Two-step engulfment of apoptotic cells

Apoptotic cells expose phosphatidylserine on their surface as an "eat me" signal, and macrophages respond by engulfing them. Although several molecules that specifically bind phosphatidylserine have been identified, the molecular mechanism that triggers engulfment remains elusive. Here, using a mouse pro-B cell line, Ba/F3, that grows in suspension, we reconstituted the engulfment of apoptotic cells. The parental Ba/F3 cells did not engulf apoptotic cells. Ba/F3 transformants expressing T cell immunoglobulin- and mucin-domain-containing molecule 4 (Tim4), a type I membrane protein that specifically binds phosphatidylserine, efficiently bound apoptotic cells in a phosphatidylserine-dependent manner but did not engulf them. However, Ba/F3 transformants expressing both Tim4 and the integrin α(v)β(3) complex bound to and engulfed apoptotic cells in the presence of milk fat globule epidermal growth factor factor VIII (MFG-E8), a secreted protein that can bind phosphatidylserine and integrin α(v)β(3). These results indicate that the engulfment of apoptotic cells proceeds in two steps: Tim4 tethers apoptotic cells, and the integrin α(v)β(3) complex mediates engulfment in coordination with MFG-E8. A similar two-step engulfment of apoptotic cells was observed with mouse resident peritoneal macrophages. Furthermore, the Tim4/integrin-mediated engulfment by the Ba/F3 cells was enhanced in cells expressing Rac1 and Rab5, suggesting that this system well reproduces the engulfment of apoptotic cells by macrophages.     

A pathway for phagosome maturation during engulfment of apoptotic cells

Removal of apoptotic cells is critical for the physiological well-being of the organism and defects in corpse removal have been linked to disease states. Genes regulating corpse recognition and internalization have been identified, but few molecules involved in the processing of internalized corpses are known. Through a combination of targeted and unbiased reverse genetic screens in Caenorhabditis elegans, and studies in mammalian cells, we have identified genes required for maturation of apoptotic-cell-containing phagosomes. We have further ordered these candidates, which include the GTPases RAB-5 and RAB-7 and the HOPS complex, into a coherent linear pathway for the maturation of apoptotic cells within phagosomes. In depth analysis of two additional candidate genes, the phosphatidylinositol 3 kinase (PI(3)K) vps-34 (A001762) and dyn-1/dynamin, showed an accumulation of internalized, but undegraded, corpses within abnormal Rab5-negative phagosomes. We ordered these candidates in our pathway, with DYN-1 functioning upstream of VPS-34 in the recruitment and/or retention of RAB-5 to the phagosome. Finally, we have also identified a previously undescribed biochemical complex containing Vps34, dynamin and Rab5(GDP), thus providing a mechanism for Rab5 recruitment to the nascent phagosome.     

Autophagy is not universally required for phosphatidyl-serine exposure and apoptotic cell engulfment during neural development

Apoptosis and autophagy are physiological processes implicated in the maintenance of cell and tissue homeostasis. We took advantage of the existence of multiple phases of

developmental cell death in the embryonic chick retina and of the availability of shortterm organotypic retinal cultures to approach the possible relationship between

apoptosis and autophagy during neural development. We examined retinas at embryonic day 5, an early stage at which cell death is related to eye morphogenesis and to retinal

ganglion cell generation, as well as at embryonic day 9, when cell death is associated with neurotrophic support of the retinal ganglion cells. Exposure to 3-methyl-adenine, a

classical inhibitor of autophagy, elicited a selective accumulation of apoptotic bodies in the dorsotemporal area of embryonic day 5 retinas where neurogenesis is taking place.

This accumulation was correlated with a blockage of phosphatidyl-serine presentation and, consequently, with a lack of engulfment of the dying cells by their neighbors. In

striking contrast, none of these phenomena were observed in association with cell death in the optic nerve and optic fissure at embryonic day 5, or in embryonic day 9 retinas.

Our data suggest that autophagy is essential for phosphatidyl-serine presentation by apoptotic cells during the phase of cell death associated to neurogenesis, but this is not a

universal requirement for all phases of cell death occurring during retinal development.     

