Using ratiometric fluorescence microscopy, the authors followed the maturation of individual macrophage phagosomes containing IgG-opsonized erythrocytes. Most of the results were uniform and unsurprising. Actin is present briefly during phagosome formation, and then all of the phagosomes sequentially associate with the small GTPases Rab5a and Rab7. The lysosomal membrane protein LAMP-1 associates with the phagosomes after Rab7, consistent with lysosomal fusion.
A marker for phosphatidylinositol 3-phosphate (PIP), however, reveals two distinct populations of erythrocyte-carrying phagosomes. In one population, PI(3)P levels spike rapidly after phagosome formation, and then fall to undetectable levels within 20 min. In the other population, PI(3)P rises slowly and persists for several hours.
The differences may reflect stochastic variations in phagosome content that are amplified by kinase/phosphatase systems. The functional significance of the two states remains unclear, although different levels of PI(3)P may be used to recruit different levels of NADPH oxidase.
If organelle maturation were driven solely by localized receptor action, then the levels of PI(3)P on a given phagosome should represent an average of all of the receptor signaling on the phagosome membrane. The complete conversion of each phagosome to one of two extreme states shows, however, that membrane chemistry is integrated over the entire organelle. This contrasts with the initial formation of the phagosome, which is driven by the zipper-like local activation of individual receptors along a pseudopod. The authors are now using FRET-based methods to examine the surface chemistries of phagosomes in more detail and hope to identify appropriate markers for the signals that integrate PI(3)P levels.