Vesicles did not colocalize with any caveolin, however it should be noted that very little caveolin was visualized in the A549 cells, consistent with reports of low levels of caveolin-1 expression in these cells [30, 31] (data not shown). These data suggest that vesicles may be associated with clathrin-coated pits, but only transiently, at an early stage in the active this website uptake process. Figure 4 Vesicles rarely
co-localize with surface-associated clathrin. AF488-S470 vesicles (2.5 μg) were incubated with A549 cells for 1 h at 37°C. Cell surface was labeled using biotin and AF633-streptavidin (blue). Cells were washed, fixed, permeabilized, and probed with mouse anti-clathrin antibodies and AF555-labeled goat anti-mouse secondary
antibody. Arrows indicate very occasional colocalization of clathrin and vesicle fluorescence at the cell surface. Internalized vesicle components colocalize with the endoplasmic reticulum We repeatedly observed internalized vesicle-associated fluorescence localized to a perinuclear region. We examined whether vesicles were trafficked to the same compartments as transferrin and cholera toxoid (CTB). Only transferrin and CTB that were perinuclear colocalized with internalized Idasanutlin cost vesicles, whereas the majority of cytosolic compartments containing transferrin and CTB did not [see Additional file 1]. To determine whether this perinuclear region corresponded to the endoplasmic reticulum (ER), we treated cells with the glycoside digitonin, which, at low concentrations, permeabilizes the plasma membrane and releases cytosolic proteins but preserves the ER membrane [32, 33]. After digitonin treatment, cells that had lost the cytoplasmic marker, β-tubulin, still retained a perinuclear halo of vesicle-associated fluorescence (data not shown). In these treated cells, vesicle fluorescence clearly colocalized with the integral ER membrane protein TRAPα (Fig. 5). These data suggest that internalized vesicle components
traffic to the ER within 1 hour of exposure. Figure 5 Vesicles co-localize MYO10 with the endoplasmic reticulum marker TRAPα. AF488-S470 vesicles (2.5 μg) were incubated with A549 cells for 1 hour at 37°C. Cell surface was labeled using biotin and AF633-streptavidin (blue). Cells were washed, fixed, permeabilized with 0.015% digitonin to release cytoplasm, and probed with anti-TRAPα primary antibody and AF555-labeled secondary antibody. PaAP promotes vesicle association with human respiratory epithelial cells We wondered whether host cell association depended on PaAP, one of the major protein components of vesicles derived from CF isolates (Fig 6A). MX69 Quantitative 2D-DIGE revealed PaAP is at least 65-fold enriched in S470 vesicles compared with PAO1 vesicles .