The ERC is localized just behind the leading lamella of motile PMNs and reorients to retain this position as the cells move.
The plasma membrane of PMNs was labeled with C6-NBD-gal as described in “Materials and methods.” The ERC was labeled by incubating C6-NBD-gal–labeled PMNs for 10 minutes at 37°C. To remove C6-NBD-gal from the plasma membrane, yet retain C6-NBD-gal labeling of the recycling compartment, cells were incubated for an additional 10 minutes on ice in back exchange medium. PMNs were plated onto fibronectin-coated coverslip dishes, stimulated with fMLP, then imaged with a Zeiss LSM510 confocal microscope. DIC and fluorescence images were acquired simultaneously at the indicated time points (numbers represent time in seconds). Outlines of the cells and the boundary between the cell body and the lamella were obtained from the DIC images, then transferred to the fluorescence images. Bar = 10 μm.
