Percent lysosome marker colocalization with internalized target.

Cells were preloaded with rhodamine dextran and then allowed to internalize opsonized erythrocytes or were stained for acid phosphatase after phagocytosis. Lysosome fusion is determined by the colocalization of the internalized target and the lysosomal marker. As shown, clones 131-3 and 173-46 expressing wild-type FcγRIIA show more than 97% of the internalized targets colocalized with one of the markers. However, internalization via tailless FcγRIIA, using CR3 to mediate phagocytosis (clones 169-8 and 169-23, n = 5 for both lines), exhibited very little colocalization of the targets with either fluorescent dextran or acid phosphatase (P < .001 comparing wild-type FcγRIIA with tailless FcγRIIA). Columns a-d represent experiments with transient transfections of the FcγRIIA constructs. FcγRIIA immunoreceptor tyrosine-based activation motif (ITAM) mutants (MFI 93) displayed no colocalization of targets and marker due to the absence of phagocytosis (column a). However, in the presence of CR3 to restore phagocytosis, FcγRIIA ITAM mutants (column b) (MFI 91) displayed near wild-type FcγRIIA (column c) (MFI 89) levels of target/marker colocalization. Tailless FcγRIIA (column d) transiently transfected (MFI 96) displayed very little colocalization of targets with dextran.