Abstract
We describe novel biochemical and electron microscopy assays to investigate in vitro fusion of latex bead phagosomes with three different endocytic organelle fractions from J774 macrophages. After formation, early phagosomes fuse avidly with early and late endosomes and for a longer period of time with lysosomes, but they subsequently become fusion-incompetent. The fusion of early, but not late, phagosomes with all three endocytic fractions could be significantly stimulated by Rab5. In contrast to other cell types investigated, this Rab is uniquely enriched on both early and late endosomes in J774 macrophages. Moreover, exogenous Rab5 stimulates homotypic fusion between both sets of organelles. This was shown by a quantitative electron microscopy fusion assay that can directly assay fusion between any combination of morphologically defined organelles. By the same approach, we discovered an unexpected 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 nonphagocytic cells.
Highlights
Phagocytosis is a process crucial for immune defense, which involves the uptake of particles larger than 0.3– 0.5 m
Preparation of Phagosomes, Early Endosomes, Late Endosomes, and Lysosomes—To study the fusion of organelles operationally defined as early endosomes, late endosomes, and lysosomes with phagosomes from J774 mouse macrophages, we established a cell-free system related to an earlier homotypic in vitro fusion assay for early endosomes [10]
The basis of this assay is the use of an avidin-biotin detection system to monitor fusion between the phagosomes enclosing avidin-conjugated beads with different endocytic organelles containing biotinylated horseradish peroxidase (bHRP)
Summary
Cell Culture and Preparation of Cytosol—J774A.1 mouse macrophages were maintained as described by Blocker et al [46]. The final volume of 182 l was incubated for 80 min at 37 °C During this time, fusion between phagosomes containing avidin beads and bHRP-loaded endocytic organelles resulted in the formation of an avidin-bHRP complex on the latex beads. Rab Recruitment to Phagosomes—50 nM of Rab5-Rab GDI complex (or 5 M Rab GDI) prepared as described [47, 48] was added to 2 ϫ 108 avidin bead phagosomes of different ages in the presence of 10 M GTP (or 10 M GDP, respectively) in membrane buffer (25 mM HEPES, pH 7.4, 115 mM KOAc, 25 mM MgOAc) and incubated for 20 min at 37 °C. The average number of points of a lattice test grid overlaying endosome profiles was used as an estimation of profile area
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