Death-receptor activation halts clathrin-dependent endocytosis

Abstract

Endocytosis is crucial for various aspects of cell homeostasis. Here, we show that proapoptotic death receptors (DRs) trigger selective destruction of the clathrin-dependent endocytosis machinery. DR stimulation induced rapid, caspase-mediated cleavage of key clathrin-pathway components, halting cellular uptake of the classic cargo protein transferrin. DR-proximal initiator caspases cleaved the clathrin adaptor subunit AP2alpha between functionally distinct domains, whereas effector caspases processed clathrin's heavy chain. DR5 underwent ligand-induced, clathrin-mediated endocytosis, suggesting that internalization of DR signaling complexes facilitates clathrin-pathway targeting by caspases. An endocytosis-blocking, temperature-sensitive dynamin-1 mutant attenuated DR internalization, enhanced caspase stimulation downstream of DRs, and increased apoptosis. Thus, DR-triggered caspase activity disrupts clathrin-dependent endocytosis, leading to amplification of programmed cell death.

Fig. 1.

Apo2L/TRAIL induces selective cleavage of the clathrin-dependent endocytosis machinery. (a) Cells were treated at 37°C with either trimeric Apo2L/TRAIL (Colo205 and HCT8) or antibody-crosslinked, tagged Apo2L/TRAIL (BJAB and HeLa-M), and cell lysates were analyzed by immunoblot for cleavage of caspase-8 (C8), caspase-3 (C3), adaptin (AP) 2α, AP1/2β (antibody does not distinguish the AP1 and 2 isoforms), CHC, or Tf receptor (TfR). (b) Colo205 cells were treated as in a and analyzed by immunoblot for processing of specific components of various types of clathrin-associated endocytic trafficking.

Fig. 2.

Involvement of different caspases in cleavage of AP2α and CHC. (a) BJAB cells were treated with the pan-caspase inhibitor zVAD-fmk (20 μM, 30 min) followed by treatment with crosslinked Apo2L/TRAIL (1 μg/ml) and analyzed by immunoblot for processing of caspase-8, caspase-3, AP2α, and CHC. Arrows with open heads indicate cleavage products, and arrows with filled heads indicate full-length proteins. (b–d) Bax^−/− or Bax^+/− HCT116 cells or caspase-3-defecient MCF-7 cells were treated with Apo2L/TRAIL and analyzed as in a.

Fig. 3.

Pretreatment with Apo2L/TRAIL inhibits Tf endocytosis. (a and b) BJAB (a) or Colo205 (b) cells were pretreated at 37°C with or without crosslinked (a) or noncrosslinked (b) Apo2L/TRAIL for the indicated times and chilled on ice. The cells were then equilibrated on ice for 30 min with Alexa-647-conjugated Tf (⁶⁴⁷TF), and uptake at 37°C was measured by flow cytometry as described in Methods. Each endocytosis rate was derived from the slope of the initial linear phase of a 4-min uptake kinetics plot (a Inset). Rates were normalized to that observed in the absence of Apo2L/TRAIL (open circles), which was comparable to that observed when ligand was excluded from the preincubation step but present during the Tf incubation phase of the assay (filled diamonds; 0 min). (c) BJAB cells were preexposed to DMSO vehicle or zVAD-fmk for 30 min, treated for 4 h with crosslinked Apo2L/TRAIL, chilled on ice, and analyzed for ⁶⁴⁷Tf endocytosis rates as in a and b. Rates were normalized to the DMSO-treated sample (±SEM).

Fig. 4.

Characterization of DR5 endocytosis. (a and b) Colo205 cells with surface-bound ⁶⁴⁷5C7 mAb were incubated on ice for 30 min in the absence (diamonds) or presence of 10 μg/ml trimeric (squares) or crosslinked (triangles) Apo2L/TRAIL, then shifted to 37°C for the indicated time and rapidly chilled on ice. Surface fluorescence was removed by acid stripping, and DR5 uptake was quantified by flow cytometry. Mean values were plotted (±SEM in b). (c) HeLa-M cells were incubated at 37°C with 5 μg/ml ⁶⁴⁷5C7 and 5 μg/ml crosslinked Apo2L/TRAIL, then processed for immunofluorescence microscopy. (Scale bar: 20 μM.) (d) Colo205 cells were preequilibrated at 37°C with unlabeled mAb 5C7 for 30 min to bind surface and recycling DR5 pools. Incubations were continued with or without 10 μg/ml Apo2L/TRAIL, and the cells were rapidly chilled on ice. Cell-surface-exposed mAb 5C7 was probed with CY5 anti-mouse IgG and quantified by flow cytometry, and means were plotted (±SEM). (e) Colo205 cells were pretreated at 37°C with 10 μg/ml Apo2L/TRAIL, then rapidly chilled on ice and assayed for endocytosis as in a (⁶⁴⁷5C7) and Fig. 3b (⁶⁴⁷Tf). Endocytosis rates were normalized to the value without Apo2L/TRAIL pretreatment (±SEM). Similar results were observed when cells were prepared as in c, and endocytosis was assayed with surface-bound CY5 anti-mouse Fab (data not shown). (f–h) Colo205 cells with surface-bound mAb 5C7 were incubated on ice with Apo2L/TRAIL, shifted to 37°C for 5 min, and fixed. Ultrathin cryosections were labeled with rabbit anti-mouse IgG antibodies and Protein A gold (10 nm). The typical electron-dense clathrin coat is indicated by arrowheads. P, plasma membrane. (Scale bars in f–h: 200 nm.)

Fig. 5.

Dynamin inactivation inhibits DR5 endocytosis. (a–d) Dyn^G273D-transduced HeLa-M cells were puromycin-selected, doxycycline-induced, preincubated for 20 min at the indicated temperature, and incubated another 20 min in the presence of Alexa-488-conjugated Tf (⁴⁸⁸Tf). Cells were then processed for immunofluorescence microscopy by using a dynamin-1-specific antibody. (e and f) Nontransduced (parental) or clonal Dyn1^G273D-transduced BJAB cells with (dox) or without (no dox) doxycycline induction were assayed for ⁴⁸⁸Tf or ⁶⁴⁷5C7 uptake over a 20-min period at 30°C (open bars) or 38°C (filled bars) by flow cytometry as described in Methods, and means were plotted (±SD).

Fig. 6.

Dynamin inactivation augments DR-mediated caspase activation and apoptosis. (a) Dyn^G273D-transduced BJAB cells with or without doxycycline induction were incubated at 38°C for 20 min to inactivate dynamin as in Fig. 5, incubated for an additional 4 h with or without crosslinked Apo2L/TRAIL, and analyzed by immunoblot for processing of the indicated proteins. (b) Dyn^G273D-transduced BJAB cells with or without doxycycline induction were incubated at 30°C or 38°C for 20 min, incubated an additional 2 h with or without crosslinked Apo2L/TRAIL, and assayed for caspase-3/7 activity as described in Methods. (c) Dyn^G273D-transduced BJAB cells with or without doxycycline induction were preincubated at 38°C for 20 min and incubated for an additional 4 h with or without crosslinked Apo2L/TRAIL or a DR5-selective Apo2L/TRAIL mutant (DR5-Sel.), and DNA fragmentation was assayed (±SEM) as described in Methods.