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Fertilization is essential for species survival. Although Izumo1 and Juno are critical for initial interaction between gametes, additional molecules necessary for sperm:egg fusion on both the sperm and the oocyte remain to be defined. Here, we show that phosphatidylserine (PtdSer) is exposed on the head region of viable and motile sperm, with PtdSer exposure progressively increasing during sperm transit through the epididymis. Functionally, masking phosphatidylserine on sperm via three different approaches inhibits fertilization. On the oocyte, phosphatidylserine recognition receptors BAI1, CD36, Tim-4, and Mer-TK contribute to fertilization. Further, oocytes lacking the cytoplasmic ELMO1, or functional disruption of RAC1 (both of which signal downstream of BAI1/BAI3), also affect sperm entry into oocytes. Intriguingly, mammalian sperm could fuse with skeletal myoblasts, requiring PtdSer on sperm and BAI1/3, ELMO2, RAC1 in myoblasts. Collectively, these data identify phosphatidylserine on viable sperm and PtdSer recognition receptors on oocytes as key players in sperm:egg fusion., Izumo and Juno are receptors on sperm and eggs respectively required for fusion, but other factors for sperm-egg fusion are poorly studied. Here, the authors report that phosphatidylserine, found mainly on cells marked for death, is also present on motile sperm, recognized by egg receptors, and is required for sperm-egg fusion.

Transmembrane protein engulfment receptors expressed on the surface of phagocytes engage ligands on apoptotic cells and debris to initiate a sequence of events culminating in material internalization and immunologically beneficial outcomes. Engulfment receptors are modular, comprised of functionally independent extracellular ligation domains and cytosolic signaling motifs. Cognate kinases, adaptors, and phosphatases regulate engulfment by controlling the degree of receptor activation in phagocyte plasma membranes, thus acting as receptor-proximal signaling modules. Here, we review recent efforts to reprogram phagocytes using modular synthetic receptors composed of antibody-based extracellular domains fused to engulfment receptor signaling domains. To aid the development of new phagocyte reprogramming methods, we then define the kinases, adaptors, and phosphatases that regulate a conserved family of engulfment receptors. Finally, we discuss current challenges and opportunities for the field.

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.

Summary Macrophages must not only be responsive to an array of different stimuli, such as infection and cellular damage, but also perform phagocytosis within the diverse and complex tissue environments found in vivo. This requires a high degree of morphological and therefore cytoskeletal plasticity. Here, we use the exceptional genetics and in vivo imaging of Drosophila embryos to study macrophage phagocytic versatility during apoptotic corpse clearance. We find that macrophage phagocytosis is highly robust, arising from their possession of two distinct modes of engulfment that utilize exclusive suites of actin-regulatory proteins. “Lamellipodial phagocytosis” is Arp2/3-complex-dependent and allows cells to migrate toward and envelop apoptotic corpses. Alternatively, Diaphanous and Ena drive filopodial phagocytosis to reach out and draw in debris. Macrophages switch to “filopodial phagocytosis” to overcome spatial constraint, providing the robust plasticity necessary to ensure that whatever obstacle they encounter in vivo, they fulfil their critical clearance function., Graphical Abstract, Highlights • Macrophages use two distinct modes of engulfment: lamellipodial and filopodial phagocytosis • Arp2/3-complex-dependent lamellipodial phagocytosis involves envelopment via a lamellipod • Filopodial phagocytosis involves phagocytic filopods extended by Dia and/or Ena • Macrophages switch to filopodial phagocytosis to overcome spatial constraint in vivo, Through the use of Drosophila genetics and the excellent in vivo imaging possible in the fly embryo, Davidson and Wood demonstrate that macrophages switch from engulfing debris via sheet-like lamellipods (lamellipodial phagocytosis) to extending phagocytic filopods (filopodial phagocytosis) when spatially confined, thus maintaining their critical clearance function in vivo.

