Local clustering of transferrin receptors promotes clathrin-coated pit initiation

Abstract

Clathrin-mediated endocytosis (CME) is the major pathway for concentrative uptake of receptors and receptor-ligand complexes (cargo). Although constitutively internalized cargos are known to accumulate into maturing clathrin-coated pits (CCPs), whether and how cargo recruitment affects the initiation and maturation of CCPs is not fully understood. Previous studies have addressed these issues by analyzing the global effects of receptor overexpression on CME or CCP dynamics. Here, we exploit a refined approach using expression of a biotinylated transferrin receptor (bTfnR) and controlling its local clustering using mono- or multivalent streptavidin. We show that local clustering of bTfnR increased CCP initiation. By tracking cargo loading in individual CCPs, we found that bTfnR clustering preceded clathrin assembly and confirmed that bTfnR-containing CCPs mature more efficiently than bTfnR-free CCPs. Although neither the clustering nor the related changes in cargo loading altered the rate of CCP maturation, bTfnR-containing CCPs exhibited significantly longer lifetimes than other CCPs within the same cell. Together these results demonstrate that cargo composition is a key source of the differential dynamics of CCPs.

Figure 1.

Experimental system for TfnR clustering. (A) Adenoviral bicistronic construct containing TfnR-AP and BirA-ER for tetracycline-repressible expression of bTfnRs. The Lys residue (marked in blue) within the AP is biotinylated by BirA-ER. (B) Schematic of site-specific biotinylation of TfnR. TfnRs with C-terminal acceptor peptide (AP) are biotinylated (bTfnR) by a coexpressed and ER-localized biotin ligase BirA. (C) SDS-PAGE of mixed streptavidin (SA) and purified heterotetrameric SA (eluted from a Ni-NTA column). The open circles and circles with a cross inside denote the A (active) and D (inactive/dead) monomers, respectively. (D) LCa-EGFP–expressing BSC1 cells infected with adenovirus encoding the TfnR-AP construct show colocalization of bound streptavidin with LCa-EGFP–labeled CCPs. Insets: enlarged channel separation of region highlighted by the white square indicating colocalization of streptavidin and LCa-EGFP (arrows). (E) Uptake of AF568-A1 in bTfnR-expressing cells in the presence or absence of 50 µg/ml Tfn assayed by flow cytometry (n = 4, average ± SD).

Figure 2.

Streptavidin clusters bTfnR at the plasma membrane. (A) Immunoblot of lysates from bTfnR-expressing cells treated with either A1 or A4 for 10 min and probed with αSA-HRP to identify SA-bound bTfnR. (B) Intensity profiles of the immunoblot in A showing multiple higher molecular weight species (arrows) in A4-treated cells. (C) Electron micrographs of the ventral surface of un-roofed bTfnR-expressing cells treated with either A1 (topl) or A4 (bottom). TfnRs were labeled with D65 immunogold particles. Small circles indicate pairs of gold particles bound to TfnR dimers; large circles indicate clusters of gold particles, exclusively found in A4-treated cells. Insets: enlarged areas highlighted by the dotted squares. (D) Thin-section electron micrographs of bTfnR-expressing cells treated with either A1 (top two panels) or A4 (bottom two panels). Representative micrographs of shallow CCPs show more gold particles in CCPs in cells treated with A4, as summarized in the table.

Figure 3.

Effect of clustering on the variation of cargo distribution within individual CCPs. (A) BTfnR-expressing cells were incubated in the presence of AF568- or AF647-labeled A1 (top panels) or A4 (bottom panels). Merged images show the extent of colocalization of the differentially labeled SA ligands. (B) Representative scatter plots of the intensities in fluorescent puncta containing both A1s (top) or both A4s (bottom) from a single cell. The spread of the intensities is larger for A4 than for A1, as indicated by the square of the correlation coefficient. (C) Simulation of intensity scatter plot assuming a uniform distribution of cargo capacity and a binomial distribution of colors. (D) Theoretical estimate of maximum cargo capacity based on correlation coefficients.

Figure 4.

CCP density increases with bTfnR clustering. (A) Box-plot of CCP density in bTfnR-expressing cells incubated with different SAs (at least 10 regions of interest from different cells). (B) Initiation density of CCPs as determined from TIRF imaging of bTfnR-expressing cells (n = 10–16) incubated with different SAs. *, P < 0.05 for t test. (C) Accumulation of AF568-A1 and LCa-EGFP in A1-treated cells in five lifetime cohorts (10–19, 20–39, 40–59, 60–79, and 80–99 s) within the ensemble of CCPs. AF568A1 (dotted line) and LCa-EGFP (blue line) accumulated together (yellow region). (D) Accumulation of AF568-A4 and LCa-EGFP in A4-treated cells. AF658A4 (dotted line) was found to precede clathrin assembly (red line), as highlighted in the yellow region. A1: N_CCP = 28,382; N_cell = 5. A4: N_CCP = 46,508; N_cell = 9.

Figure 5.

Internalization of A4 is more rapid than A1. (A) Uptake assay of AF647 A4 (blue) and A1 (red) in bTfnR-expressing LCa-EGFP BSC1 cells measured by flow cytometry (n = 7, average ± SD). For each experiment, the fraction internalized was normalized to the uptake of A1 at 10 min in order to compare multiple experiments. *, P < 0.05 by paired t test. (B) Competition of AF647 A1 uptake by the presence of different unlabeled SAs as measured by flow cytometry of 10,000 cells per condition. Error bars denote SEM.

Figure 6.

Distinct dynamic behaviors of bTfnR-containing CCPs. (A) Segregation of CCPs into bTfnR-containing and residual CCPs. (Left panel) Shown is the accumulation of AF568-A1 in five lifetime cohorts within the ensemble of EGFP-Cla–labeled CCPs. (Right panel) Cargo-based deconvolution was used to parse ensemble CCPs into either bTfnR-containing (blue) or residual CCPs (dotted). (B and C) Fraction of CCPs found in each lifetime cohort for bTfnR-containing and residual CCPs for A1- and A4-treated cells, respectively. The fraction unaccounted for in the summations are CCPs with lifetimes >100 s. All pairwise comparison CCP fractions in all lifetime cohorts were significantly different as determined by paired t test (P < 0.05), except for the 40–60-s cohort for A1-treated cells. (D and E) Survival functions of CCPs in cells expressing bTfnR and incubated with either A1 (D, blue) or A4 (E, red). BTfnR-containing and residual CCPs are shown in solid and dotted lines, respectively. A1: N_CCP = 28,382; N_cell = 5. A4: N_CCP = 46,508; N_cell = 9.

Figure 7.

BTfnR-containing CCPs recruit more AP-2 complexes and are larger than residual CCPs. Intensity analyses based on the LCa-EGFP (A and B) or σ2-EGFP (C and D) channels in cells incubated with either A1 (A and C; blue) or A4 (B and D; red). BTfnR-containing CCPs (solid lines) were parsed out from residual CCPs (dotted lines) for five lifetime cohorts: 11–20, 21–40, 41–60, 61–80, and 81–100 s. (E) Maximum σ2-EGFP intensity plateau for each cohort (average of top five intensities ± SD) of residual (shaded blue) and bTfnR-containing (solid blue) CCPs for A1-treated cells. (F) Maximum σ2-EGFP intensity plateau of CCP cohorts in A1- (blue) and A4 (red)-containing CCPs. For LCa-EGFP, A1: N_CCP = 28,382; N_cell = 5. A4: N_CCP = 46,508; N_cell = 9. For σ2-EGFP, A1: 25,670; N_cell = 6. A4: 21,028; N_cell = 6. **, P < 0.01.