The recognition and clearance of dead cells is a process that must occur efficiently to prevent an autoimmune or inflammatory response. Recently, a process was identified wherein the autophagy machinery is recruited to pathogen-containing phagosomes, termed MAPLC3A (LC3)-associated phagocytosis (LAP), which results in optimal degradation of the phagocytosed cargo. Here, we describe the engagement of LAP upon uptake of apoptotic, necrotic, and RIPK3-dependent necrotic cells by macrophages. This process is dependent on some members of the classical autophagy pathway, including Beclin1, ATG5, and ATG7. In contrast, ULK1, despite being required for autophagy, is dispensable for LAP induced by uptake of microbes or dead cells. LAP is required for efficient degradation of the engulfed corpse, and in the absence of LAP, engulfment of dead cells results in increased production of proinflammatory cytokines and decreased production of anti-inflammatory cytokines. LAP is triggered by engagement of the TIM4 receptor by either phosphatidylserine (PtdSer)-displaying dead cells or PtdSer-containing liposomes. Therefore, the consequence of phagocytosis of dead cells is strongly affected by those components of the autophagy pathway involved in LAP.inflammation | autoimmunity | apoptosis | necrosis
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...
SummaryDuring development, the processes of cell division, differentiation and apoptosis must be precisely coordinated in order to maintain tissue homeostasis. The nematode C. elegans is a powerful model system in which to study cell death and its control. C. elegans apoptotic cells condense and form refractile corpses under differential interference contrast (DIC) microscopy. Activation of the GTPase CED-10 (Rac) in a neighbouring cell mediates the recognition and engulfment of the cell corpse. After inclusion of the engulfed corpse in a phagosome, different proteins are sequentially recruited onto this organelle to promote its acidification and fusion with lysosomes, leading to the enzymatic degradation of the cell corpse. We show that CCZ-1, a protein conserved from yeasts to humans, mediates the digestion of these apoptotic corpses. CCZ-1 seems to act in lysosome biogenesis and phagosome maturation by recruiting the GTPase RAB-7 over the phagosome.
SUMMARYMulticellular organisms use programmed cell death to eliminate unwanted or potentially harmful cells. Improper cell corpse removal can lead to autoimmune diseases. The development of interventional therapies that increase engulfment activity could represent an attractive approach to treat such diseases. Here, we describe mtm-1, the Caenorhabditis elegans homolog of human myotubularin 1, as a potential negative regulator of apoptotic cell corpse clearance. Loss of mtm-1 function leads to substantially reduced numbers of persistent cell corpses in engulfment mutants, which is a result of a restoration of engulfment function rather than of impaired or delayed programmed cell death. Epistatic analyses place mtm-1 upstream of the ternary GEF complex, which consists of ced-2, ced-5 and ced-12, and parallel to mig-2. Over-activation of engulfment results in the removal of viable cells that have been brought to the verge of death under limiting caspase activity. In addition, mtm-1 also promotes phagosome maturation in the hermaphrodite gonad, potentially through CED-1 receptor recycling. Finally, we show that the CED-12 PH domain can bind to PtdIns(3,5)P 2 (one target of MTM-1 phosphatase activity), suggesting that MTM-1 might regulate CED-12 recruitment to the plasma membrane.
Clearance of apoptotic cells is of key importance during development, tissue homeostasis and wound healing in multi-cellular animals. Genetic studies in the nematode Caenorhabditis elegans have identified a set of genes involved in the early steps of cell clearance, in particular the recognition and internalization of apoptotic cells. A pathway that orchestrates the maturation of phagosomes containing ingested apoptotic cells in the worm has recently been described. However, many steps in this pathway remain elusive. Here we show that the C. elegans SNX9-family member LST-4 (lateral signaling target) and its closest mammalian orthologue SNX33 play an evolutionary conserved role during apoptotic cell corpse clearance. In lst-4 deficient worms, internalized apoptotic cells accumulated within non-acidified, DYN-1-positive but RAB-5-negative phagosomes. Genetically, we show that LST-4 functions at the same step as DYN-1 during corpse removal, upstream of the GTPase RAB-5. We further show that mammalian SNX33 rescue C. elegans lst-4 mutants and that overexpression of truncated SNX33 fragments interfered with phagosome maturation in a mammalian cell system. Taken together, our genetic and cell biological analyses suggest that LST-4 is recruited through a combined activity of DYN-1 and VPS-34 to the early phagosome membrane, where it cooperates with DYN-1 to promote recruitment/retention of RAB-5 on the early phagosomal membrane during cell corpse clearance. The functional conservation between LST-4 and SNX33 indicate that these early steps of apoptotic phagosome maturation are likely conserved through evolution.
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