Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling

Abstract

Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.

Keywords: AP2; EGFR; clathrin-coated pits; endocytic adaptors; endocytosis; eps15; epsin; receptor degradation; recycling; signaling; transcription.

Graphical abstract

Figure 1

Live TIRF Imaging of CCPs in SUM159 and AP2-WT MEF Cells (A) Cumulative frequency distribution of the initial mean square displacement (MSD) of clathrin-coated structures containing or not AP2 in SUM159 cells imaged by TIRF. Clathrin events with initial MSD larger than 0.01 μm² (dotted line) were excluded from the plots displaying fluorescence intensity cohorts (B). (B) Automated analysis of clathrin-coated structure formation at the plasma membrane of SUM159 cells obtained from 13 cells and ∼350 clathrin traces. The analysis for traces excluding events with lifetimes < 20 s shows average fluorescence intensity plots (mean ± SD) grouped as cohorts according to their lifetimes. The left panel corresponds to 235 events containing CLTA-TagRFP and AP2-σ2-EGFP (clathrin+/AP2+), while the right panel corresponds to 112 events devoid of AP2-σ2-EGFP (clathrin+/AP2−). (C) Cumulative frequency distribution of the initial MSD of clathrin-coated structures containing or not AP2 in MEF AP2-WT cells imaged by TIRF. Clathrin events with initial MSD larger than 0.01 μm² (dotted line) were excluded in the plots displaying fluorescence intensity cohorts (D). (D) Automated analysis of clathrin-coated structure formation at the plasma membrane from 8 cells and ∼670 clathrin traces from MEF WT cells. (E) Representative TIRF microscopy time series acquired every 2 s from the bottom surface of MEF cells, stably expressing clathrin light chain A tagged with TagRFP (CLTA-TagRFP) together with σ2 of AP2 tagged with EGFP (AP2σ-EGFP). The TIRF snapshots (left) were recorded at 224 and 138 s, and the corresponding right panels are kymographs from the complete time series. The yellow tracings display the path used to generate the kymographs. The green channels in the kymographs were shifted upward by 5 pixels. The majority of the endocytic clathrin structures contained clathrin together with AP2 in the WT cells (e.g., pits 1 and 4), and few contained only clathrin (e.g., pits 2 and 3).

Figure 2

Live TIRF Imaging of CCPs in AP2 KO MEF Cells (A) MEFs from conditional AP2μ^fl/fl mice (Figure S2A) were treated in vitro with CRE recombinase, as indicated, followed by immunoblotting (IB) as shown. The lower band in the AP2μ IB is nonspecific; the specific AP2μ band is indicated by an arrow. In all subsequent experiments, AP2μ^fl/fl MEFs were either left untreated or treated with CRE for 14 days-two rounds (henceforth referred as AP2-WT and AP2-KO, respectively). (B) AP2-WT and AP2-KO MEFs were analyzed for mRNA levels of Ap2m1 and Cltc using qRT-PCR. mRNA levels are reported relative to untreated controls and normalized to the 18S gene. Error bars are calculated on technical replicates (n = 3). (C) Cumulative frequency distribution of the initial MSD of clathrin-coated structures in MEF AP2-WT and AP2-KO cells imaged by TIRF. Clathrin events with initial MSD larger than 0.01 μm² (dotted line) were excluded in the plots displaying fluorescence intensity cohorts (D). (D) Automated analysis of clathrin-coated structure formation at the plasma membrane from 12 cells and ∼439 clathrin traces from MEF KO cells. (E) Representative TIRF microscopy time series acquired every 2 s from the bottom surface of MEF AP2-KO cells, stably expressing CLTA-TagRFP together with AP2σ-EGFP. The TIRF snapshots (left) were recorded at 224 and 138 s, and the corresponding right panels are kymographs from the complete time series. The yellow tracings display the path used to generate the kymographs. The green channels in the kymographs were shifted upward by 5 pixels. Endocytic “clathrin-only” structures are present (e.g., pits 1 and 2).

