Publications

During an animal's life, many cells undergo apoptosis, a form of genetically programmed cell death. These cells are swiftly engulfed by other cells through phagocytosis and subsequently degraded inside phagosomes. Phagocytosis and macroautophagy/autophagy are two different cellular events: whereas phagocytosis is a cell-eat-cell event, autophagy, or "self-eating", occurs within one cell, resulting in the enveloping of protein aggregates or damaged organelles within double-membrane autophagosomes. Despite this critical difference, these two events share common features: (1) both are means of safe garbage disposal; (2) both phagosomes and autophagosomes fuse to lysosomes, which drive the degradation of their contents; and (3) both events facilitate the recycling of biological materials. Previously, whether autophagosomes per se directly participate in the degradation of apoptotic cells was unknown, although autophagy proteins were implicated in apoptotic cell clearance. We recently discovered that autophagosomes fuse with phagosomes and contribute to the degradation of apoptotic cells.

Autophagosomes are double-membrane intracellular vesicles that degrade protein aggregates, intracellular organelles, and other cellular components. During the development of the nematode Caenorhabditis elegans, many somatic and germ cells undergo apoptosis. These cells are engulfed and degraded by their neighboring cells. We discovered a novel role of autophagosomes in facilitating the degradation of apoptotic cells using a real-time imaging technique. Specifically, the double-membrane autophagosomes in engulfing cells are recruited to the surfaces of phagosomes containing apoptotic cells and subsequently fuse to phagosomes, allowing the inner vesicle to enter the phagosomal lumen. Mutants defective in the production of autophagosomes display significant defects in the degradation of apoptotic cells, demonstrating the importance of autophagosomes to this process. The signaling pathway led by the phagocytic receptor CED-1, the adaptor protein CED-6, and the large GTPase dynamin (DYN-1) promotes the recruitment of autophagosomes to phagosomes. Moreover, the subsequent fusion of autophagosomes with phagosomes requires the functions of the small GTPase RAB-7 and the HOPS complex components. Further observations suggest that autophagosomes provide apoptotic cell-degradation activities in addition to and in parallel of lysosomes. Our findings reveal that, unlike the single-membrane, LC3-associated phagocytosis (LAP) vesicles reported for mammalian phagocytes, the canonical double-membrane autophagosomes facilitate the clearance of C. elegans apoptotic cells. These findings add autophagosomes to the collection of intracellular organelles that contribute to phagosome maturation, identify novel crosstalk between the autophagy and phagosome maturation pathways, and discover the upstream signaling molecules that initiate this crosstalk.

Calcium ions trigger many cellular events, including the release of neurotransmitters at the synaptic terminal and excitotoxic cell death. Recently, we have discovered that a transient increase in the level of cytoplasmic Ca2+ triggers the exposure of phosphatidylserine (PS) on the surfaces of necrotic cells in the nematode Caenorhabditis elegans. PS serves as an "eat me" signal that attracts engulfing cells to engulf and degrade necrotic cells. During the above study, we developed a microscopic imaging protocol for real-time monitoring the levels of cytoplasmic Ca2+ and cell surface PS in Caenorhabditis elegans touch neurons. Previously, Ca2+ dynamics was monitored in neurons in Caenorhabditis elegans larvae in time periods ranging from milliseconds to seconds. Methods for monitoring Ca2+ dynamics for a relatively long period of time during embryonic development were not available, let alone for simultaneous monitoring Ca2+ and PS dynamics. The protocol reported here utilizes a deconvolution imaging system with an optimized experimental setting that reduces photo-damage and allows the proper development of embryos during the real-time imaging process. This protocol enables the simultaneous measurement of cytosolic Ca2+ and cell surface PS levels in necrotic touch neurons during embryonic development in a period longer than six hours. Our method provides an easy and sensitive approach to perform long-time Ca2+ and PS recording in living animals, simultaneously or individually. This protocol can be applied to study various cellular and developmental events that involve the dynamic regulation of Ca2+ and/or PS.