Annexin I is an endogenous ligand that mediates apoptotic cell engulfment

Engulfment of apoptotic cells requires presentation of new cell surface ligands by the dying cells. Using a differential proteomics technology, we identify that annexin I is a caspase-dependent engulfment ligand; it is recruited from the cytosol and exported to the outer plasma membrane leaflet, colocalizes with phosphatidylserine, and is required for efficient clearance of apoptotic cells. Furthermore, phosphatidylserine receptor (PSR) clustering around apoptotic cells indicates a requirement for annexin I. In the nematode Caenorhabditis elegans, downregulation of the annexin homolog prevents efficient engulfment of pharyngeal cell corpses. These results provide novel mechanistic insights into how apoptotic cells are removed and may explain a pathogenic mechanism of chronic inflammatory diseases where annexin I autoantibodies have been described.     

Thymosin beta4 is involved in stabilin-2-mediated apoptotic cell engulfment

Stabilin-2 was recently identified as a novel receptor for membrane phosphatidylserine of apoptotic cells. To identify proteins that were candidates for stabilin-2 cytoplasmic domain binding, we screened a human spleen cDNA library using the yeast two-hybrid system. We found that thymosin beta4 interacts with the stabilin-2 cytoplasmic domain and is co-localized with stabilin-2 at the phagocytic cup. Knockdown of thymosin beta4 significantly decreased the phagocytic activity of stabilin-2, whereas overexpression of thymosin beta4 increased this activity. Additionally, amino acids 2504-2514 of stabilin-2 cytoplasmic domain were found to be responsible for the interaction with thymosin beta4. Taken together, these results suggest that thymosin beta4 is a downstream molecule of stabilin-2 that plays a role in stabilin-2-mediated cell corpse clearance.     

Cues for apoptotic cell engulfment: eat-me, don't eat-me and come-get-me signals

Efficient elimination of cells undergoing programmed cell death is crucial for normal tissue homeostasis and for the regulation of immune responses. This review examines unique signals presented by apoptotic cells and the mechanisms by which phagocytes recognize and respond to these signals to orchestrate the selective and rapid removal of apoptotic cells. Such unique signals include direct and indirect ‘eat-me’ markers on the apoptotic cell surface, the absence of ‘don't eat-me’ markers normally found on living cells and soluble ‘come-get-me’ signals secreted by apoptotic cells to attract phagocytes to sites of apoptotic cell death. Once apoptotic cells are identified, their uptake by phagocytes further depends on the molecular machinery highly conserved from Caenorhabditis elegans to mammals.     

ABCA1 and the engulfment of apoptotic cells

Programmed cell death is one of the major devices controlling cellular homeostasis. However, the generation of cell debris that follows the execution phase of apoptosis has to be backed up by their efficient removal by phagocyte. This highly dynamic process requires the concerted action of a number of surface molecules able to recognize early signals of membrane modifications on the apoptotic prey. Among those, the loss of phospholipid asymmetry and exposure of phosphatidylserine on the prey to be is determinant to engage phagocyte receptors and trigger the removal of corpses. A loss of membrane lipid asymmetry occurs also on the phagocyte determining its efficiency as an undertaker. Here we will discuss how, in our mind, the ATP binding cassette transporter, ABCA1, by its action on the arrangement of lipids at the phagocyte membrane, may actively promote their competence to engulf.     

Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis

Axon pruning is a common phenomenon in neural circuit development. Previous studies demonstrate that the engulfing action of glial cells is essential in this process. The underlying molecular mechanisms, however, remain unknown. We show that draper (drpr) and ced-6, which are essential for the clearance of apoptotic cells in C. elegans, function in the glial engulfment of larval axons during Drosophila metamorphosis. The drpr mutation and glia-specific knockdown of drpr and ced-6 by RNA interference suppress glial engulfment, resulting in the inhibition of axon pruning. drpr and ced-6 interact genetically in the glial action. Disruption of the microtubule cytoskeleton in the axons to be pruned occurs via ecdysone signaling, independent of glial engulfment. These findings suggest that glial cells engulf degenerating axons through drpr and ced-6. We propose that apoptotic cells and degenerating axons of living neurons are removed by a similar molecular mechanism.     