Figure 3

Morphological Characterization of CCPs in AP2-WT and AP2-KO Cells (A) Plasma membrane sheets (PMSs) of AP2-WT and AP2-KO MEFs showing examples of clathrin-coated structures (arrowheads, flat clathrin lattices; big arrows, CCPs). Bar, 100 nm. (B) Top: CCS density in AP2-WT and AP2-KO MEFs. Bottom: CCS number was normalized for surface area (Figure S3A; STAR Methods) and expressed relative to control cells. N represents the number of random images analyzed. Data are represented as mean ± SEM. p values were calculated using two-tailed Student’s t test (^∗∗∗p < 0.001). (C) Left: size distribution of CCSs in AP2-WT and AP2-KO MEFs (STAR Methods; Grove et al., 2014). Right: analysis of distribution of CCP areas in AP2-WT and AP2-KO MEFs. Only CCPs < 0.03 μm² were included in the analysis. N represents the number of CCSs analyzed. p values were calculated using two-tailed Student’s t test (^∗∗∗p < 0.001). (D) Transmission electron microscopy (TEM) analysis of CCPs in AP2-WT and AP2-KO MEFs. In AP2-KO cells, CCPs appear smaller compared with AP2-WT cells (arrows and insets), as also shown by the morphometric analysis in the right panel. N represents the number of random images analyzed. Bar, 100 nm. p values were calculated using two-tailed Student’s t test (^∗∗∗p < 0.001).

Figure 4

EGF Internalization in AP2-KO MEFs or upon AP2-KD in Different Cell Contexts (A) ¹²⁵I-EGF (left) and ¹²⁵I-Tf (right) internalization in AP2-WT and AP2-KO MEFs in the presence or absence of clathrin KD. Internalization constants (K_e) are the mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test (^∗∗∗p < 0.001). (B) Analysis of the impact of AP2μ KD versus clathrin KD in different cell lines as indicated. The number of EGFRs per cell in the different cell lines was measured using ¹²⁵I-EGF saturation binding assay. Kinetics of ¹²⁵I-EGF (1.5 ng/mL) were measured and are reported as internalization constants (K_e). The percentage of AP2-independent EGFR internalization was calculated from the residual K_e in AP2-KD cells relative to the K_e in control cells (after subtracting the residual internalization in clathrin KD cells; Sigismund et al., 2013). (C) TEM analysis of EGFR internalization. AP2-WT and AP2-KO MEFs expressing EGFR under a doxycycline-inducible promoter (AP2-EGFR) were induced with doxycycline. Cells were then subjected to in vivo immunolabeling with anti-EGFR 13A9 antibody and 10 nm protein A-gold, stimulated 5 min with EGF (30 ng/mL), and fixed in the presence of ruthenium red, to distinguish PM-connected CCPs (ruthenium red positive) and internalized CCVs (ruthenium red negative) structures. Bar, 100 nm. (D) Morphometrical analysis of (C). Left: CCP number was normalized for the difference in PM length between AP2-KO MEFs versus control (∼1.5-fold increase; see Figure S3A and STAR Methods) and expressed as relative to control cells. Right: number of gold particles per CCS (CCPs + CCVs). N represents the numbers of random images (left) and CCSs (right) analyzed. Data are expressed as mean ± SEM. p values were calculated using two-tailed Student’s t test (^∗p < 0.05 and ^∗∗p < 0.01).