The clearance of dead cells is a fundamental process in the maintenance of tissue homeostasis. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signaling pathways that act redundantly to regulate this engulfment process: the ced-1/-6/-7 and ced-2/-5/-12 pathways. Of these engulfment genes, only the ced-7/ABCA1 ortholog remains to be identified in D. melanogaster. Homology searches have revealed a family of putative ced-7/ABCA1 homologs encoding ATP-binding cassette (ABC) transporters in D. melanogaster. To determine which of these genes functions similarly to ced-7/ABCA1, we analyzed mutants for engulfment phenotypes in oogenesis, during which nurse cells (NCs) in each egg chamber undergo programmed cell death (PCD) and are removed by neighboring phagocytic follicle cells (FCs). Our genetic analyses indicate that one of the ABC transporter genes, which we have named Eato (Engulfment ABC Transporter in the ovary), is required for NC clearance in the ovary and acts in the same pathways as drpr, the ced-1 ortholog, and in parallel to Ced-12 in the FCs. Additionally, we show that Eato acts in the FCs to promote accumulation of the transmembrane receptor Drpr, and promote membrane extensions around the NCs for their clearance. Since ABCA class transporters, such as CED-7 and ABCA1, are known to be involved in lipid trafficking, we propose that Eato acts to transport membrane material to the growing phagocytic cup for cell corpse clearance. Our work presented here identifies Eato as the ced-7/ABCA1 ortholog in D. melanogaster, and demonstrates a role for Eato in Drpr accumulation and phagocytic membrane extensions during NC clearance in the ovary.

Macrophages are specialized phagocytic cells involved in clearing invading pathogens. Previously we reported that engulfment and cell motility protein 1 (ELMO1) in macrophages mediates bacterial internalization and intestinal inflammation. Here we studied the role of ELMO1 in the fate of internalized targets. ELMO1 is present in the intracellular vesicles and enhances accumulation of the protein LC3B following engulfment of Salmonella or treatment with autophagy-inducing rapamycin. The protein ATG5 and the kinase ULK1 are involved in classical autophagy, while LC3-associated phagocytosis is ULK1 independent. ATG5 but not ULK1 cooperated with ELMO1 in LC3 accumulation after infection, suggesting the ELMO1 preferentially regulated LC3-associated phagocytosis. Because LC3-associated phagocytosis delivers cargo for degradation, the contribution of ELMO1 to the lysosome degradation pathways was evaluated by studying pH and cathepsin B activity. ELMO1-depleted macrophages showed a time-dependent increase in pH and a decrease in cathepsin B activity associated with bacterial survival. Together, ELMO1 regulates LC3B accumulation and antimicrobial responses involved in the clearance of enteric pathogens. This paper investigated how innate immune pathways involving ELMO1 work in a coordinated fashion to eliminate bacterial threats. ELMO1 is present in the phagosome and enhances bacterial clearance by differential regulation of lysosomal acidification and enzymatic activity.

SUMMARY Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that is deposited at the site of gonad-to-cloaca fusion and is removed during mating. Inhibition of lobe scission inhibits linker cell death, demonstrating that the linker cell invades its host while alive. Our findings demonstrate a developmental function for entosis: to eliminate a migrating cell and facilitate gonad-to-cloaca fusion, which is required for fertility., Graphical Abstract, In Brief Entosis is a cell death mechanism, previously observed in cancer cell populations, that involves the invasion of live cells into their neighbors. Lee et al. now show that entosis has a developmental function in C. elegans, clearing the linker cell during gonad formation.

Efferocytosis, the clearing of dead or dying cells from living tissues, is a highly programmed, vital process to maintain the healthy functioning of every organism. Disorders of efferocytosis have been linked to several chronic diseases including atherosclerosis and auto-immune diseases. To date several different assays to determine phagocytosis, using microscopy or FACS analysis with labelled targets, have been developed. However, many of these are unable to differentiate between cells that have truly been phagocytosed and those still present on the surface of the macrophages hindering exact assessment of efferocytotic capacity. We herein describe AnxA5-pHrodo and its negative control M1234-pHrodo as new molecular probes to measure in vitro as well as ex-vivo efferocytotic capacity.

