Infection induces CHC phosphorylation. (a) Quantification of the intensity of chemiluminescence of three independent Western blots of Jeg3 cells treated as indicated or HeLa cells infected with EPEC for the indicated times. After each treatment, CHC was immunoprecipitated and tyrosine phosphorylation was assayed with the anti-phosphotyrosine antibody PY20. Error bars indicate mean ± SD. (b) MALDI-TOF mass spectrometry spectrum of in vitro phosphorylated CHC fragment (1074–1675). Src kinase was incubated with recombinant CHC followed by digestion with trypsin. Tyr 1487 is found within a peptide with a predicted monoisotopic mass of 1942.9 D and within the phosphopeptide with a predicted mass of 2022.9 D. Tyr 1477 is found within a peptide with a predicted monoisotopic mass of 2355.1 D and within the phosphopeptide with a predicted mass of 2435.1 D. Arrows indicate the native and phosphorylated peptides. (c) Purified recombinant his-tagged-hub fragment of CHC (residues 1074–1675) was treated with or without recombinantly purified Src kinase for 3 h at room temperature. Samples were immunoblotted with antibody against the histidine tag on the hub (His, as loading control), 4G10 (for phospho-tyrosine), and anti-phospho-CHC antibody (pCHC) purified by affinity column and by membrane-based depletion. (d) Anti-pCHC (affinity purified by column) was tested for binding to the 1477&1487-phospho-peptide in the presence of the nonphospho-peptide, 1477-phospho-peptide, 1487-phospho-peptide, or 1477&1487-phospho-peptide at the indicated dilutions by ELISA. (e) Jeg3 cells were infected with L. innocua(InlA) for 30 min and HeLa cells were infected with L. innocua(InlB) for 5 min or with EPEC for 6 h. After infections, cells were fixed and labeled for total CHC (CHC, red) and phospho-CHC (pCHC, green), and F-actin was labeled with fluorescent phalloidin (actin, blue). Arrows indicate colocalization between bacteria, pCHC, and actin. Bars 10 µm.

CHC phosphorylation is required for bacterial infection and pedestal formation. (a) Representative Western blot of Jeg3 cells transfected with a control siRNA or with a CHC-targeted siRNA alone or in combination with the overexpression of wt CHC-GFP or the Y1477, 1487F mutant of CHC-GFP (non-pCHC-GFP). Comparable results were obtained in HeLa cells. Note that the GFP tag on the rescue constructs interferes with reactivity of the TD.1 antibody against the CHC N-terminal domain (Näthke et al., 1992) used for immunoblotting. (b and c) Jeg3 cells and HeLa cells were then infected with L. monocytogenes and HeLa cells with EPEC. Bacterial internalization and EPEC pedestal formation were quantified by immunofluorescence. In all cases, values are means ± SD of three independent experiments (error bars).

Ultrastructure of clathrin coats during bacterial infections. (a–c and e) Jeg3 cells were incubated with L. innocua(InlA) (a and b) and HeLa cells with L. innocua(InlB) (c) or EPEC (e), fixed, and processed for electron microscopy. (d) HeLa cells were infected with L. monocytogenes and processed for immuno-labeling on thawed cryosections where clathrin was localized by antibody labeling. Enlarged views of the boxed regions are shown on the right. Bars, 1 µm.

Recruitment of actin-organizing machinery during infection. Jeg3 cells were incubated with L. innocua(InlA, red) for 15, 30, and 45 min, and HeLa cells were incubated with L. innocua(InlB, red) for 5, 15, and 30 min or with EPEC for 4, 5, and 6 h, then fixed and processed for immunofluorescence with antibodies recognizing Dab2, myosin VI, or Hip1R (all green). Actin was labeled with fluorescent phalloidin (blue). At each time point of infection, the frequency of protein recruitment at bacteria adhesion sites was evaluated by immunofluorescence. Arrows point to sites of colocalization between bacteria, the labeled protein, and actin. Values are means (± SD) of three independent experiments where 100 bacteria were counted for each condition. Error bars indicate mean ± SD. Bars, 10 µm.

Dab2, Myosin VI, and Hip1R are required during bacterial infections. (a–c) Jeg3 cells and HeLa cells were transfected with two siRNA sequences (si1 and si2) targeted to Dab2 (a), Myosin VI (b), or Hip1R (c). L. monocytogenes was then used to infect Jeg3 and HeLa cells to follow the InlA or InlB internalization pathway, respectively, by a gentamicin survival assay. HeLa cells were infected with EPEC to follow actin-based pedestals formation by immunofluorescence where ∼200 pedestals were counted for each experiment. Values are means ± SD of three independent experiments (error bars). Asterisks represent P-values (**, P ≤ 0.01; ***, P ≤ 0.001; Student’s t test).

Ordered recruitment of the clathrin-actin machinery established by siRNA treatment. (a–f) Jeg3 and HeLa cells were transfected with siRNA targeted to either Dab2 (a), CHC (b), CLC (c), Myosin VI (d), or Hip1R (e); or incubated with cytochalasin D for 30 min (f). Cells were incubated with L. innocua(InlA), L. innocua(InlB), or EPEC, then fixed and processed by immunofluorescence. The frequency of recruitment of the different component of the clathrin/actin machinery at the bacterial adhesion sites was evaluated by fluorescence microscopy and normalized to that of control cells. Values are means (± SD) of three independent experiments where ∼100 bacteria were counted for each condition. Red horizontal lines mark the control level of protein recruitment. Error bars indicate mean ± SD.

CHC phosphorylation is required to assemble the clathrin-actin machinery. (a–c) Jeg3 cells were transfected with a control siRNA or with a CHC-targeted siRNA, alone or in combination with the overexpression of wt CHC-GFP or the Y1477, 1487F mutant of CHC-GFP (non-p-CHC). Cells were then incubated with L. innocua(InlA), L. innocua(InlB), or EPEC, then fixed and processed by immunofluorescence. The frequency of recruitment of the different component of the clathrin–actin machinery at the bacterial adhesion sites was evaluated by fluorescence microscopy and normalized to that of control cells. Values are means ± SD of three independent experiments (error bars) where ∼100 bacteria were counted for each condition.

Proposed model for the clathrin-actin machinery triggered by bacterial infection. Bacteria activate host receptor downstream signaling, which results in the recruitment of the clathrin adaptor Dab2 and of the CHC–CLC complex. Clathrin coats are then stabilized by the tyrosine phosphorylation of CHC (P), followed by the sequential recruitment of Hip1R and actin. Myosin VI is also recruited in the case of invading bacteria, probably providing the pulling force for bacterial internalization, whereas it is excluded from EPEC-induced actin pedestals. CHC remains phosphorylated at the bacterial adhesion or internalization sites where clathrin and actin are colocalized.