Figure 5

Mechanism of AP2-Independent EGFR-CME: Role of eps15/L1 and epsin1 (A) Transient KD of the μ subunit of the indicated AP complexes was performed in AP2-KO MEFs, and K_e of ¹²⁵I-EGF internalization was calculated. Data are mean ± SD (two replicates). (B) K_e of ¹²⁵I-EGF internalization in stable eps15/eps15L1-KD HeLa cells transiently depleted of epsin1, alone or in combination with AP2μ, in comparison with AP2-KD and clathrin-KD HeLa cells. Results are mean ± SD of two to eight independent experiments. p values were calculated using each pair Student’s t test (^∗∗p < 0.01 and ^∗∗∗p < 0.001). (C) AP2-WT and AP2-KO MEFs were transiently depleted as indicated. K_e values of ¹²⁵I-EGF internalization are shown as mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test (^∗∗p < 0.01 and ^∗∗∗p < 0.001). (D) TEM analysis of CCSs in AP2-WT-EGFR MEFs, control or triple KD for eps15, eps15L1, and epsin1, induced with doxycycline (to allow EGFR expression) and stimulated with EGF (30 ng/mL). Morphometrical analysis was performed on EGFR gold-positive structures. Left: CCP number was normalized for the difference in PM length between eps15/eps15L1/epsin1 KD MEFs versus control (∼0.8 decrease; see Figure S3A and STAR Methods) and expressed relative to control cells. Right: mean number of gold particle per CCS (CCPs + CCVs). N represents the numbers of random images (left panel) and numbers of CCSs (center and right panels) analyzed. Data are expressed as mean ± SEM. p values were calculated using each pair Student’s t test (^∗p < 0.05). (E) Automated analysis of clathrin-coated structure formation at the plasma membrane from 196 traces containing CLTA-TagRFP and AP2σ-EGFP (clathrin+/AP2+; left panel), 200 traces devoid of AP2σ-EGFP (clathrin+/AP2−; middle panel) from 10 MEF AP2-WT/triple-KD, or 154 traces from 10 MEF AP2-KO/triple-KD cells (right panel).

Figure 6

EGF-Dependent Signaling and Migration in AP2-Depleted Cells (A) HeLa cells were subjected to AP2 KD or eps15/L1/epsin1 KD followed by ¹²⁵I-EGF degradation assay at low EGF concentration (1.5 ng/mL; see STAR Methods). At the indicated time points, (1) degraded EGF (top) represents the TCA soluble fraction of ¹²⁵I-EGF recovered in the medium and intracellularly, and (2) recycled EGF (bottom) represents the TCA insoluble fraction of ¹²⁵I-EGF recovered in the medium. Results are mean ± SD of two independent experiments. p values were calculated using each pair Student’s t test (^∗p < 0.05 and ^∗∗p < 0.01). (B) Top: AP2-WT and AP2-KO MEFs were stimulated with low dose EGF (1.5 ng/mL) for the indicated times. Lysates were subjected to IB with the indicated antibodies. Bottom: quantitation of phosphoAKT signal normalized to total AKT (pAKT/AKT) and represented as percentage of signal in WT cells at 5 min of EGF stimulation. Results are mean ± SD of three independent experiments. p values were calculated using two-tailed Student’s t test (^∗p < 0.05 versus WT at each time point). (C) Left: HeLa cells were subjected to AP2 KD and/or eps15/L1/epsin1 KD in different combinations, followed by stimulation with low dose EGF (1.5 ng/mL) for the indicated times. IB was as shown. Right: quantitation of phosphoAKT signal normalized to total AKT (pAKT/AKT) and represented as percentage of signal in control cells at 5 min of EGF stimulation. Results are mean ± SD of three independent experiments. p values were calculated using two-tailed Student’s t test. ^∗p < 0.05 versus control at each time point. (D) Transwell migration assay of AP2 KD and eps15/L1/epsin1 KD HeLa cells under serum-starved (SS) conditions or in the presence of low-dose EGF (1.5 ng/mL) or 10% serum as indicated. Right: quantitation of migrating cells per field. Results are the mean ± SD of two to four independent experiments. p values were calculated using two-tailed Student’s t test (^∗p < 0.05 versus control in each condition).

Figure 7

EGF-Dependent Transcriptional Output in AP2-Depleted Cells (A) Control or AP2 KD HeLa cells were serum starved overnight followed by stimulation with low-dose EGF (1.5 ng/mL) or with complete medium (10% serum) for the indicated time points. Reported is the mRNA level, assessed by RT-qPCR, of the indicated genes, relative to the unstimulated control and normalized to the levels of the RPLP0 housekeeping gene. Mean ± SD of two independent experiments with duplicates points is shown. (B) Control or AP2 KO MEFs were serum starved overnight, followed by stimulation with low-dose EGF (1.5 ng/mL) for the indicated time points. Data are expressed as in (A). Mean ± SD of three independent experiments with duplicate points is shown. p values in (A) and (B) were calculated using each pair Student’s t test (^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001).