IgG autoantibodies against deposited C3 inhibit macrophage-mediated apoptotic cell engulfment in systemic autoimmunity

Defective clearance of apoptotic cells has been shown in systemic lupus erythematosus (SLE) and is postulated to enhance autoimmune responses by increasing access to intracellular autoantigens. Until now, research has emphasized inherited rather than acquired impairment of apoptotic cell engulfment in the pathogenesis of SLE. In this study, we confirm previous results that efficient removal of apoptotic cells (efferocytosis) is bolstered in the presence of wild-type mouse serum, through the C3 deposition on the apoptotic cell surface. In contrast, sera from three mouse models of SLE, Mer(KD), MRL(lpr), and New Zealand Black/WF1 did not support and in fact actively inhibited apoptotic cell uptake. IgG autoantibodies were responsible for the inhibition, through the blockade of C3 recognition by macrophages. Consistent with this, IgG removal reversed the inhibitory activity within autoimmune serum, and purified autoimmune IgG blocked both the detection of C3 on apoptotic cells and C3-dependent efferocytosis. Sera from SLE patients demonstrated elevated anti-C3b IgG that blocked detection of C3 on apoptotic cells, activity that was not found in healthy controls or patients with rheumatoid arthritis, nor in mice prior to the onset of autoimmunity. We propose that the suppression of apoptotic cell disposal by Abs against deposited C3 may contribute to increasing severity and/or exacerbations in SLE.     

Metabolic signatures in apoptotic human cancer cell lines

Cancer cells have several specific metabolic features, which have been explored for targeted therapies. Agents that promote apoptosis in tumors are currently considered as a powerful tool for cancer therapeutics. The present study aimed to design a fast, reliable and robust system for metabolite measurements in cells lines to observe impact of apoptosis on the metabolome. For that purpose the NBS (newborn screen) mass spectrometry-based metabolomics assay was adapted for cell culture approach. In HEK 293 and in cancer cell lines HepG2, PC3, and MCF7 we searched for metabolic biomarkers of apoptosis differing from that of necrosis. Already nontreated cell lines revealed distinct concentrations of metabolites. Several metabolites indicative for apoptotic processes in cell culture including aspartate, glutamate, methionine, alanine, glycine, propionyl carnitine (C3-carnitine), and malonyl carnitine (C3DC-carnitine) were observed. In some cell lines metabolite changes were visible as early as 4?h after apoptosis induction and preceeding the detection by caspase 3/7 assay. We demonstrated for the first time that the metabolomic signatures might be used in the tests of efficacy of agents causing apoptosis in cell culture. These signatures could be obtained in fast high-throughput screening.     

Involvement of Beclin 1 in engulfment of apoptotic cells

Efficient apoptotic cell engulfment is important for both tissue homeostasis and immune response in mammals. In the present study, we report that Beclin 1 (a regulator of autophagy) is required for apoptotic cell engulfment. The engulfment process was largely abolished in Beclin 1 knock-out cells, and Beclin 1 knockdown significantly decreased apoptotic cell internalization in macrophage and fibroblast cell lines. Beclin 1 was recruited to the early phagocytic cup along with the generation of phosphatidylinositol 3-phosphate and Rac1, which regulates actin dynamics in lamellipodia. No lamellipodia were formed in Beclin 1 knock-out cells, and Beclin 1 knockdown completely inhibited the promotion of engulfment by ectopic expression of Rac1. Beclin 1 was co-immunoprecipitated with Rac1. These data indicate that Beclin 1 coordinates actin dynamics and membrane phospholipid synthesis to promote efficient apoptotic cell engulfment.     

Two alternative mechanisms that regulate the presentation of apoptotic cell engulfment signal in Caenorhabditis elegans

