A-B: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Clathrin Heavy Chain (CHC), the ubiquitin ligase E3 Cbl (Cbl), Grb2, AP-1, AP-2, β-arrestin-1 (β-Arr-1), β-arrestin-2 (β-Arr-2) or Dab2. The efficiency of siRNAs was analyzed on cell extracts (40 µg of proteins) by SDS-PAGE and western blotting (Fig. S1). Cells were incubated with 500 ng/ml PA63 and 100 ng/ml LF for 1 hour at 4°C and different times at 37°C and cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA SDS-resistant heptamer (pPA^7mer) and N-terminus of MEK1 (MEK1 (N)). B: Levels of pPA^7mer and full length MEK1 were quantified using the Typhoon scanner and normalized to 1 and 100% respectively at time 0 (1 hour at 4°C). The plot represents the means of 4 independent experiments. Errors represent standard deviations. C: Hela cells were transfected 72 hours with CMG2-V5 and control siRNAs or siRNAs against AP-1, AP-2 or β-arrestin-2 (β-Arr-2). Cell extracts were subsequently analyzed by SDS-PAGE and western blotting to reveal the different forms of PA, CMG2-V5 and N-terminus of MEK1 (MEK1 (N)).

Hela cells were treated 45 min at 37°C with or without Latrunculin A prior to addition of 1 µg/ml of PA63 for 1 hr at 4°C (red) followed by different incubation times at 37°C. A: Cells were then submitted to FACS analysis. B: The plot represents the mean of the percentage of PA at the cell surface for 4 experiments. Errors represent standard deviations.

Hela cells were transfected 24 hrs with TEM8/1-HA (ABC) or TEM8/2-HA (B) or CMG2 (B) or constructs CTT (B) or CCT (B) or TEM8/1 Y383C-HA (C). Cells were then treated or not with 1 µg/ml of PA83 WT (ABC) or mutant resistant to furin cleavage (C) for 1 hr at 4°C. A: Cells were subsequently incubated 5 or 20 minutes at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Actin, Talin, Vinculin, TEM8-HA and PA. B: Cells were subsequently incubated 10 minutes at 37°C. Immunoprecipitates against HA were analyzed by SDS-PAGE and western blotting against Vinculin, Actin and HA. C: Cells were subsequently incubated 10 minutes at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Actin, TEM8-HA and PA.

CHO^ΔATR cells were transfected 48 hrs with TEM8/1-HA (A) or TEM8/2-HA (B) or CMG2/4-V5 (C) or empty pCDNA3 plasmid (control). Cells were then treated 45 min at 37°C with (filled symbols) or without (open symbols) Latrunculin A, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. Total cell extracts were either analyzed directly by SDS-PAGE and western blotting against PA or first treated 10 min at room temperature with acid buffer to reveal the total PA63 heptameric population (surface + intracellular: dashed lines). PA^7mer and pPA^7mer levels were quantified using the Typhoon scanner and normalized to 1 at the level at time 0 (1 hour at 4°C). The plot represents the mean of 3 independent experiments. Errors represent standard deviations.

A: Hela cells were transfected 72 hrs with control siRNAs or siRNAs against Clathrin Heavy Chain (CHC), the dynamin 2 (Dyn-2) or Epidermal growth factor receptor pathway substrate 15 (Eps 15). Cells were incubated with 500 ng/ml PA63 for 1 hr at 4°C and different times at 37°C and cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA monomer and SDS-resistant heptamer (pPA^7mer). B: Cell extracts (40 µg of protein) from Raw, BHK and Hela–transfected or not with mouse CMG2-4–cells were analyzed by SDS-PAGE and Western blotting for the expression of CMG2. C: Stable BHK21-tTA/anti-CHC cells maintained in tetracycline (control cells) or not (CHC AS) were incubated with 500 ng/ml PA63 for 1 hr at 4°C and different times at 37°C and extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal PA63 and SDS-resistant heptamer (pPA^7mer). DE: Hela cells were transfected 72 hrs with human CMG2-V5 and with control siRNAs or siRNAs against CHC. Cells were then treated as in A. Cells extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal the different forms of PA, CMG2-V5 and the N-terminus of the LF target MEK1 (MEK1 (N)).

A: Hela cells were transfected 72 hours with control siRNAs or siRNAs against Cbl, AP-1, AP-2, β-arrestin-1 (β-Arr-1), or β-arrestin-2 (β-Arr-2). Cells were treated with 1 µg/ml of PA63 for 1 hr at 4°C (red) and 30 min at 37°C (blue) and subsequently submitted to FACS analysis. B: Hela cells were transfected 72 hrs with TEM8/1-HA and with control siRNAs or siRNAs against β-arrestin 2. Cells were then treated or not with 1 µg/ml of PA63 WT for 1 hr at 4°C and different times at 37°C. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and western blotting against Ubiquitin, TEM8-HA and PA. C: Hela cells were transfected or not for 24 hrs with TEM8/1-HA. Immunoprecipitates against β-arrestin were analyzed by SDS-PAGE and western blotting against Cbl, TEM8-HA and β-arrestin. D: Hela cells were transfected 72 hours with human CMG2-HA and with control siRNAs or siRNAs against β-arrestin 2. Cells were then treated or not with 1 µg/ml of PA63 WT for 1 hr at 4°C and different times at 37°C. Immunoprecipitates against CMG2-HA were analyzed by SDS-PAGE and western blotting against Ubiquitin, CMG2-HA and PA.

A: Hela cells were treated 45 min at 37°C with or without Latrunculin A, prior to the addition of 500 ng/ml of PA63 and 100 ng/ml LF for 1 hr at 4°C followed by different incubation times at 37°C. Cell extracts (40 µg of proteins) were analyzed by SDS-PAGE and western blotting to reveal pPA^7mer, PA63 and the N-terminus of MEK1 (MEK1 (N)). Tubulin is the equal loading control. B: Cell extracts (40 µg of proteins) described in A were treated 10 min at room temperature with acid buffer pH 4.5 and analyzed by SDS-PAGE and western blotting to reveal the total heptameric PA63 (PA^7mer) population and PA63 monomer.

Hela cells were transfected 48 hrs with TEM8-1-GFP (AC) or CMG2-GFP (BD) and treated (red curves) or not (blue curves) with Latrunculin A. AB: Cells were submitted to FRAP analysis in the absence of toxin treatment. CD: Cells were incubated with 3 µg/ml of mutant PA, resistant to furin cleavage and thus defective in heptamerization, at 4°C for 1 hr and subsequently warmed back to room temperature, in the presence or absence of latrunculin A, for FRAP analysis. Each curve is the average of at least 9 different cells. Error bars represent standard deviations of the mean at each time point.

TEM8-1 is pre-organized at the cell surface by the cortical actin cytoskeleton while CMG2 is not. Upon PA binding, processing and oligomerization, the toxin receptor complex moves to lipid rafts. There, β-arrestin mediates the recruitment of the E3 ligase Cbl to the cytoplasmic tail of the receptor. The ubiquinated receptor subsequently recruits the heterotetrameric adaptor AP-1 and finally clathrin. Completion of the endocytic process and pinching off of the toxin containing clathrin-coated vesicle requires both actin and dynamin.