We recently identified the novel function of the small GTPase RAB-35 in apoptotic cell clearance in Caenorhabditis elegans, a process in which dying cells are engulfed and degraded inside phagosomes. We have found that RAB-35 functions in two separate steps of cell corpse clearance, cell corpse recognition and the initiation of phagosome maturation. During the latter process, RAB-35 facilitates the removal of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) from the membranes of nascent phagosomes and the simultaneous production of phosphatidylinositol-3-P (PI(3)P) on these same membranes, a process that we have coined the PI(4,5)P2 to PI(3)P shift. RAB-35 also promotes the recruitment of the small GTPase RAB-5 to the phagosomal surface. During these processes, the activity of RAB-35 is controlled by the candidate GTPase-activating protein (GAP) TBC-10 and the candidate guanine nucleotide exchange factor (GEF) FLCN-1. Overall, RAB-35 leads a third pathway during cell corpse clearance that functions in parallel to the two known pathways, one led by the phagocytic receptor CED-1 and the other led by the CED-10/Rac1 GTPase. Here, we further report that RAB-35 acts as a robustness factor that maintains the clearance activity and embryonic viability under conditions of heat stress. Moreover, we obtained additional evidence suggesting that RAB-35 acts upstream of RAB-5 and RAB-7. To establish a precise temporal pattern for its own dissociation from phagosomal surfaces, RAB-35 controls the removal of its own GAP. We propose that RAB-35 defines a largely unexplored initial phase of phagosome maturation.

Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an "eat-me" signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a "two-step" pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

In metazoans, apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Multiple small GTPases in the Rab family are known to function in phagosome maturation by regulating vesicle trafficking. We discovered rab-35 as a new gene important for apoptotic cell clearance from a genetic screen targeting putative Rab GTPases in Caenorhabditis elegans. We further identified TBC-10 as a putative GTPase-activating protein (GAP), and FLCN-1 and RME-4 as two putative Guanine Nucleotide Exchange Factors (GEFs), for RAB-35. We found that RAB-35 was required for the efficient incorporation of early endosomes to phagosomes and for the timely degradation of apoptotic cell corpses. More specifically, RAB-35 promotes two essential events that initiate phagosome maturation: the switch of phagosomal membrane phosphatidylinositol species from PtdIns(4,5)P2 to PtdIns(3)P, and the recruitment of the small GTPase RAB-5 to phagosomal surfaces. These functions of RAB-35 were previously unknown. Remarkably, although the phagocytic receptor CED-1 regulates these same events, RAB-35 and CED-1 appear to function independently. Upstream of degradation, RAB-35 also facilitates the recognition of apoptotic cells independently of the known CED-1 and CED-5 pathways. RAB-35 localizes to extending pseudopods and is further enriched on nascent phagosomes, consistent with its dual roles in regulating apoptotic cell-recognition and phagosome maturation. Epistasis analyses indicate that rab-35 acts in parallel to both of the canonical ced-1/6/7 and ced-2/5/10/12 clearance pathways. We propose that RAB-35 acts as a robustness factor, defining a novel pathway that aids these canonical pathways in both the recognition and degradation of apoptotic cells.

The fidelity of epigenetic inheritance or, the precision by which epigenetic information is passed along, is an essential parameter for measuring the effectiveness of the process. How the precision of the process is achieved or modulated, however, remains largely elusive. We have performed quantitative measurement of epigenetic fidelity, using position effect variegation (PEV) in Schizosaccharomyces pombe as readout, to explore whether replication perturbation affects nucleosome-mediated epigenetic inheritance. We show that replication stresses, due to either hydroxyurea treatment or various forms of genetic lesions of the replication machinery, reduce the inheritance accuracy of CENP-A/Cnp1 nucleosome positioning within centromere. Mechanistically, we demonstrate that excessive formation of single-stranded DNA, a common molecular abnormality under these conditions, might have correlation with the reduction in fidelity of centromeric chromatin duplication. Furthermore, we show that replication stress broadly changes chromatin structure at various loci in the genome, such as telomere heterochromatin expanding and mating type locus heterochromatin spreading out of the boundaries. Interestingly, the levels of inheritable expanding at sub-telomeric heterochromatin regions are highly variable among independent cell populations. Finally, we show that HU treatment of the multi-cellular organisms C. elegans and D. melanogaster affects epigenetically programmed development and PEV, illustrating the evolutionary conservation of the phenomenon. Replication stress, in addition to its demonstrated role in genetic instability, promotes variable epigenetic instability throughout the epigenome.