Apoptotic cells expressing phosphatidylserine (PS) on their cell surface are directly or indirectly recognized by phagocytes through PS-binding proteins. The PS-binding protein Tim-4 secures apoptotic cells to phagocytes to facilitate the engulfment of apoptotic cells. However, the molecular mechanism by which Tim-4 transduces signals to phagocytes during Tim-4-mediated efferocytosis is incompletely understood. Here, we report that Tim-4 collaborates with Mertk during efferocytosis through a biochemical interaction with Mertk. Proximal localization between the two proteins in phagocytes was observed by immunofluorescence and proximal ligation assays. Physical association between Tim-4 and Mertk, which was mediated by an interaction between the IgV domain of Tim-4 and the fibronectin type-III domain of Mertk, was also detected with immunoprecipitation. Furthermore, the effect of Mertk on Tim-4-mediated efferocytosis was abolished by GST-MertkFnIII, a soluble form of the fibronectin type-III domain of Mertk that disrupts the interaction between Tim-4 and Mertk. Taken together, the results from our study suggest that a physical interaction between Tim-4 and Mertk is necessary for Mertk to enhance efferocytosis mediated by Tim-4.

International audience; Phagocytosis and macroautophagy/autophagy are 2 processes involved in lysosome-mediated clearance of extracellular and intracellular components, respectively. Recent studies have identified the recruitment of the autophagic protein LC3 during phagocytosis of apoptotic corpses in what is now called LC3-associated phagocytosis (LAP). LAP is a distinct process from autophagy but it relies on some members of the autophagy pathway to allow efficient degradation of the phagocytosed cargo. We investigated whether both LC3/LGG-2 and GABARAP/LGG-1 are involved in phagocytosis of apoptotic corpses during embryonic development of Caenorhabditis elegans. We discovered that both LGG-1 and LGG-2 are involved in the correct elimination of apoptotic corpses, but that they have different functions. lgg-1 and lgg-2 mutants present a delay in phagocytosis of apoptotic cells but genetic analyses indicate that LGG-1 and LGG-2 act upstream and downstream of the engulfment pathways, respectively. Moreover, LGG-1 and LGG-2 display different cellular localizations with enrichment in apoptotic corpses and phagocytic cells, respectively. For both LGG-1 and LGG-2, subcellular localization is vesicular and dependent on UNC-51/ULK1, BEC-1/BECN1 and the lipidation machinery, indicating that their functions during phagocytosis of apoptotic corpses mainly rely on autophagy. Finally, we show that LGG-1 is involved in the exposure of the 'eat-me signal' phosphatidylserine at the surface of the apoptotic cell to allow its recognition by the phagocytic cell, whereas LGG-2 is involved in later steps of phagocytosis to allow efficient cell corpse clearance by mediating the maturation/degradation of the phagosome.

Involution is the process of post-lactational mammary gland regression to quiescence and it involves secretory epithelial cell death, stroma remodeling and gland repopulation by adipocytes. Though reportedly accompanying apoptosis, the role of autophagy in involution has not yet been determined. We now report that autophagy-related (ATG) proteins mediate dead cell clearance and suppress inflammation during mammary involution. In vivo, Becn1(+/-) and Atg7-deficient mammary epithelial cells (MECs) produced 'competent' apoptotic bodies, but were defective phagocytes in association with reduced expression of the MERTK and ITGB5 receptors, thus pointing to defective apoptotic body engulfment. Atg-deficient tissues exhibited higher levels of involution-associated inflammation, which could be indicative of a tumor-modulating microenvironment, and developed ductal ectasia, a manifestation of deregulated post-involution gland remodeling. In vitro, ATG (BECN1 or ATG7) knockdown compromised MEC-mediated apoptotic body clearance in association with decreased RAC1 activation, thus confirming that, in addition to the defective phagocytic processing reported by other studies, ATG protein defects also impair dead cell engulfment. Using two different mouse models with mammary gland-associated Atg deficiencies, our studies shed light on the essential role of ATG proteins in MEC-mediated efferocytosis during mammary involution and provide novel insights into this important developmental process. This work also raises the possibility that a regulatory feedback loop exists, by which the efficacy of phagocytic cargo processing in turn regulates the rate of engulfment and ultimately determines the kinetics of phagocytosis and dead cell clearance.

