Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells

Ac, Tosello-Trampont; Kinchen, Jason M.; Brugnera, Enrico; Lb, Haney; Hengartner, Michael O.; Ks, Ravichandran

doi:10.1038/sj.cdd.4402094

Cited by 48 publications

(46 citation statements)

References 38 publications

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“…For siRNA experiments, cells were transfected using Amaxa program U-30 and Kit R for NIH/ 3T3 cells or T-21 and Kit V for J774 macrophages (Amaxa, Germany) with an siRNA SMARTpool containing 4 siRNAs targeting mouse dynamin-2 (Dharmacon cat # M-044919-01), elmo1 (M-041254-00) or a noncoding SMARTpool (Dharmacon cat # D-001206-13) using 1.2 Ãg of total siRNA (0.3 Ãg of each individual siRNA) as previously described 83 , then incubated 48 hours to recover. Images were acquired using a Zeiss 510-UV laser scanning confocal microscope with 405, 488, 543, and 633 lasers (Zeiss AG, Germany).…”

Section: Immunofluorescence In Mammalian Cellsmentioning

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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Section: Immunofluorescence In Mammalian Cellsmentioning

confidence: 99%

A pathway for phagosome maturation during engulfment of apoptotic cells

et al. 2008

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“…In vitro phagocytosis assay. Various cell lines (LR73 Chinese hamster ovary cells, J774 macrophages and NIH 3T3 cells) and primary cells (mouse astrocytes) were transiently or stably transfected before the phagocytosis assays 20 . Phagocytic cells were incubated with fluorescently labelled targets such as apoptotic thymocytes, 2-mm carboxylate-modified latex beads, or 2-mm phospholipid containing microbubbles.…”

Section: Methods Summarymentioning

confidence: 99%

“…The HF7C yeast strain (with His, Trp and Leu as selection markers) 25 was used for the yeast two-hybrid screen against a mouse embryo library. Immunoprecipitation and GST pull-down assays were used to detect proteinprotein interactions 20 . Protein expressions and the presence of bound proteins were confirmed by western blotting.…”

Section: Methods Summarymentioning

confidence: 99%

“…LR73 fibroblasts transiently transfected with BAI1 carrying different tags also enhanced the engulfment of apoptotic thymocytes ( Supplementary Fig. 4) or fluorescently labelled 2-mm carboxylate-modified beads, surrogate targets that mimic apoptotic cells 19,20 (a fold increase of 2.62 6 0.20 for green fluorescent protein (GFP)-labelled BAI1, P , 0.001, n 5 16; Fig. 2c).…”

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BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module

Park

Tosello‐Trampont

Elliott

et al. 2007

Nature

738

741

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Engulfment and subsequent degradation of apoptotic cells is an essential step that occurs throughout life in all multicellular organisms [1][2][3] . ELMO/Dock180/Rac proteins are a conserved signalling module for promoting the internalization of apoptotic cell corpses 4,5 ; ELMO and Dock180 function together as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac, and thereby regulate the phagocyte actin cytoskeleton during engulfment [4][5][6] . However, the receptor(s) upstream of the ELMO/ Dock180/Rac module are still unknown. Here we identify brainspecific angiogenesis inhibitor 1 (BAI1) as a receptor upstream of ELMO and as a receptor that can bind phosphatidylserine on apoptotic cells. BAI1 is a seven-transmembrane protein belonging to the adhesion-type G-protein-coupled receptor family, with an extended extracellular region 7-9 and no known ligands. We show that BAI1 functions as an engulfment receptor in both the recognition and subsequent internalization of apoptotic cells. Through multiple lines of investigation, we identify phosphatidylserine, a key 'eat-me' signal exposed on apoptotic cells 10-13 , as a ligand for BAI1. The thrombospondin type 1 repeats within the extracellular region of BAI1 mediate direct binding to phosphatidylserine. As with intracellular signalling, BAI1 forms a trimeric complex with ELMO and Dock180, and functional studies suggest that BAI1 cooperates with ELMO/Dock180/Rac to promote maximal engulfment of apoptotic cells. Last, decreased BAI1 expression or interference with BAI1 function inhibits the engulfment of apoptotic targets ex vivo and in vivo. Thus, BAI1 is a phosphatidylserine recognition receptor that can directly recruit a Rac-GEF complex to mediate the uptake of apoptotic cells.Previous studies revealed two 'functional' regions within ELMO1 and its Caenorhabditis elegans homologue CED-12 during phagocytosis 5,14-17 . The amino-terminal 558 amino-acid residues (N-term) were necessary for targeting of the ELMO-Dock180 complex to the membrane 14,17 , whereas the carboxy-terminal 196 residues (C-term) were necessary for binding Dock180 and for optimal Rac activation 15,16 . Because the receptor(s) upstream of ELMO1 during engulfment were not known, we performed a yeast two-hybrid screen, with N-term as bait. After screening more than 1.1 3 10 7 colonies from a mouse embryo library, followed by several subscreens for specificity, we identified a single membrane protein, BAI1.BAI1 belongs to subgroup VII of the adhesion-type G-proteincoupled receptor (GPCR) family 7-9 , with extended extracellular termini containing multiple domains and motifs that are thought to function in cell-cell or cell-matrix interactions 9 . BAI1 (1,584 residues) has an 943-residue extracellular region, a seven-transmembrane

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“…Also, a recent report by Iioka et al indicates that paxillin, a CrkII-binding protein important for cytoskeletal rearrangement, is ubiquitylated and associates with the proteasome complex via a RING finger protein (XRNF185) in Xenopus embryos to regulate mesodermal cell motility and adhesion during gastrulation 23 . Although the importance of paxillin in engulfment is poorly understood, this finding raises the possibility that CrkII may be another potential target for ubiquitylation, which could help explain the somewhat enigmatic role of CrkII in engulfment 24 .…”

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confidence: 99%

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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Identification of two signaling submodules within the CrkII/ELMO/Dock180 pathway regulating engulfment of apoptotic cells

Cited by 48 publications

References 38 publications

A pathway for phagosome maturation during engulfment of apoptotic cells

A pathway for phagosome maturation during engulfment of apoptotic cells

BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/Dock180/Rac module

Pallbearer and friends: lending a hand in apoptotic cell clearance

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