Endophilin-A2-dependent tubular endocytosis promotes plasma membrane repair and parasite invasion

Abstract

Endocytosis of caveolae has previously been implicated in the repair of plasma membrane wounds. Here, we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway and have a reduced capacity to reseal after permeabilization with pore-forming toxins compared with wild-type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the parasites. These findings reveal a novel crosstalk between caveolin-1 and endophilin-A2 in the regulation of clathrin-independent endocytosis and plasma membrane repair, a process that is subverted by T. cruzi parasites for cell invasion.

Keywords: Endocytosis; Endophilin; Plasma membrane repair.

Fig. 1.

Cav1 knockout MEFs have a reduced PM repair capacity. (A) Flow cytometry of WT (blue) and Cav1 KO MEFs (red) exposed to 50 ng/ml SLO in the presence or absence of Ca²⁺ at 37°C for 5 min and stained with propidium iodide (PI) to detect permeabilized cells. The results are representative of three independent experiments. (B) Flow cytometry of WT (blue) and Cav1 KO MEFs (red) exposed to increasing concentrations of SLO (50–300 ng/ml) in the presence of Ca²⁺ at 37°C for 1 min and stained with PI. The results are representative of four independent experiments. In A and B the vertical dashed lines represent the gate used to calculate cell population percentages. (C) Fluorescence microscopy images of attached WT and Cav1 KO MEFs treated with increasing concentrations of SLO (ng/ml) for 1 min in the presence of Ca²⁺ and stained with DAPI (blue) and PI (red). Scale bars: 50 µm. The graph shows the mean±s.e.m. percentage of PI-positive cells determined for each condition in three independent experiments. *P=0.04; ****P<0.0001 (unpaired two-tailed Student's t-test).

Fig. 2.

CTxB endocytosis occurs predominantly through caveolae-like small vesicles in WT MEFs but shifts to tubular endosomes in Cav1 KO MEFS. (A) TEM images of WT and Cav1 KO MEFs treated or not with SLO in the presence of CTxB–gold for 1 min at 37°C. Small arrows: <100 nm vesicles containing CTxB–gold. Arrowheads: tubular endosomes. Large arrows: CCVs. Scale bars: 100 nm. (B) Quantification of <100 nm vesicles, >100 nm vesicles, tubular endosomes and CCVs containing CTxB–gold in WT and Cav1 KO MEFs treated as in A. All vesicles containing CTxB–gold were counted in 20 individual cell sections/sample. The data represent the mean±s.e.m. of CTxB–gold-containing vesicles/cell section and are representative of two independent experiments. *P=0.029; **P<0.01; ***P<0.001 (unpaired two-tailed Student's t-test comparing each sample with the WT no SLO control condition).

Fig. 3.

EndoA2 promotes fission of tubular endosomes and PM repair in Cav1 KO MEFs. (A) Western blot using antibodies against EndoA2 and tubulin (loading control) of WT and Cav1 KO MEF lysates treated with control (−) or EndoA2 (+) siRNA. (B) Flow cytometry of WT (blue) and Cav1 KO MEFs (red) treated with control (dashed lines) or EndoA2 (full lines) siRNA for 48 h, exposed to increasing concentrations of SLO (50–100 ng/ml) in the presence of Ca²⁺ at 37°C for 2 min, and stained with PI to detect permeabilized cells. The vertical dashed lines represent the gate utilized to calculate cell population percentages (light color, control siRNA; dark color, EndoA2 siRNA). The results are representative of six independent experiments. (C) TEM images of tubular endosomes in WT and Cav1 KO MEFs treated with control or EndoA2 siRNA for 48 h and incubated with CTxB–gold for 1 min at 37°C. Arrowheads: CTxB–gold. Arrows: open tubular endosomes. Scale bars: 100 nm. (D) Quantification of total, apparently closed and open tubular endosomes containing CTxB–gold in WT and Cav1 KO MEFs treated as in C. All tubular endosomes containing CTxB–gold were counted in 20 individual cell sections/sample. The data represent the mean±s.e.m. of CTxB–gold-containing tubular endosomes/cell section, and are representative of two independent experiments. *P=0.015–0.02; **P<0.01; ***P<0.001 (unpaired two-tailed Student's t-test comparing each sample with the WT control siRNA condition). (E) Flow cytometry of WT (blue) and Cav1 KO MEFs (red) treated with control (dashed lines) or EndoA2 (full lines) siRNA for 48 h and exposed to SLO (25 and 100 ng/ml) or not (no SLO) in the presence of Alexa Fluor 488–CTxB for 10 min at 37°C, followed by quenching with anti-Alexa Fluor 488 antibodies and Trypan Blue for 20 min at 4°C. Negative controls (gray dashed lines) represent WT or Cav1 KO MEFs incubated with Alexa Fluor 488–CTxB at 4°C followed by quenching at 4°C, without stimulating endocytosis at 37°C. The results are representative of three independent experiments.

