Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2

Abstract

The critical initiation phase of clathrin-mediated endocytosis (CME) determines where and when endocytosis occurs. Heterotetrameric adaptor protein 2 (AP2) complexes, which initiate clathrin-coated pit (CCP) assembly, are activated by conformational changes in response to phosphatidylinositol-4,5-bisphosphate (PIP2) and cargo binding at multiple sites. However, the functional hierarchy of interactions and how these conformational changes relate to distinct steps in CCP formation in living cells remains unknown. We used quantitative live-cell analyses to measure discrete early stages of CME and show how sequential, allosterically regulated conformational changes activate AP2 to drive both nucleation and subsequent stabilization of nascent CCPs. Our data establish that cargoes containing Yxxφ motif, but not dileucine motif, play a critical role in the earliest stages of AP2 activation and CCP nucleation. Interestingly, these cargo and PIP2 interactions are not conserved in yeast. Thus, we speculate that AP2 has evolved as a key regulatory node to coordinate CCP formation and cargo sorting and ensure high spatial and temporal regulation of CME.

Figure 1.

AP2 mutants and TIRFM-based assays used in this study. (A) Table listing the in vitro biochemical phenotypes of mutant AP2 subunits, their designations, and the residues mutated. (B) Schematic representation showing the positions of mutated residues with respect to the closed (left) and open (right) conformations of AP2. (C) Schematic representation of the CCP parameters measured by TIRF imaging and quantitative image analysis in this work. Tyr, tyrosine.

Figure 2.

Binding of PIP2 to the α subunit is essential for allosteric activation of AP2 to trigger clathrin polymerization and CCP initiation. (A) Schematic representation of the possible early roles of α–PIP2 interactions. The question mark and double arrowheads point to potential roles in enhancing the rates and or extents of clathrin polymerization, CCP nucleation, cargo recruitment, and CCP maturation. (B) Initiation density of all detected subthreshold CLSs and bona fide CCPs for the indicated wt or mutant cells (≥15 cells per condition, #CCPs α^wt 32,320, #CCPs α^PIP2− 20,510). Box plots show medians, 25th and 75th percentiles, and outermost data points. ***, P ≤ 0.001, unpaired t test. (C) Mean clathrin fluorescence intensity traces of lifetime cohorts of CCPs from α^wt (gray) and α^PIP2− (blue) cells. Intensities are shown as mean ± SE calculated from ≥15 cells per condition. (D) The slope of intensity trace (averaged in the time interval 3–8 s of the elapsed lifetime) in α^wt and α^PIP2− cells. A.U., arbitrary units; Fluo., fluorescence.

Figure 3.

Sustained binding of PIP2 to AP2 α subunit is required for CCP maturation. (A) Fraction of CCPs found in short-lived versus longer-lived lifetime cohorts in α^WT (gray) and α^PIP2− (blue) cells. Data shown are mean ± SD (n > 100,000 CCPs from three independent experiments); n.s., not significant; ***, P < 0.001. Lifetime distributions of all bona fide CCPs (black lines), dynamin-2 (DYN2)-positive CCPs (green lines), and DYN2-negative CCPs (blue lines) in α^WT (B) and α^PIP2− (C) cells. (D) The ratio of epifluorescence (EPI):TIRF intensity levels for individual CCPs is indicative of curvature acquisition. EPI:TIRF ratio for individual CCPs plotted as a function of CCP lifetime in α^WT (E) and α^PIP2− (F) cells. Heatmap indicates frequency. EM images of “unroofed” htertRPE cells reconstituted with either α^WT (G) or α^PIP2− (H). Bottom panels show higher-magnification view of the indicated area. Bars: (top) 500 nm; (bottom) 200 nm.

Figure 4.

Binding of PIP2 to AP2 β subunit is equally essential for efficient CCP initiation and clathrin polymerization. (A) Initiation density of subthreshold CLSs and bona fide CCPs for the indicated wt or mutant cells (18 cells per condition). Box plots show medians, 25th and 75th percentiles, and outermost data points. ***, P ≤ 0.001, unpaired t test. (B) Mean clathrin fluorescence intensity traces in lifetime cohorts of CCPs from β2^wt (gray) and β2^PIP2- (blue) reconstituted cells. Intensities are shown as mean ± SE calculated from 18 cells per condition. A.U., arbitrary units; Fluo., fluorescence. (C) Fraction of CCPs found in short-lived versus longer-lived cohorts for β^WT (gray) and β^PIP2− (blue) cells. ***, P < 0.001. (D) Transferrin receptor internalization was measured in β^WT, α^PIP2−, and β^PIP2− cells using a monoclonal anti-TfnR antibody as ligand. Percentage of TfnR uptake was calculated relative to the initial total of surface-bound antibody. Data represent mean ± SD, n = 4. ***, P ≤ 0.005, unpaired t test.

Figure 5.

