Apoptotic cells release ‘find-me’ signals at the earliest stages of death to recruit phagocytes1. The nucleotides ATP and UTP represent one class of find-me signals2, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 over-expression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the ‘selective’ plasma membrane permeability of early apoptotic cells to specific dyes3. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

The C. elegans genes ced-2, ced-5, and ced-10, and their mammalian homologs crkII, dock180, and rac1, mediate cytoskeletal rearrangements during phagocytosis of apoptotic cells and cell motility. Here, we describe an additional member of this signaling pathway, ced-12, and its mammalian homologs, elmo1 and elmo2. In C. elegans, CED-12 is required for engulfment of dying cells and for cell migrations. In mammalian cells, ELMO1 functionally cooperates with CrkII and Dock180 to promote phagocytosis and cell shape changes. CED-12/ELMO-1 binds directly to CED-5/Dock180; this evolutionarily conserved complex stimulates a Rac-GEF, leading to Rac1 activation and cytoskeletal rearrangements. These studies identify CED-12/ELMO as an upstream regulator of Rac1 that affects engulfment and cell migration from C. elegans to mammals.

Phagosome maturation is the process by which internalized particles (such as bacteria and apoptotic cells) are trafficked into a series of increasingly acidified membrane-bound structures, ultimately leading to particle degradation. Studies in model organisms and mammals, along with characterization of the phagosomal proteome have led to the identification of numerous candidate proteins that cooperate to control the maturation of phagosomes containing different particles. A subset of these candidates make up the first pathway that has been identified for maturation of apoptotic cell-containing phagosomes. suggesting the use of a machinery that is distinct from receptor-mediated endocytosis.Eukaryotic cells internalize a variety of particles during their lifetime. The uptake of particles >0.5 μm in size by cells is considered phagocytosis, whereas particles <0.5 μm are taken up by receptor-mediated endocytosis or pinocytosis. There are distinct types of phagocytosis, which tend to be ligand specific: bacteria (~0.5-3 μm) or yeast (~3-4 μm) are internalized by macrophages via scavenger receptors; microorganisms can also be coated with serum components (eg. complement) or antibodies and then taken up via complement 1, 2 or Fc receptors 3, 4 , respectively. Cells undergoing apoptosis, which can range in size from 5-50 μm, must also be removed (Box 1): in fact, we turn over ~200 billion cells each day of our life, perhaps making apoptotic cell removal one of the most common type of phagocytosis occurring throughout life 5 . Thus, understanding how apoptotic cells are phagocytosed and processed is a fundamentally important biological problem. Apoptotic cell turnover begins with the induction of an apoptotic programme or other cellular changes that mark them for removal 6,7 . The subsequent recognition of altered features by phagocytes leads to their highly efficient and immunologically silent removal 8 . Apoptotic cells can be taken up either by neighbouring cells or by professional phagocytes such as macrophages 9-11 and dendritic cells 12,13 ; the contribution of each type of phagocyte to clearance in vivo has not been extensively addressed.Phagosome maturation can be viewed as the end of the phagocytic process, when internalized apoptotic cells or bacteria must be efficiently degraded. Once a particle is internalized, it "matures" through a series of increasingly acidified membrane-bound Corresponding authors: Kodi S. Ravichandran

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...

Graphical Abstract Highlights d Engulfment of dying cells elevates macrophage fatty acids and oxygen consumption d Mitochondrial respiration during efferocytosis is required for NAD homeostasis d NAD mobilization is required for anti-inflammatory responses in macrophages d Defects in myeloid mitochondrial complex III impair wound healing

The removal of apoptotic cells is essential for the physiological well being of the organism. In Caenorhabditis elegans, two conserved, partially redundant genetic pathways regulate this process. In the first pathway, the proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively) function to activate CED-10 (Rac1). In the second group, the candidate receptor CED-1 (CD91/LRP/SREC) probably recognizes an unknown ligand on the apoptotic cell and signals via its cytoplasmic tail to the adaptor protein CED-6 (hCED-6/GULP), whereas CED-7 (ABCA1) is thought to play a role in membrane dynamics. Molecular understanding of how the second pathway promotes engulfment of the apoptotic cell is lacking. Here, we show that CED-1, CED-6 and CED-7 are required for actin reorganization around the apoptotic cell corpse, and that CED-1 and CED-6 colocalize with each other and with actin around the dead cell. Furthermore, we find that the CED-10(Rac) GTPase acts genetically downstream of these proteins to mediate corpse removal, functionally linking the two engulfment pathways and identifying the CED-1, -6 and -7 signalling module as upstream regulators of Rac activation.

SUMMARY Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue level autonomy, has never been ascertained in vivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions, specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ~ 10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks and light contributes to tissue-autonomous clock function.

Skeletal muscle arises from the fusion of precursor myoblasts into multinucleated myofibers1,2. While conserved transcription factors and signaling proteins involved in myogenesis have been identified, upstream regulators are less well understood. Here, we report an unexpected discovery that the membrane protein BAI1, previously linked to recognition of apoptotic cells by phagocytes3, promotes myoblast fusion. Endogenous BAI1 expression increased during myoblast fusion, and BAI1 overexpression enhanced myoblast fusion via signaling through ELMO/Dock180/Rac1 proteins4. During myoblast fusion, a fraction of myoblasts underwent apoptosis and exposed phosphatidylserine (PtdSer), an established ligand for BAI13. Blocking apoptosis potently impaired myoblast fusion, and adding back apoptotic myoblasts restored fusion. Furthermore, primary human myoblasts could be induced to form myotubes by adding apoptotic myoblasts, even under normal growth conditions. In vivo, myofibers from Bai1−/− mice are smaller than wild-type littermates. Muscle regeneration after injury was also impaired in Bai1−/− mice, highlighting a role for BAI1 in mammalian myogenesis. Collectively, these data identify signaling via the phosphatidylserine receptor BAI1 and apoptotic cells as novel promoters of myoblast fusion, with significant implications for muscle development and repair.