Role of COPI in Phagosome Maturation

Abstract

Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in ⑀-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human Fc␥RIIA receptors. Following incubation at the restrictive temperature, ⑀-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of ⑀-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of ⑀-COP, while clearance of the Fc␥RIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in ⑀-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.

Azithromycin, a Lysosomotropic Antibiotic, Has Distinct Effects on Fluid-Phase and Receptor-Mediated Endocytosis, but Does Not Impair Phagocytosis in J774 Macrophages

“…In particular, azithromycin does not inhibit phagocytosis nor alter the accessibility of phagocytosed latex beads into lysosomes, in contrast with chloroquine [68,69], but similar to -COP-defective cells [70]. Moreover, interference with acidification and/or vacuolation do not directly account for two distinct properties of azithromycin that, it should be noted, could be evidenced even several hours after drug withdrawal.…”

“…A third candidate is the COP complex. Some features of azithromycin-treated cells are indeed reminiscent of -COP inactivation in CHO cells, which (i) selectively inhibits fluid-phase endocytosis when measured after long intervals, (ii) may inhibit receptor-mediated endocytosis [76], (iii) does not inhibit phagocytosis, and (iv) blocks transfer of a pinocytosed tracer to lysosomes, but not maturation of phagosomes [64,70,77]. However, -COP inactivation does not delay receptor-mediated endocytosis [76] (but see also [77]).…”

Induction of p38 Mitogen-activated Protein Kinase Reduces Early Endosome Autoantigen 1 (EEA1) Recruitment to Phagosomal Membranes

“…Confocal fluorescence microscopy images were collected and processed using the PerkinElmer Life Sciences UltraView confocal microscopy system. Colocalization of endocytic markers with phagosomes were determined using the previously described methods of unbiased counting (11,32).…”

Differential Role of Actin, Clathrin, and Dynamin in Fcγ Receptor-mediated Endocytosis and Phagocytosis

“…Effect of Dyn I K44A on Phagosome Maturation-The ability of RAW 264.7 cells to engulf IgG-opsonized particles despite the expression of dominant-negative dynamin I enabled us to analyze the role of clathrin-dependent fission in phagosome maturation. The transition between the early and intermediate stages of maturation is marked by the removal of Tf receptors from the phagosomal membrane, a process that in RAW cells is completed ϳ30 min after completion of phagocytosis (38). We therefore analyzed the rate of depletion of Tf receptors from phagosomes formed in cells expressing Dyn I K44A.…”