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Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells

¹ Department of Medicine, Johns Hopkins University, Baltimore, MD, USA
² Department of Biology, Johns Hopkins University, Baltimore, MD, USA

View larger version (73K): [in a new window]   Fig. 1. Co-localization of Stx1B receptors and CTB receptors GM1 on the apical surface of Caco-2 intestinal epithelial cells. Confocal optical section through the apical surface of Caco-2 monolayers. Cells were double labeled with Stx1B-Oregon Green 488 (green) and CTB-TMR (red). Overlay panel shows co-localization of both receptors (yellow). Note, not all cells in confluent Caco-2 monolayers bind CTB and Stx1B due to the cell heterogeneity.

View larger version (99K): [in a new window]   Fig. 2. Typical example of FRET between donor GM1 and acceptor Gb3 molecules. (D1) Donor Cy3-CTB-GM1 image before acceptor photobleaching. (A1) Acceptor Cy5-Stx1B-Gb3 image before photobleaching. (A2) Acceptor image after photobleaching. (D2) Donor image after acceptor photobleaching. Energy transfer can be detected from the increase in donor fluorescence (difference between D2 and D1 intensity fluorescence). In this experiment, the cells were labeled with donor- and acceptor-conjugated CTB and Stx1B at D:A=1:3 ratio. Colors reflect the fluorescence intensity of 8-bit images from lowest (black, 0 gray levels) to highest (red, 255 gray levels). The increase in D2 vs D1 fluorescence intensity is due to FRET. Note that neighboring cells, which are black on the D1 image because CTB intensity was below threshold level due to lower GM1 expression or due to a difference in cell thickness, appeared in blue color on the D2 image due to FRET. (B) Donor fluorescence intensity Cy3-CTB before and after (C) the bleaching period (30 seconds) in the absence of acceptor Cy5-Stx1B. Note that no donor bleaching or FRET occurred in that time period in the absence of acceptor.

View larger version (21K): [in a new window]   Fig. 3. Experimental FRET between donor GM1 and acceptor Gb3 molecules. E increased as the concentration of acceptor Cy5-Stx1B used to label the cell was increased from D:A 1:1 (b, closed circles) to D:A 1:3 (a, open circles). In these experiments E increased from 15% to 33%, as D:A was increased. FRET does not occur between GM1 and Gb3 molecules after MßCD treatment (c, open triangles).

View larger version (67K): [in a new window]   Fig. 4. Effect of cholesterol on Stx1B interaction with the apical surface of Caco-2 cells. (A) Stx1B and CTB co-localization in cells with disrupted LR. Confocal optical section through the apical surface of Caco-2 monolayers exposed to MßCD. Cells were double-labeled with Stx1B-Oregon Green 488 (green) and CTB-TMR (red). The overlay panel shows co-localization of both receptors (yellow). Bar, 5 µm. (B) Effect of cholesterol depletion on Stx1B binding plus internalization by Caco-2 cells. (C) effect of cholesterol depletion on Stx1B binding only by Caco-2 cells. (1) Control monolayers (n=7) with intact LR; (2) cells treated with filipin (n=7); (3) cells treated with MßCD (n=7); (4) cells incubated with cholesterol oxidase (n=7).

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