Microglia, the resident CNS macrophages, have been implicated in the pathogenesis of Rett Syndrome (RTT), an X-linked neurodevelopmental disorder. However, the mechanism by which microglia contribute to the disorder is unclear and recent data suggest that microglia do not play a causative role. Here, we use the retinogeniculate system to determine if and how microglia contribute to pathogenesis in a RTT mouse model, the Mecp2 null mouse (Mecp2tm1.1Bird/y). We demonstrate that microglia contribute to pathogenesis by excessively engulfing, thereby eliminating, presynaptic inputs at end stages of disease (≥P56 Mecp2 null mice) concomitant with synapse loss. Furthermore, loss or gain of Mecp2 expression specifically in microglia (Cx3cr1CreER;Mecp2fl/yor Cx3cr1CreER; Mecp2LSL/y) had little effect on excessive engulfment, synapse loss, or phenotypic abnormalities. Taken together, our data suggest that microglia contribute to end stages of disease by dismantling neural circuits rendered vulnerable by loss of Mecp2 in other CNS cell types. DOI: http://dx.doi.org/10.7554/eLife.15224.001, eLife digest Rett Syndrome is a neurodevelopmental disorder with symptoms that typically begin in girls between 6 and 18 months old. Those affected developmentally stagnate and regress – during which they lose some of their previously acquired skills and develop an array of physical impairments. Mutations in a gene called Mecp2 on the X chromosome cause most cases of Rett Syndrome. Mice that lack the Mecp2 gene develop symptoms similar to those seen in people with Rett Syndrome, and so such “Mecp2 null” mice are often used to study the disorder. Microglia, the resident immune cells of the central nervous system, have been implicated in the development of Rett Syndrome. Introducing microglia that carry the Mecp2 gene into Mecp2 null mice has been shown to reduce several disease-associated abnormalities. However, exactly how microglia contribute to these changes remains unknown. In addition, a more recent report failed to reproduce these findings, and instead obtained results suggesting that microglia do not affect the development of Rett syndrome. Schafer et al. now use the mouse visual system as a model to determine if and how microglia contribute to the development of Rett Syndrome. Like many other brain regions, the developing visual system initially has a surplus of connections between neurons, or synapses, which are subsequently pruned back. Schafer et al. previously showed in the developing visual system of early postnatal (5 days after birth) control mice (who express the Mecp2 gene) that microglia contribute to this pruning by engulfing and eliminating a subset of these excessive synaptic connections. The new experiments by Schafer et al. show that another wave of microglia-mediated synaptic pruning occurs in 40-day-old juvenile control mice. Because Mecp2 null mice begin to display features of Rett Syndrome when they’re about 40 days old, Schafer et al. tested whether the microglia of these animals inappropriately prune synaptic connections. While this process occurred normally in neonatal and juvenile Mecp2 null mice, microglia began to excessively engulf cells in Mecp2 null mice when they were around 56 days old. Unexpectedly, deleting or reintroducing the Mecp2 gene solely in the microglia of these mice had little effect on pruning activity of the microglia, and failed to affect Rett-syndrome-like symptoms in the mice. Taken together, the data presented by Schafer et al. suggest how microglia contribute to the final stages of Rett Syndrome: by dismantling circuits of neurons that are rendered vulnerable by the loss of the Mecp2 gene in other cell types. DOI: http://dx.doi.org/10.7554/eLife.15224.002

Background Programmed cell deaths in the nematode Caenorhabditis elegans are generally considered suicides. Dying cells are engulfed by neighboring cells in a process of phagocytosis. To better understand the interaction between the engulfment and death processes, we analyzed B.al/rapaav cell death, which has been previously described as engulfment-dependent and hence as a possible murder. Results We found that B.al/rapaav is resistant to caspase-pathway activation: the caspase-mediated suicide pathway initiates the cell-death process but is insufficient to cause B.al/rapaav death without the subsequent assistance of engulfment. When the engulfing cell P12.pa is absent, other typically non-phagocytic cells can display cryptic engulfment potential and facilitate this death. Conclusions We term this death an “assisted suicide” and propose that assisted suicides likely occur in other organisms. The study of assisted suicides might provide insight into non-cell autonomous influences on cell death. Understanding the mechanism that causes B.al/rapaav to be resistant to activation of the caspase pathway might reveal the basis of differences in the sensitivity to apoptotic stimuli of tumor and normal cells, a key issue in the field of cancer therapeutics., Howard Hughes Medical Institute, National Institutes of Health (U.S.) (Pre-Doctoral Training Grant T32GM007287)