Phosphatidylserine exposed on the surface of apoptotic mammalian cells is considered an "eat-me" signal that attracts phagocytes. The generality of using phosphatidylserine as a clearance signal for apoptotic cells in animals and the regulation of this event remain uncertain. Using ectopically expressed mouse MFG-E8, a secreted phosphatidylserine-binding protein, we detected specific exposure of phosphatidylserine on the surface of apoptotic cells in Caenorhabditis elegans. Masking the surface phosphatidylserine inhibits apoptotic cell engulfment. CED-7, an ATP-binding cassette (ABC) transporter, is necessary for the efficient exposure of phosphatidylserine on apoptotic somatic cells, and for the recognition of these cells by phagocytic receptor CED-1. Alternatively, phosphatidylserine exposure on apoptotic germ cells is not CED-7 dependent, but instead requires phospholipid scramblase PLSC-1, a homologue of mammalian phospholipid scramblases. Moreover, deleting plsc-1 results in the accumulation of apoptotic germ cells but not apoptotic somatic cells. These observations suggest that phosphatidylserine might be recognized by CED-1 and act as a conserved eat-me signal from nematodes to mammals. Furthermore, the two different biochemical activities used in somatic cells (ABC transporter) and germ cells (phospholipid scramblase) suggest an increased complexity in the regulation of phosphatidylserine presentation in response to apoptotic signals in different tissues and during different developmental stages.     

The active role of corpse engulfment pathways during cell competition

Cell competition was first described in imaginal discs of genetically-mosaic Drosophila. In extreme cases, cell competition can replace entire compartments with the descendents of a single cell. We recently identified five genes that are required by wild-type epithelial cells to kill neighboring Minute cells during cell competition. These draper, wasp, phosphatidyl-serine receptor, MBC/DOCK180 and Rac1 genes, were each previously implicated in the engulfment of apoptotic corpses. The results draw attention to the active, killing role of engulfing cells during cell competition. Here we discuss the contributions of these engulfment genes to Minute competition in more detail, and compare Minute competition with competition between cells expressing different levels of Myc, or of Warts pathway genes. We also speculate about how cell interactions at clone boundaries may initiate cell competition.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.Key words: autophagy, Draper, programmed cell death, engulfment, developmentProgrammed cell death is required for animal development and tissue homeostasis. Improper cell death leads to pathologies including autoimmunity and cancer. Several morphological forms of cell death occur during animal development, including apoptosis and autophagic cell death. Autophagic cell death is characterized by the presence of autophagosomes in dying cells that are not known to be engulfed by phagocytes. Autophagic cell death is observed during several types of mammalian developmental cell death, including regression of the corpus luteum and involution of mammary and prostate glands.During macroautophagy (autophagy), cytoplasmic components are sequestered by autophagosomes and delivered to the lysosome for degradation. Autophagy is a cellular response to stress required for survival in response to starvation. Whereas autophagy has been associated with cell death, it is unknown how autophagy is distinguished during cell death and cell survival. Autophagy is induced in Drosophila in response to starvation in the fat body where it promotes cell survival, while autophagy is induced by the steroid hormone ecdysone in salivary glands where it promotes cell death. This allows studies of autophagy in different cell types and in response to different stimuli.Drosophila larval salivary glands die with autophagic cell death morphology and autophagy is required for their degradation. Expression of the caspase inhibitor p35 enhances salivary gland persistence in Atg mutants, suggesting that caspases and autophagy function in parallel during salivary gland degradation. Either activation of caspases or Atg1 misexpression is sufficient to induce ectopic salivary gland clearance. We queried genome-wide microarray data from purified dying salivary glands and noted the induction of engulfment genes, those required for a phagocyte to consume and degrade a dying cell. We also noted few detectable changes in engulfment genes in Drosophila larvae during starvation.We found that Drpr, the Drosophila orthologue of C. elegans engulfment receptor CED-1, is enriched in dying salivary glands, and drpr null mutants have persistent salivary glands. Interestingly, whereas knockdown of drpr in phagocytic blood cells fails to influence salivary gland clearance, expression of drpr-RNAi in salivary glands prevents gland clearance. Drosophila drpr is alternatively spliced to produce three isoforms. We found that drpr-I-specific knockdown prevents salivary gland degradation and Drpr-I expression in salivary glands of drpr null mutants rescues salivary gland persistence. Therefore, drpr is autonomously required for salivary gland clearance. However, how Drpr is induced or activated during hormone-regulated cell death remains to be determined.drpr knockdown fails to influence caspase activation, and caspase inhibitor p35 expression in drpr null mutants enhances salivary gland persistence, suggesting that Drpr functions downstream or parallel to caspases in dying salivary glands. Interestingly, we found that drpr knockdown in salivary glands prevents the formation of GFP-LC3 puncta. Further, Atg1 misexpression in salivary glands of drpr null mutants suppresses salivary gland persistence. drpr is therefore required for autophagy induction in salivary glands, and Atg1 functions downstream of Drpr in this tissue. We found that several other engulfment genes are required for salivary gland degradation. However, the Drpr signaling mechanism leading to autophagy induction in salivary glands remains to be elucidated.We tested whether drpr is a general regulator of autophagy. The Drosophila fat body is a nutrient storage and mobilization organ akin to the mammalian liver, and is a well-established model to study starvation-induced autophagy. We found that drpr-RNAi expression in fat body clone cells fails to prevent GFP-Atg8 puncta formation in response to starvation. Similarly, drpr null fat body clone cells form Cherry-Atg8 puncta after starvation. Strikingly, drpr-RNAi expression in salivary gland clone cells inhibits the formation of GFP-Atg8 puncta. Therefore, drpr is cell-autonomously required for autophagy induction in dying salivary gland cells, but not for autophagy induction in fat body cells after starvation. These findings suggest that distinct signaling mechanisms regulate autophagy in response to nutrient deprivation compared to steroid hormone induction. Little is known about what distinguishes autophagy function in cell survival versus death. It is possible that varying levels of autophagy are induced during specific cell contexts and that high levels of autophagy could overwhelm a cell—leading to cell death. Autophagic degradation of specific cargo, such as cell death inhibitors, could also contribute to cell death.Given recent interest in manipulation of autophagy for therapies, it is possible that factors such as Drpr could be used as biomarkers to distinguish autophagy leading to cell death versus cell survival. While it is generally accepted that augmentation of protein clearance by autophagy during neurodegeneration would be beneficial, the role of autophagy in tumor progression is less clear. For example, monoallelic loss of the human Atg6 homolog beclin 1 is prevalent in human cancers, suggesting that autophagy is a tumorsuppressive mechanism. Thus, autophagy enhancers have been proposed for cancer prevention. However, autophagy occurs in tumor cells as a survival mechanism, and autophagy inhibitors have been proposed for anti-cancer therapies. Understanding how autophagy is regulated in different contexts is critical for appropriate therapeutic strategies.     

