siRNA to reduce AP2 levels, infected with CD8/LDLR adenovirus, and incubated with or without tet as indicated. The mean efficiency CD8/LDLR uptake as determined in at least two independent experiments is shown. Of note: to follow basal endocytic uptake of CD8, minimal exogenous expression of CD8/LDLR was necessary (i.e., overnight culture in the presence of 75 ng/ml tet). ***, confidence levels of P < 0.001 relative to wt control, 0 tet.

Figure 7.

Figure 7.

CD8/LDLRs prolong the lifetimes of productive CCPs. (A) TIR-FM time-lapse movies have been recorded and analyzed as described previously (Loerke et al., 2009). (A) Relative contributions and (B) lifetimes of CCP subpopulations labeled by LCa-EGFP are shown. Error bars represent cell-to-cell variation; the height of the lifetime bars in B denotes the range around the mean lifetime that contains 50% of the data. Brackets indicate confidence levels P < 10−8 (Kolmogorov-Smirnov test). The number of CCP trajectories (n) and cells (k) for each condition are: LCa-EGFP control (n = 100,823; k = 65); LCa-EGFP + CD8/LDLR (n = 70,955; k = 38); LCa-EGFP + ARH + CD8/LDLR (n = 23,394; k = 17).

Figure 8.

Figure 8.

CD8/LDLR and adaptors affect CCP size. TIR-FM time-lapse movies have been recorded under various conditions, following the indicated fluorescently tagged protein. CCP cohorts of given lifetimes have been binned and their intensity profiles (A, B, D, and E) determined as a measure for CCP growth. The number of CCP trajectories for each condition, which ranges from 626 to 20,000, is shown in

Table S1

. The maximum plateau level of the LCa-EGFP (C) or σ2-EGFP (F) is plotted against the mean cohort lifetime for the indicated cells.

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