A systems biology analysis of the Drosophila phagosome

Stuart, Lynda M.; Boulais, Jonathan; Charrière, Guillaume M.; Hennessy, Elizabeth J.; Brunet, Sylvain; Jutras, Isabelle; Goyette, Guillaume; Rondeau, Christiane; Letarte, Simon; Huang, Hailiang; Ye, Pei-Chen; Morales, F.; Kocks, Christine; Bader, Joel S.; Desjardins, Michel; Ezekowitz, R. A. B.

doi:10.1038/nature05380

Cited by 212 publications

(224 citation statements)

References 29 publications

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“…Proteomic approaches using latex beads internalized by cultured cells have identified a large number of phagosome-associated proteins 31 . How these candidates coordinately function in phagosome maturation and processing remains to be determined.…”

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confidence: 99%

A pathway for phagosome maturation during engulfment of apoptotic cells

et al. 2008

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The efficient removal of apoptotic cells is critical for the physiological well-being of the organism 1 Ã 4 ; defects in corpse removal have been linked to autoimmune disease 4,5 . While several players regulating the early steps of corpse recognition and internalization have been characterized 6 , the molecules and mechanisms relevant to the subsequent processing of the internalized corpses are poorly understood. Here, we identify a novel pathway for the processing of internalized apoptotic cells in C. elegans and in mammals. First, we show that RAB-5 and RAB-7 are sequentially recruited to phagosomes containing apoptotic corpses as they mature within phagocytes, and that both proteins are required for efficient corpse clearance. We then used targeted genetic screens to identify players regulating the recruitment and/or retention of Rab5 and Rab7 to phagosomes. Seven members of the HOPS complex (a Rab7 activator/effector complex) were required for Rab7 localization or retention on phagosomes. In an effort to identify factors that regulate Rab5 recruitment, we undertook an unbiased reverse genetic screen and identified 61 genes potentially required for corpse removal. In-depth analysis of two candidate genes, vps-34 and dyn-1/ dynamin, showed accumulation of internalized, but undegraded corpses within abnormal phagosomes that are defective in RAB-5 recruitment. Using a series of genetic and biochemical experiments in worms and mammalian cells, we ordered these proteins in a pathway, with DYN-1 functioning upstream of VPS-34, in the recruitment/retention of Rab5 to the nascent phagosome. Further, we identified a novel biochemical complex containing Vps34, dynamin and Rab5 GDP , providing a mechanism for Rab5 recruitment to the nascent phagosome.Removal of apoptotic cells (engulfment) is an essential process that occurs throughout life in multi-cellular animals as part of development, homeostasis, and wound healing1Ã4 , 7 , 8. Engulfment) can be broken down into a series of steps, comprising recognition, internalization, phagosome maturation and finally lysosomal degradation of the apoptotic cell by the phagocyte. In mammals, impaired clearance of apoptotic cell corpses can lead to exposure of autoantigens, resulting in onset of autoimmune diseases, such as systemic lupus erythematosus 4,9,10 . Modulation of the engulfment process is therefore a potential therapeutic target in these conditions. One of the fundamental challenges in understanding how defects in engulfment of apoptotic cells translates into diseased states is the identification of critical players involved in corpse removal and how these proteins orchestrate the different stages of engulfment.The nematode C. elegans represents a powerful genetic tool for the study of programmed cell death 11,12 . Large numbers of cells are induced to die during two periods in the life of a worm: during embryonic and larval morphogenesis and during germ cell development 13 . Genetic studies have identified two evolutionarily conserved signaling pathways invol...

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A pathway for phagosome maturation during engulfment of apoptotic cells

et al. 2008

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“…In addition to known factors, RNAi screens were the first to identify the coatamer complexes (COPI and COPII) in uptake [26,[31][32][33]35,36]. The COPI and COPII complexes are required for vesicular trafficking between the ER and golgi [37], are found to associate with the phagosome, and are required in S2 cells for the uptake of a wide variety of pathogens, although the mechanism of this requirement has not yet been established.…”

Section: Microbial Recognition and Survival Screensmentioning

confidence: 99%

“…The COPI and COPII complexes are required for vesicular trafficking between the ER and golgi [37], are found to associate with the phagosome, and are required in S2 cells for the uptake of a wide variety of pathogens, although the mechanism of this requirement has not yet been established. Another novel and general finding of the RNAi screens was the requirement for the exocyst in phagocytosis [26,32,33,35,36]. This complex plays essential roles in secretory vesicle targeting and docking at the plasma membrane for exocytosis [38].…”

Section: Microbial Recognition and Survival Screensmentioning

confidence: 99%

Genomic RNAi screening in Drosophila S2 cells: what have we learned about host–pathogen interactions?