Identification of TLT2 as an engulfment receptor for apoptotic cells

Clearance of apoptotic cells (efferocytosis) is critical to the homeostasis of the immune system by restraining inflammation and autoimmune response to intracellular Ags released from dying cells. TLRs-mediated innate immunity plays an important role in pathogen clearance and in regulation of the adaptive immune response. However, the regulation of efferocytosis by activation of TLRs has not been well characterized. In this study, we found that activation of TLR3 or TLR9, but not of TLR2, enhances engulfment of apoptotic cells by macrophages. We found that the activation of TLR3 upregulates the expression of triggering receptor expressed on myeloid cells (TREM)-like protein 2 (TLT2), a member of the TREM receptor family, on the surface of macrophages. Blocking TLT2 on the macrophage surface by either specific anti-TLT2 Ab or soluble TLT2 extracellular domain attenuated the enhanced ability of macrophages with TLR3 activation to engulf apoptotic cells. To the contrary, overexpression of TLT2 increased the phagocytosis of apoptotic cells. We found that TLT2 specifically binds to phosphatidylserine, a major "eat me" signal that is exposed on the surface of apoptotic cells. Furthermore, we found that TLT2 mediates phagocytosis of apoptotic cells in vivo. Thus, our studies identified TLT2 as an engulfment receptor for apoptotic cells. Our data also suggest a novel mechanism by which TREM receptors regulate inflammation and autoimmune response.     

The engulfment receptor Draper is required for autophagy during cell death

Autophagy is a process to degrade and recycle cytoplasmic contents. Autophagy is required for survival in response to starvation, but has also been associated with cell death. How autophagy functions during cell survival in some contexts and cell death in others is unknown. Drosophila larval salivary glands undergo programmed cell death requiring autophagy genes, and are cleared in the absence of known phagocytosis. Recently, we demonstrated that Draper (Drpr), the Drosophila homolog of C. elegans engulfment receptor CED-1, is required for autophagy induction

during cell death, but not during cell survival. drpr mutants fail to clear salivary glands. drpr knockdown in salivary glands prevents the induction of autophagy, and Atg1 misexpression in drpr null mutants suppresses salivary gland persistence. Surprisingly, drpr knockdown cell-autonomously prevents autophagy induction in dying salivary gland cells, but not in larval fat body cells following starvation. This is the first engulfment factor shown to function in cellular self-clearance, and the first report of a cell-death-specific autophagy regulator.     

