Figure 1.
Inhibition of dynamin with dynasore and a dominant-negative mutant.
Dynasore-pretreated and non-pretreated HeLa cells were incubated with (A) 5 nM diphtheria toxin for 180 min or (B) 4 pM toxin B for 75 min, prior to microscopical analysis of the cell morphology. (C) HeLa cells preincubated with dynasore or with solvent only were treated with 4 pM toxin B for 75 min, 1.5 nM toxin A for 105 min, 4 nM lethal toxin for 75 min or 1.5 nM α-toxin for 60 min. The percentage of rounded cells was quantified and data are given +/− SD (n = 3) and from a minimum of 200 cells in total. (D) Dynasore-pretreated HT-29 cells and untreated cells were intoxicated with 40 pM toxin. After onset of cell rounding (150 min), glucosylation status of Rac1 in cell lysates was analyzed with antibodies recognizing either only unmodified Rac1 (α-Rac1non-Glc) or all Rac1 (α-Rac1total). (E) HeLa cells expressing dominant-negative dynamin (HA-dynaminK44A) for 24 h or mock-transfected cells were intoxicated with 4 pM toxin B for 75 min and cell morphology analyzed microscopically. (F) Selective expression of HA-dynaminK44A in plasmid-transfected HeLa cells, but not in mock-transfected cells, was detected in cell lysates with an anti-HA antibody. Antibody detection of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) served as a control for equal loading of lysate samples.
Figure 2.
Pharmacological inhibition of clathrin assembly with chlorpromazine.
HeLa cells were preincubated with chlorpromazine (Cp) or left untreated, prior to addition of (A) 5 nM diphtheria toxin (DT) and incubation for 180 min, (B) 3.5 nM CNF1 and incubation for 150 min or (C) 4 pM toxin B and incubation for 75 min. (D) The percentage of cell rounding in chlorpromazine-pretreated or non-pretreated HeLa cells after intoxication with 4 pM toxin B for 75 min, 1.5 nM toxin A for 105 min, 4 nM lethal toxin for 75 min or 1.5 nM α-toxin for 60 min was quantified and data are given +/− SD (n = 3) and from a minimum of 200 cells in total. (E) Human intestinal epithelial cells (CaCo-2) were grown to confluency on filters and were preincubated with chlorpromazine (Cp) or left untreated. A subset of cells was intoxicated with 40 pM toxin B and transepithelial electrical resistance (TER) was measured at indicated time points, where starting resistance was set to 100% and TER values are calculated as relative TER in % from starting resistance (+/− SD, n = 3).
Figure 3.
RNAi-mediated gene silencing of the clathrin heavy chain.
HeLa cells were transfected with siRNA against the clathrin heavy chain (siclathrin). (A) Two days after transfection, cell lysates from siclathrin-transfected cells and mock-transfected cells were analyzed for clathrin heavy chain expression using a specific antibody (α-clathrin(HC)). Antibody detection of the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (α-GAPDH) served as a control for equal loading of lysate samples. (B) After addition of toxin B (4 pM), Rac1 glucosylation status was compared in lysates from siclathrin- and mock-transfected HeLa cells at indicated time points with antibodies recognizing either only unmodified Rac1 (α-Rac1non-Glc) or all Rac1 (α-Rac1total).
Figure 4.
Expression of dominant-negative Eps15 and Cav-1.
(A) HeLa cells, transfected with plasmids, encoding dominant-negative forms of Eps15 or Cav-1 fused to a GFP moiety, were eventually intoxicated for 75 min with 4 pM toxin B or left untreated. Toxin B-induced alterations in cell morphology were analyzed in Eps15- (Eps15 DN::EGFP), Cav-1- (GFP::Cav-1 DN) and mock-transfected cells after actin staining with FITC-phalloidin (red and grey signals) and by applying fluorescence microscopy. Green signals derived from cells expressing GFP-tagged, dominant-negative Eps15 or Cav-1, respectively. (B) HeLa cells were transfected with a plasmid encoding Eps15 DN::EGFP, intoxicated with 4 pM toxin B for 30 min and subsequently subjected to fluorescence-assisted cell sorting. GFP excitation of the Eps15 DN::EGFP-transfected cells resulted in two cell populations (left panel, blue curve, bordered with dashed line). One population represents non-transfected cells, with overlapping background fluorescence as obtained in mock-transfected cells (left panel, red curve). The other population represents Eps15 DN::EGFP-expressing cells. Equal number of cells from both cell populations (non-transf. and Eps15 DN::EGFP) where subjected to cell lysis and analysis of the Rac1 glucosylation status with antibodies recognizing either only unmodified Rac1 (α-Rac1non-Glc) or all Rac1 (α-Rac1total). (C) Procedure was performed essentially as described in (B), but with HeLa cells transfected with a plasmid encoding GFP::Cav-1.
Figure 5.
Disintegration of lipid rafts by exogenous sphingomyelin depletion with SMase.
HeLa cells were pretreated with SMase or left untreated prior to intoxication with (A) 4 pM toxin B for 75 min, 5 nM toxin A for 300 min, 5 nM lethal toxin for 180 min, 5 nM α-toxin for 120 min, (B) mock incubation for 300 min, or (C) 50 nM VacA toxin for 300 min. Images were obtained by microscopy, upon onset of intoxication characteristics. (D) SMase-preincubated or non-preincubated HeLa cells were intoxicated with 4 pM toxin B or left untreated. The percentage of cell rounding was quantified from three independent experiments at indicated time points (data are given +/− SD).