Figure 1.
Stx increases formation of clathrin-coated pits.
(A) Representative electron micrographs of immunogold-labeled HeLa cells showing Stx associated with clathrin-coated profiles (arrows in a–d) as well as associated to non-specialized regions of the plasma membrane (a–e). Bar 200 nm. (B) Exposure of HeLa cells to Stx for 10 min increased the number of clathrin-coated pits at the plasma membrane with statistical significance by 28±4% (mean ± SE, p = 0.008, n = 3). (C) Exposure of HEp-2 cells to Stx for 10 min resulted in a 38±2% statistically significant increase in the number of clathrin-coated pits at the plasma membrane (mean ± SE, p = 0.004, n = 3). Data were obtained from 20–30 cell profiles, containing from 38 to 63 pits altogether, per experiment and condition. Data are displayed in the plots as mean ± SD.
Figure 2.
The number of AP-2-containing structures increases after incubation with StxB.
Time-series obtained by spinning disk confocal microscopy of the ventral surface of HeLa cells stably expressing σ2-EGFP were recorded before and after addition of StxB. The σ2-EGFP positive spots are fiduciaries of endocytic clathrin-coated structures. The number of clathrin-coated structures obtained from 9 independent experiments was determined as described in Materials and Methods. The maximum number of AP-2 spots in each cell was set equal to 100%, and the number of AP-2 spots at other time points was then calculated as fractions of the maximum number. Results are displayed ± SE or average deviation. The raw data for each cell are displayed in table 1 (cells 1 to 9).
Table 1.
Number of AP-2 spots in 16 cells before and after incubation with StxB for different periods.
Figure 3.
StxB increases the activity of AP-2 structures.
Representative time-series were obtained by live-cell spinning disk confocal imaging of a HeLa cell stably expressing σ2-EGFP before (upper panel) and after (lower panel) exposure to StxB for 10 min. Arrows point to examples of diffraction-limited AP-2 spots appearing and disappearing within the time-series. Images were acquired every 3 s, and are shown with 9 s between images. Laplacian 2D and Gaussian filtering were applied on all images in order to facilitate their graphical display. The time series corresponding to this experiment is available as Supporting Information (Movie S1).
Figure 4.
Lifetime and maximum intensity distributions of AP-2 spots are shifted upon StxB treatment.
Representative results obtained from a HeLa cell stably expressing σ2-EGFP are shown. A control time-lapse series was first recorded for 3 min, and then another 3 min time-lapse series was acquired after 10 min of incubation with StxB. Images were acquired every 3 s. A total of 92 (control) and 162 (after StxB-treatment) AP-2 spots were recorded and analyzed. Only spots appearing and disappearing within the time-lapse period of 3 min and typically lasting between 7 and 30 timeframes (21 to 90 s) were included in the analysis. (A) Effect of StxB on the lifetime distribution of the AP-2 spots. White and grey bars represent the number of AP-2 clusters in the different lifetime intervals of the histogram for the control and the StxB-data set, respectively. Lifetime was defined as the period from appearance to disappearance of the AP-2 spot. (B) Effect of StxB on the distribution of maximum fluorescence intensity for AP-2 spots. White and grey bars represent the number of AP-2 spots in the different maximum intensity intervals of the histogram for the control and the StxB data set, respectively. Maximum fluorescence intensity is expressed in arbitrary fluorescence units and was measured just prior to dissolution of the AP-2 signal due to un-coating of the clathrin/AP-2 coat.
Figure 5.
Rate of Tf internalization in HeLa cells is not affected by the addition of StxB.
At different time points 125I-labeled Tf was added to all wells in the presence or absence of StxB. The 125I-signals corresponding to endocytosed and surface bound fractions were determined. Data are expressed as the mean ± average deviation of results from two independent experiments done in duplicate.
Figure 6.
Inhibition of Syk by piceatannol inhibits the Stx-induced increase in the number of AP-2 spots.
HeLa cells stably expressing σ2-EGFP were pre-incubated with piceatannol (50 µM) for 30 min. A time-lapse series was acquired by spinning disk confocal microscopy with an image obtained every 3 s for 1–2 min. The cells were then exposed to StxB for 10 min, followed by the acquisition of a second time-lapse series of 1–2 min. Six independent experiments, with 1–4 cells per experiment, were analyzed, and the result is expressed relative to piceatannol only. There was no statistically significant increase (p = 0.2) in the number of AP-2 spots when adding StxB upon piceatannol treatment. For comparison, the number of AP-2 spots before (control) and after 10 min incubation with StxB is shown as determined from table 1; the result is expressed relative to the control and shows a statistically significant increase of 57±16% (mean ± SE, p = 0.002).