A common set of engulfment genes mediates removal of both apoptotic and necrotic cell corpses in C. elegans

Similar to mammalian excitotoxic cell death, necrotic-like cell death (NCD) in Caenorhabditis elegans can be initiated by hyperactive ion channels. Here we investigate the requirements for genes that execute and regulate programmed cell death (PCD) in necrotic-like neuronal death caused by a toxic MEC-4 channel. Neither the kinetics of necrosis onset nor the total number of necrotic corpses generated is altered by any C. elegans mutation known to block PCD, which provides genetic evidence that the activating mechanisms for NCD and apoptotic cell death are distinct. In contrast, all previously reported ced genes required for phagocytotic removal of apoptotic corpses, as well as ced-12, a new engulfment gene we have identified, are required for efficient elimination of corpses generated by distinct necrosis-inducing stimuli. Our results show that a common set of genes acts to eliminate cell corpses irrespective of the mode of cell death, and provide the first identification of the C. elegans genes that are required for orderly removal of necrotic cells. As phagocytotic mechanisms seem to be conserved from nematodes to humans, our findings indicate that injured necrotic cells in higher organisms might also be eliminated before lysis through a controlled process of corpse removal, a hypothesis that has significant therapeutic implications.     

Requirement of adaptor protein GULP during stabilin-2-mediated cell corpse engulfment

The prompt clearance of cells undergoing apoptosis is critical during embryonic development and normal tissue turnover, as well as during inflammation and autoimmune responses. We recently demonstrated that stabilin-2 is a phosphatidylserine receptor that mediates the clearance of apoptotic cells, thereby releasing the anti-inflammatory cytokine, transforming growth factor-beta. However, the downstream signaling components of stabilin-2-mediated phagocytosis are not known. Here, we provide evidence that the adaptor protein, GULP, physically and functionally interacts with the stabilin-2 cytoplasmic tail. Using fluorescent resonance energy transfer analysis and biochemical approaches, we show that GULP directly binds to the cytoplasmic tail of stabilin-2. Knockdown of endogenous GULP expression significantly decreased stabilin-2-mediated phagocytosis. Conversely, overexpression of GULP caused an increase in aged cell engulfment. The phosphotyrosine binding (PTB) domain of GULP was sufficient for the interaction with stabilin-2; therefore, transduction of TAT fusion PTB domain acts as a dominant negative, resulting in impaired engulfment of aged red blood cells in stabilin-2 expressing cells. In addition, the PTB domain of GULP was able to specifically interact with the NPXY motif of the stabilin-2 cytoplasmic tail. Taken together, these results indicate that GULP is a likely downstream molecule in the stabilin-2-mediated signaling pathway and plays an important role in stabilin-2-mediated phagocytosis.     

ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine

ATP-binding-cassette transporter 1 (ABC1) has been implicated in processes related to membrane-lipid turnover. Here, using in vivo loss-of-function and in vitro gain-of-function models, we show that ABC1 promotes Ca2+-induced exposure of phosphatidylserine at the membrane, as determined by a prothrombinase assay, membrane microvesiculation and measurement of transbilayer redistribution of spin-labelled phospholipids. That ABC1 promotes engulfment of dead cells is shown by the impaired ability of ABC1-deficient macrophages to engulf apoptotic preys and by the acquisition of phagocytic behaviour by ABC1 transfectants. Release of membrane phospholipids and cholesterol to apo-AI, the protein core of the cholesterol-shuttling high-density lipoprotein (HDL) particle, is also ABC1-dependent. We propose that both the efficiency of apoptotic-cell engulfment and the efflux of cellular lipids depend on ABC1-induced perturbation of membrane phosphatidylserine turnover. Transient local exposure of anionic phospholipids in the outer membrane leaflet may be sufficient to alter the general properties of the membrane and thus influence discrete physiological functions.     

A conserved role for SNX9-family members in the regulation of phagosome maturation during engulfment of apoptotic cells

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.