When a phagocyte encounters a pathogen, the triggering of cell-surface receptors leads to pathogen uptake in a membrane-bound vacuole (the phagosome). The simplest way to imagine the process is as an invagination of the plasma membrane, but a paper published three years ago by Desjardins and colleagues (see Further reading) concluded that the endoplasmic reticulum (ER) also contributes membrane to the phagosome. Now, Touret et al. have re-examined this finding and conclude from the results of various independent methods that the ER does not make a significant contribution to phagosome formation.

Using glycosylphosphatidylinositol (GPI)-linked green fluorescent protein (GFP) as a marker of the plasma membrane in a macrophage cell line phagocytosing latex beads, they showed that the plasma membrane constitutes a large proportion of the early phagosome membrane, limiting any potential contribution of the ER. By contrast, in macrophages expressing the ER marker GFP–KDEL, there was no significant overlap of this marker with phagosome markers. This observation that the ER is not a significant component of the phagosome was also made for various combinations of ER and phagosome markers and for mouse dendritic cells.

Immuno-electron microscopy of macrophage sections that were labelled with calnexin-specific antibodies could not detect the ER-resident protein calnexin on the phagosome membrane, and immunocytochemical staining could not detect the ER marker glucose 6-phosphatase in phagosomes. The possibility that the ER makes a minor contribution to the phagosome membrane by transient fusion was analysed for macrophages that had been stably transfected with the ER marker GFP–KDEL and were exposed to latex beads in the presence of high extracellular concentrations of the dye FM4-64. FM4-64 stained the plasma and phagosome membranes but did not reach the ER membrane (as shown by lack of colocalization with GFP–KDEL), even when phagocytosis was arrested by wortmannin to stabilize any transient connections that might form between the phagosome and the ER.

Biochemical assessment of ER–phagosome fusion was carried out using macrophages that had been stably transfected with the soluble ER marker avidin–KDEL and were allowed to ingest biotinylated beads. Immunostaining of isolated phagosomes with avidin-specific antibodies failed to detect association of avidin with the phagocytosed beads. Finally, the pH sensitivity of GFP (and its derivatives such as yellow fluorescent protein, YFP) was used to analyse the association of phagosomes with the ER on the basis that ER components delivered to the acidic phagosome would experience a decrease in pH. Addition of the weak base ammonia increased the fluorescence of GPI–YFP in phagosomes but had no effect on the fluorescence of GFP–KDEL, showing that this ER marker is not exposed to the acidic phagosome environment.

All of these experiments fail to provide any evidence in support of fusion of the ER with phagosomes, although the authors point out that they cannot definitively rule out the involvement of the ER in phagocytosis of particles of a different size or in phagocytosis using different cell-surface receptors.