Cherry

2008

Current Opinion in Microbiology

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The détente between pathogen and host has been of keen interest to researchers in spite of being exceedingly difficult to probe. Recently, new RNA interference (RNAi) technologies, in particular in Drosophila tissue culture cells, have made it possible to interrogate the genetics of host organisms rapidly, with nearly complete genomic coverage and high fidelity. Therefore, it is not surprising that the applications of RNAi to the study of host-pathogen interactions were amongst the first to be published, and have already revealed many new insights into the hosts' role in infection. This review will highlight the application of RNAi screening to pathogen-host interactions in Drosophila cells and will reveal some of the lessons learned from this approach.

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“…Currently, we do not know whether the kinetics of phagosomal maturation are affected by the pall mutation, which could prevent efficient uptake by an as yet unknown feedback mechanism. Although it has been shown that components of the proteasome associate with the phagosome there has been no conclusive link established between phagosome maturation and phagocytic efficiency 9,18 .…”

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Pallbearer and friends: lending a hand in apoptotic cell clearance

Elliott

Ravichandran

2008

Trends in Cell Biology

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Engulfment and prompt removal of apoptotic cells occurs from embryogenesis throughout the lifespan of multi-cellular organisms. A new player, Pallbearer, has recently been identified in Drosophila as being important for efficient engulfment by macrophages. Pallbearer is a component of the SCF E3 ubiquitin ligase complex involved in ubiquitylation of proteins targeted for proteasomal degradation. This work provides the first link between the cellular processes of ubiquitylation/proteasomal degradation and the ability to efficiently clear apoptotic cells.The efficient engulfment and removal of apoptotic cells is important for tissue homeostasis and immunological self-tolerance 1 . Such engulfment is carried out by competent neighboring cells or by professional phagocytes, such as macrophages and immature dendritic cells. In recent years, the molecular underpinnings of this process have begun to be revealed through studies in the model organisms C. elegans and Drosophila, along with molecular and biochemical studies in mammalian cells and via knockout mice 2, 3 . Three key steps in the phagocytic removal of apoptotic cells are recognition, internalization and degradation. Apoptotic cells display several "eat-me" markers that serve as molecular cues for phagocyte recognition and to initiate engulfment via receptors on the phagocyte. The most prominent of these "eat-me" signals is the exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane 4 . Multiple modes of PS recognition have been defined, including the recent identification of direct PS-binding receptors Bai1, Tim4 and Stabilin-2 5-7 . Upon encountering the apoptotic target, rearrangement of the actin cytoskeleton occurs in the phagocyte, driven primarily by the Rho GTPase Rac 8 . This leads to the formation of an actin-rich phagocytic cup that envelops the apoptotic cell. After internalization, the corpse proceeds through the phagolysosomal pathway where the remnants of the apoptotic cell are degraded and processed. Although the degradative step remains poorly understood, this process is clearly important for antigen presentation and protein recycling and may impact the efficiency of corpse engulfment 9, 10 .The post-translational modification of proteins by ubiquitylation has a well-documented role in targeting proteins for proteasomal degradation and regulation of numerous cellular processes including protein transport, cell cycle progression, DNA repair and inflammation [11][12][13] . Ubiquitylation is carried out through a series of enzymatic steps that results in the covalent attachment of the 8 kDa ubiquitin (Ub) protein to lysine residues on substrate proteins. The Ub-activating enzyme (E1) transfers Ub to Ub-conjugating enzymes (E2). The Ub-ligases (E3) bind to substrate proteins and promote transfer of the Ub from E2 to specific lysine residues on the target protein. Substrate specificity for ubiquitylation is determined by

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A systems biology analysis of the Drosophila phagosome

Cited by 212 publications

References 29 publications

A pathway for phagosome maturation during engulfment of apoptotic cells

A pathway for phagosome maturation during engulfment of apoptotic cells

Genomic RNAi screening in Drosophila S2 cells: what have we learned about host–pathogen interactions?

Pallbearer and friends: lending a hand in apoptotic cell clearance

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