IN-VITRO FUSION OF PHAGOSOMES WITH DIFFERENT ENDOCYTIC ORGANELLES FROM J774 MACROPHAGES

We describe novel biochemical and electron microscopy assays to invest igate in vitro fusion of latex bead phagosomes with three different en docytic organelle fractions from J774 macrophages. After formation, ea rly phagosomes fuse avidly with early and late endosomes and for a lon ger period of time with lysosomes, but they subsequently become fusion -incompetent. The fusion of early, but not late, phagosomes with all t hree endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enric hed on both early and late endosomes in J774 macrophages. Moreover, ex ogenous Rab5 stimulates homotypic fusion between both sets of organell es. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologica lly defined organelles. By the same approach, we discovered an unexpec ted Rab5-stimulatable fusion between early and late endosomes in J774, but not in BHK cells. Thus, in J774 cells both Rab5 and the endocytic pathway seem to have evolved additional functions not yet seen in non phagocytic cells.