1. Laboratory of Developmental Immunology, Massachusetts General Hospital/ Harvard Medical School, 55 Fruit Street, Boston, Massachusetts 02114, USA
2. MRC Centre for Inflammation Research, The University of Edinburgh, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, UK
3. Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada
4. Department of Biomedical Engineering and High-Throughput Biology Center, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA
5. Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B4, Canada
6. Present address: Merck Research Laboratories, RY80K-107, PO Box 2000, Rahway, New Jersey 07065, USA.

Correspondence to: L. M. Stuart^1,2 Correspondence and requests for materials should be addressed to L.M.S. (Email: lstuart@partners.org).

Phagocytes have a critical function in remodelling tissues during embryogenesis and thereafter are central effectors of immune defence^{1, 2}. During phagocytosis, particles are internalized into 'phagosomes', organelles from which immune processes such as microbial destruction and antigen presentation are initiated³. Certain pathogens have evolved mechanisms to evade the immune system and persist undetected within phagocytes, and it is therefore evident that a detailed knowledge of this process is essential to an understanding of many aspects of innate and adaptive immunity. However, despite the crucial role of phagosomes in immunity, their components and organization are not fully defined. Here we present a systems biology analysis of phagosomes isolated from cells derived from the genetically tractable model organism Drosophila melanogaster and address the complex dynamic interactions between proteins within this organelle and their involvement in particle engulfment. Proteomic analysis identified 617 proteins potentially associated with Drosophila phagosomes; these were organized by protein–protein interactions to generate the 'phagosome interactome', a detailed protein–protein interaction network of this subcellular compartment. These networks predicted both the architecture of the phagosome and putative biomodules. The contribution of each protein and complex to bacterial internalization was tested by RNA-mediated interference and identified known components of the phagocytic machinery. In addition, the prediction and validation of regulators of phagocytosis such as the 'exocyst'⁴, a macromolecular complex required for exocytosis but not previously implicated in phagocytosis, validates this strategy. In generating this 'systems-based model', we show the power of applying this approach to the study of complex cellular processes and organelles and expect that this detailed model of the phagosome will provide a new framework for studying host–pathogen interactions and innate immunity.

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