Angiogenic endothelium plays a crucial role in tumor growth. During angiogenesis, complex alterations in the microenvironment occur. In response, the endothelium undergoes phenotypic changes, for example overexpression of αv-integrins. Here, we show that the overexpression of αv-integrins on angiogenic endothelial cells is engaged in phagocytic actions involving binding (“tethering”) and uptake (“tickling”) of lactadherin (also termed MFG-E8)–opsonized particles. Phosphatidylserine (PS)–exposing multilamellar vesicles, “aged” erythrocytes, and apoptotic melanoma cells incubated with lactadherin were all phagocytosed by angiogenic endothelial cells in vitro. Furthermore, we demonstrated lactadherin expression in and around tumor blood vessels making opsonization in situ plausible. By engineering the surface of erythrocytes with covalently coupled cyclic Arg-Gly-Asp (RGD) peptides—mimicking lactadherin opsonization—we could induce phagocytosis by angiogenic endothelial cells both in vitro and in vivo. In vitro, this was confirmed by cytochalasin D preincubation. When RGD-erythrocytes were administered intravenously in tumor-bearing mice, blood vessel congestion followed by tumor core necrosis was seen. Moreover, RGD-erythrocytes could delay tumor growth in a murine melanoma model, possibly through induction of tumor infarctions. These results reveal that angiogenic endothelial cells have phagocytic properties for lactadherin-opsonized large particles and apoptotic cells. Implications of our findings for diagnostic and therapy of angiogenesis-driven diseases are discussed.

Apoptotic cell death is an integral part of cell turnover in many tissues, and proper corpse clearance is vital to maintaining tissue homeostasis in all multicellular organisms. Even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. In Caenorhabditis elegans, two parallel and partly redundant conserved pathways act in cell corpse engulfment. The pathway for cytoskeletal rearrangement requires the small GTPase CED-10 Rac1 acting for an efficient surround of the dead cell. The CED-10 Rac pathway is also required for the proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. Parkin, the mammalian homolog of the C. elegans PDR-1, interacts with Rac1 in aged human brain and it is also implicated with actin dynamics and cytoskeletal rearrangements in Parkinsons's disease, suggesting that it might act on engulfment. Our genetic and biochemical studies indicate that PDR-1 inhibits apoptotic cell engulfment and DTC migration by ubiquitylating CED-10 for degradation.

Autophagy is a catabolic process through which damaged organelles and protein aggregates are delivered to lysosomes for degradation. Autophagy genes are reported to promote exposure of "eat me" signals on the surface of apoptotic cells, but whether they function in engulfing cells is not clear. Recently, we found that the autophagy mutants atg-18 and epg-5 are defective in removing apoptotic cells derived from the C. elegans Q neuroblast, a phenotype that can be fully rescued by expression of ATG-18 and EPG-5 in the engulfing cell. Loss of ATG-18 or EPG-5 does not affect cell corpse engulfment but causes defects in phagosomal recruitment of RAB-5 and RAB-7 and formation of phagolysosomes. EPG-5, ATG-18 and LGG-1 are sequentially recruited to phagosomes, suggesting that they function at different steps of phagosomal maturation. Our studies indicate that autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell. © 2012 Landes Bioscience.

9 páginas, 6 figuras -- PAGS nros. 964-972, 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 short-term organotypic retinal cultures to explore 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, This research was supported by grants from the Spanish Ministerio de Ciencia e Innovación (BFU2006-00508 to P.B. and SAF2007-66175 to E.J.dl.R.) and the Comunidad de Madrid (CCG06-CSIC/SAL-0821 to P.B.). M.A.M. is a FPU Fellow and P.B. is a Ramón y Cajal Fellow (both Ministerio de Ciencia e Innovación programs)

Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that is deposited at the site of gonad-to-cloaca fusion and is removed during mating. Inhibition of lobe scission inhibits linker cell death, demonstrating that the linker cell invades its host while alive. Our findings demonstrate a developmental function for entosis: to eliminate a migrating cell and facilitate gonad-to-cloaca fusion, which is required for fertility.