C6-NBD-gal on the surface of PMNs is endocytosed and trafficked through an endocytic recycling compartment (ERC) before being transported back out to the cell surface.
PMNs were labeled with the fluorescent lipid analogue C6-NBD-gal (C-F) or with a Cy3-conjugated, nonfunction-blocking monoclonal antibody to α5 (Cy3-VC5; A, B), then maintained at 4°C (A-D) or warmed to 37°C for 30 minutes (E-H). After this warm-up, cells were incubated for an additional 2 minutes (E, F) or 30 minutes (G, H) in back exchange medium. Cy3-VC5 and C6-NBD-gal are initially found on the plasma membrane (A-D). After a 30-minute incubation at 37°C, both Cy3-VC5 and C6-NBD-gal accumulate in a central compartment (see Figure8). Two minutes after back exchange of C6-NBD-gal from the plasma membrane, a central compartment is clearly visible in the cells (large arrows, panel F) and in some cases C6-NBD-gal–labeled vesicles (small arrows, panel F) appear to be emanating from the central compartment. By 30 minutes after warm-up, almost all of the C6-NBD-gal has been returned to the plasma membrane and been back exchanged into the medium (H). DIC images are shown in (A, C, E, and G). Bar = 10 μm.