Fig. 4.

EndoA2 partially colocalizes with endocytosed CTxB in WT and Cav1 KO MEFs. (A) Confocal fluorescence microscopy images of WT and Cav1 KO MEFs incubated with fluorescent CTxB (red) for 15 s at 37°C and stained with anti-EndoA2 antibodies (green). The arrowheads point to EndoA2 puncta that partially colocalize with CTxB. Scale bars: 2 µm. The images are representative of three independent experiments. (B) Quantification of juxtaposed EndoA2 and CTxB puncta. The data represent the mean±s.e.m. of juxtaposition events detected in >20 cells, in three independent experiments. ***P<0.001 (unpaired two-tailed Student's t-test).

Fig. 5.

Cell invasion by T. cruzi is inhibited by EndoA2 depletion, and leads to host cell death when combined with Cav1 deficiency. (A–D) Cells were incubated with T. cruzi for various periods of time, fixed and stained with anti-T. cruzi antibodies and DAPI to quantify intracellular parasites. (A) Intracellular T. cruzi in WT (blue) or Cav1 KO (red) MEFs over time, expressed as intracellular parasites/100 cells. The data represents the mean±s.e.m. of triplicates. **P<0.01 (unpaired Student's t-tests comparing WT and Cav1 KO samples). (B) Cell density in T. cruzi-infected cultures, as in A. DAPI-stained cells in 20 microscopic fields (1000× magnification) were counted. The data represents the mean±s.e.m. of triplicates. Unpaired, two-tailed Student's t-tests were performed comparing each value with the corresponding 0.5 h time point; P-values were not significant (>0.05). (C) Intracellular T. cruzi after 1 or 2 h of exposure to WT (blue) or Cav1 KO (red) MEFs pre-treated with control (light color) or EndoA2 siRNA (dark color). The data represent the mean±s.e.m. of triplicate quantification of intracellular parasites in 100 cells. The arrow indicates the condition (EndoA2 siRNA, 2 h infection) where invasion was not quantified due to cell loss. *P<0.05; **P<0.01 (unpaired Student's t-test). (D) Cell density in T. cruzi-infected cultures, as in C. DAPI-stained cells in 20 microscopic fields (1000× magnification) were counted. The data represent the mean±s.e.m. of triplicates. *P<0.05; **P<0.01 (unpaired Student's t-test).

Fig. 6.

EndoA2 is recruited to nascent parasite-containing vacuoles. Confocal fluorescence microscopy images of EndoA2 staining (green) in WT and Cav1 KO MEFs infected with T. cruzi for 15 min. Extracellular or partially internalized T. cruzi parasites are stained with extracellularly-added anti-T. cruzi antibodies (red). Nuclei and parasite kinetoplast DNA are stained with DAPI (blue). Arrowheads: extracellular portions of T. cruzi. Small arrows: intracellular portions of T. cruzi within PM invaginations stained for EndoA2. Large arrows: intracellular T. cruzi showing fainter EndoA2 staining. Scale bars: 5 µm. Images are representative of three independent experiments.