Binding of PIP2 to the µ2 subunit is required downstream of initial AP2 activation for later stages of CCP maturation. (A) Schematic representation of the possible early roles of α–PIP2 interactions. The question mark and double arrowheads point to potential roles in enhancing the rates and or extents of clathrin polymerization, CCP nucleation, cargo recruitment, and CCP maturation. (B) Initiation density of all subthreshold CLSs and bona fide CCPs for the indicated wt or mutant cells (≥22 cells per condition, #CCPs µ2^wt 27,943, #CCPs µ2^PIP2− 36,523). Box plots show medians, 25th and 75th percentiles, and outermost data points. ***, P ≤ 0.0005, unpaired t test. n.s., not significant. (C) Mean clathrin fluorescence intensity traces in lifetime cohorts of CCPs from µ2^wt (gray) and µ2^PIP2− (blue) reconstituted cells. Intensities are shown as mean ± SE calculated from 17 cells per condition. (D) Slope of intensity trace (averaged in the time interval 3–8 s of the elapsed lifetime) in µ2^wt (gray) and µ2^PIP2− (blue) cells. (E) Fraction of CCPs found in short-lived versus longer-lived lifetime cohorts in µ2^WT (gray) and µ2^PIP2− (blue) cells. ***, P < 0.001. (F) Lifetime distributions of all bona fide CCPs (black lines), dynamin-2 (DYN2)-positive CCPs (green lines), and Dyn2-negative CCPs (blue lines) in µ2^PIP2− cells.

Figure 6.

Increased phosphorylation of µ2 at T156 compensates to maintain efficient TfnR internalization in µ2^PIP2− cells. (A) Schematic representation of the potential roles for AAK1 activation and phosphorylation of T156 on µ2. The question mark and arrows point to possible compensatory mechanisms and a positive feed-forward loop that can restore efficient CME in µ2^PIP2− cells. (B) Quantification (mean ± SD, n = 3; two-tailed Student’s t tests were used to assess statistical significance: *, P < 0.05) of total and phosphorylated µ2^pT156 subunit in µ2^WT, µ2^PIP2-, and α^PIP2− cells (see also Fig. S5 B). (C) TfnR uptake measured at 10 min in control, µ2^PIP2−, and α^PIP2− cells with or without treatment with the 10-µM AAK1 inhibitor (inhib), Compound 2. Data shown are mean ± SD, n = 3; normalized to total surface bound; *, P < 0.05; ***, P < 0.0005. n.s., not significant.

Figure 7.

Activation of AP2 by binding to YXXφ sorting signals is necessary for CCP nucleation. (A) Schematic representation of the potential role of σ2 and µ2 interactions with their cognate (diLeu and YXXφ, respectively) or orthogonal cargo on AP2 activation and CCP nucleation. (B) Internalization of CD8 chimeras containing either the YXXφ (cognate for µ2, orthogonal for σ2) or diLeu (cognate for σ2, orthogonal for µ2) sorting signals was followed using CD8 mAb (data shown are mean ± SD, n = 3; normalized to total surface bound; **, P < 0.05; ***, P < 0.005). (C) Heatmap representing the changes in Pearson correlation coefficient (PC) between EGFP-CLCa, respective CD8 chimeras, and other CME cargo showing efficiency of cargo loading into CCPs in µ2^cargo− and σ2^cargo− cells. (D) Initiation density of all detected CLSs and bona fide CCPs for the indicated wt or mutant cells (≥35 cells per condition, #CCPs µ2^wt 45,054, #CCPs µ2^cargo− 38,120). Box plots show medians, 25th and 75th percentiles, and outermost data points. ***, P < 0.0005; ****, P < 0.0001, t test. n.s., not significant. (E) Mean clathrin fluorescence (fluo.) intensity traces in lifetime cohorts of CCPs from µ2^WT (gray) and µ2^cargo− (blue) reconstituted cells. Intensities are shown as mean ± SE calculated from 20 cells per condition. A.U., arbitrary units. (F) Slope of intensity trace (averaged in the time interval 3–8 s of the elapsed lifetime) in µ2^wt (gray) and µ2^cargo− (blue) cells.

Figure 8.

Selective role of YXXφ-bearing cargo for AP2 activation and model for sequential allosteric regulation of AP2 by PIP2 and cargo interactions. (A) Overexpression of CD8 chimera containing a diLeu sorting motif does not rescue initiation of CCPs in µ2^cargo− cells (≥16 cells per condition; CCPs µ2^wt 18,235, #CCPs µ2^cargo− 16,573, #CCPs µ2^cargo−[CD8-EAAALL] 15,836). n.s., not significant. (B) Bioinformatics search for human transmembrane proteins containing YXXφ- or diLeu-based sorting motif revealed overrepresentation of YXXφ motif–bearing cargo. (C) Model for regulation of CCP nucleation and maturation through allosteric activation of AP2 by sequential and hierarchical interactions with its ligands, PIP2, and YXXφ-bearing cargo. Initiation (step 1) involves interactions between surface-exposed PIP2-binding sites on both the α and β2 subunits, as well as interactions between YXXφ-bearing cargo and the µ2 subunit to allow AP2 to recruit clathrin. Rapidly assembling clathrin (step 2) can activate AAK1 kinase, which phosphorylates µ2, creating a positive feed-forward loop that drives efficient CCP maturation. Full activation of AP2, which is required for CCP growth and stabilization, occurs when the µ2 subunit also engages PIP2. Early CCP intermediates formed by AP2 mutants defective in α–, β2–, or µ2–PIP2 binding are impaired in curvature generation and the recruitment of dynamin-2 (Dyn2) and exhibit a greater tendency to abort.