A pathway for phagosome maturation during engulfment of apoptotic cells

Abstract

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...

Do not let death do us part: ‘find-me’ signals in communication between dying cells and the phagocytes

“…19,20 Once internalized, phagolysosome maturation occurs through the action of several Rab-family GTPases eventually leading to the degradation of the ingested cell corpse. 21 'Find-me' signals Both non-professional phagocytes and resident professional phagocytes can clear dying cells under homeostatic conditions. Although neighboring cells may not need to 'migrate' toward an apoptotic cell to engulf, motile phagocytes such as a resident macrophage likely do.…”

Involvement of Beclin 1 in Engulfment of Apoptotic Cells

“…PI(3)P signaling is involved in the formation and maturation of both autophagosomes and phagosomes (13,14). Vps34 also regulates apoptotic cell engulfment (15). Although it has been reported that Beclin 1 is involved in the clearance of apoptotic cells during embryonic development by regulating the expression of PS (16), whether or not Beclin 1 plays a role in apoptotic cell engulfment by phagocytes is unclear.…”

“…It has been reported that PI(3)P is generated by the VPS34-PI3K complex on the phagosome membrane during apoptotic cell engulfment (15). The FYVE zinc-finger domain binds to PI(3)P. Therefore, a fluorescent protein-fused FYVE domain is widely used as a marker for PI(3)P. Accordingly, we examined the co-localization of an mCherry-fused FYVE domain (mCherry-FYVE) with Beclin 1. mCherry-FYVE accumulation was observed around the phagocytic cups, and there was prominent co-localization with Beclin 1 (Fig.…”

Nucleoside diphosphate kinases (NDPKs) in animal development

“…2b). DYN-1 is also essential for the degradation of apoptotic cells through its fundamental role in phagosome maturation: DYN-1 is necessary for recruitment and fusion of endosomes and lysosomes to phagosomes [121,123,124] (Fig. 2b).…”

“…2b). Recruitment of two endosomal markers, RAB-5 and RAB-7, also relies on DYN-1 activity [122][123][124]. RAB-5 is important for promoting the maturation of phagosomes containing apoptotic cells [124,125].…”