Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors

Downey, Gregory P.; Botelho, Roberto J.; Butler, Jeffrey R.; Moltyaner, Yuri; Chien, Paul; Schreiber, Alan D.; Grinstein, Sergio

doi:10.1074/jbc.274.40.28436

Cited by 111 publications

(103 citation statements)

References 52 publications

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“…More importantly, FcR-ldl cells were able to internalize opsonized RBC (Fig. 1C), as described earlier for Fc␥RIIA-transfected wild-type CHO cells (56). In fact, the phagocytic efficiency of the FcR-ldl clone selected for the subsequent experiments was greater than reported for Fc␥RIIA-transfected CHO cells (Ͼ50 versus Ͻ30%).…”

Section: Ldlf Cells Expressing the Human Fc␥riia Receptor Aresupporting

confidence: 81%

“…To measure intraphagosomal pH, zymosan was labeled with both Oregon Green 514 and FITC as described (56). Labeled zymosan was then opsonized with 1 mg/ml human IgG for 1 h at 37°C and washed three times.…”

Section: Methodsmentioning

confidence: 99%

“…It was shown earlier that expression of Fc␥ receptors suffices to confer phagocytic capabilities to nonmyeloid cells such as COS and CHO (54,55). Such transfectants not only internalize IgG-opsonized particles, but the resulting phagosomes undergo a maturation process that faithfully recapitulates the maturation sequence described in professional phagocytes (56). Comparison of the rate and extent of maturation when Fc␥RIIA-transfected ldlF cells were incubated at the permissive or restrictive temperatures allowed us to assess the role of COPI in vesicular fission from phagosomes.…”

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confidence: 89%

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Role of COPI in Phagosome Maturation

Botelho¹,

Hackam²,

Schreiber³

et al. 2000

Journal of Biological Chemistry

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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.

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Section: Ldlf Cells Expressing the Human Fc␥riia Receptor Aresupporting

confidence: 81%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 89%

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Role of COPI in Phagosome Maturation

Botelho¹,

Hackam²,

Schreiber³

et al. 2000

Journal of Biological Chemistry

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“…Transfection of Fc␥RIIa renders CHO cells phagocytic and capable of phagolysosome formation (26). Cells were transfected for 20 h and incubated in the absence or presence of IgG beads.…”

Section: Resultsmentioning

confidence: 99%

Immunoglobulin G signaling activates lysosome/phagosome docking

Trivedi

Zhang

Castoreno

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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An important role of IgG antibodies in the defense against microbial infections is to promote the ingestion and killing of microbes by phagocytes. Here, we developed in vivo and in vitro approaches to ask whether opsonization of particles with IgG enhances intracellular targeting of lysosomes to phagosomes. To eliminate the effect of IgG on the ingestion process, cells were exposed to latex beads at 15-20°C, which allows engulfment of both IgG-coated and uncoated beads but prevents the fusion of lysosomes with phagosomes. Upon shifting the temperature to 37°C, phagosomes containing IgG beads matured significantly faster into phagolysosomes as judged by colocalization with lysosomal markers. The IgG effect was independent of other particle-associated antigens or serum factors. Lysosome/phagosome attachment was also quantified biochemically with a cytosol-dependent scintillation proximity assay. Interactions were enhanced significantly in reactions containing cytosol from mouse macrophages that had been exposed to IgG-coated beads, indicating that IgG signaling modulates the cytosolic-targeting machinery. Similar results were obtained with cytosol from primary human monocytes, human U-937 histiocytic lymphoma cells and from Chinese hamster ovary (CHO) cells transfected with a human IgG (Fc␥) receptor. IgG-induced activation is shown to affect the actin-dependent tethering/docking stage of the targeting process and to proceed through a pathway involving protein kinase C. These results provide a rare example of an extracellular signal controlling membrane targeting on the level of tethering and docking. We propose that this pathway contributes to the role of antibodies in the protection against microbial infections. membrane fusion ͉ protein kinase C ͉ scintillation proximity assay ͉ Fc receptor ͉ macrophages

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“…Subsequent phosphorylation of Syk kinase leads to the recruitment and activation of various downstream effector molecules associated with phagosome maturation (5)(6)(7)(8)(9)(10). Once internalized, phagosomes fuse with lysosomes containing the enzymes necessary for target degradation (1,(11)(12)(13). Phagolysosome formation is believed to require proteins such as Rab5, EEA1, rabaptin, Rab7, and lysosome-associated protein 1 (LAMP-1), as well as intracellular calcium signaling (14,15).…”

mentioning

confidence: 99%

Signal sequence within FcγRIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion

Worth

Kim²,

Kindzelskii³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Calcium oscillations and traveling calcium waves have been observed in living cells, although amino acid sequences regulating wave directionality and downstream cell functions have not been reported. In this study we identify an amino acid sequence within the cytoplasmic domain of the leukocyte IgG receptor Fc␥RIIA that affects the amplitude of calcium spikes and the spatiotemporal dynamics of calcium waves in the vicinity of phagosomes. By using high-speed microscopy to map calcium-signaling routes within cells, we have discovered that bound IgG-coated targets trigger two calcium waves traveling in opposite directions about the perimeter of cells expressing Fc␥RIIA. After phagocytosis, one calcium wave propagates around the plasma membrane to the site of phagocytosis where it splits into two calcium signals: one traveling to and encircling the phagosome once, and the second continuing around the plasma membrane to the point of origin. However, in a genetically engineered form of Fc␥RIIA containing a mutation in the cytoplasmic L-T-L motif, the calcium signal travels around the plasma membrane, but is not properly routed to the phagosome. Furthermore, these calcium pattern-deficient mutants were unable to support phagolysosome fusion, although recruitment of phagolysosome-associated proteins lysosome-associated protein 1, Rab5, and Rab7 were normal. Our findings suggest that: (i) calcium signaling is a late step in phagolysosome fusion, (ii) a line of communication exists between the plasma membrane and phagosome, and (iii) the L-T-L motif is a signal sequence for calcium signal routing to the phagosome.

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Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors

Cited by 111 publications

References 52 publications

Role of COPI in Phagosome Maturation

Role of COPI in Phagosome Maturation

Immunoglobulin G signaling activates lysosome/phagosome docking

Signal sequence within FcγRIIA controls calcium wave propagation patterns: Apparent role in phagolysosome fusion

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