Necrosis is a type of cell death often caused by cell injury and is linked to human diseases including neuron degeneration, stroke, and cancer. Cells undergoing necrosis are engulfed and degraded by engulfing cells, their predators. The mechanisms by which necrotic cells are recognized and removed remain elusive. Here we comment on our recent findings that reveal new molecular mechanisms of necrotic-cell recognition. Through studying the C. elegans touch neurons undergoing excitotoxic necrosis, we identified a receptor/ligand pair that enables engulfing cells to recognize necrotic neurons. The phagocytic receptor CED-1 is activated through interaction with its ligand phosphatidylserine (PS), exposed on the surface of necrotic cells. Furthermore, against the common belief that necrotic cells have ruptured plasma membrane, we found that necrotic C. elegans touch neurons actively present PS on their outer surfaces while maintaining plasma membrane integrity. We further identified 2 mechanisms governing the presentation of PS, one of which is shared with cells undergoing apoptosis, a "cell suicide" event, whereas the other is unique to necrotic neurons. The influx of Ca(2+), a key necrosis-triggering factor, is implicated in activating a neuronal PS-scramblase for PS exposure. We propose that the mechanisms controlling PS-exposure and necrotic-cell recognition by engulfing cells are likely conserved from worms to humans.

Necrosis, a kind of cell death closely associated with pathogenesis and genetic programs, is distinct from apoptosis in both morphology and mechanism. Like apoptotic cells, necrotic cells are swiftly removed from animal bodies to prevent harmful inflammatory and autoimmune responses. In the nematode Caenorhabditis elegans, gain-of-function mutations in certain ion channel subunits result in the excitotoxic necrosis of six touch neurons and their subsequent engulfment and degradation inside engulfing cells. How necrotic cells are recognized by engulfing cells is unclear. Phosphatidylserine (PS) is an important apoptotic-cell surface signal that attracts engulfing cells. Here we observed PS exposure on the surface of necrotic touch neurons. In addition, the phagocytic receptor CED-1 clusters around necrotic cells and promotes their engulfment. The extracellular domain of CED-1 associates with PS in vitro. We further identified a necrotic cell-specific function of CED-7, a member of the ATP-binding cassette (ABC) transporter family, in promoting PS exposure. In addition to CED-7, anoctamin homolog-1 (ANOH-1), the C. elegans homolog of the mammalian Ca(2+)-dependent phospholipid scramblase TMEM16F, plays an independent role in promoting PS exposure on necrotic cells. The combined activities from CED-7 and ANOH-1 ensure efficient exposure of PS on necrotic cells to attract their phagocytes. In addition, CED-8, the C. elegans homolog of mammalian Xk-related protein 8 also makes a contribution to necrotic cell-removal at the first larval stage. Our work indicates that cells killed by different mechanisms (necrosis or apoptosis) expose a common "eat me" signal to attract their phagocytic receptor(s); furthermore, unlike what was previously believed, necrotic cells actively present PS on their outer surfaces through at least two distinct molecular mechanisms rather than leaking out PS passively.

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.

Efficient apoptotic corpse clearance is essential for metazoan development and adult tissue homeostasis. Several autophagy proteins have been previously shown to function in apoptotic cell clearance; however, it remains unknown whether autophagy genes are essential for efficient apoptotic corpse clearance in the developing embryo. Here we show that, in Caenorhabditis elegans embryos, loss-of-function mutations in several autophagy genes that act at distinct steps in the autophagy pathway resulted in increased numbers of cell corpses and delayed cell corpse clearance. Further analysis of embryos with a null mutation in bec- 1, the C. elegans ortholog of yeast VPS30/ATG6/mammalian beclin 1 (BECN1), revealed normal phosphatidylserine exposure on dying cells. Moreover, the corpse clearance defects of bec- 1(ok691) embryos were rescued by BEC-1 expression in engulfing cells, and bec- 1(ok691) enhanced corpse clearance defects in nematodes with simultaneous mutations in the engulfment genes, ced- 1, ced- 6 or ced- 12. Together, these data demonstrate that autophagy proteins play an important role in cell corpse clearance during nematode embryonic development, and likely function in parallel to known pathways involved in corpse removal.

The nematode Caenorhabditis elegans is an excellent model organism for studying the mechanisms -controlling cell death, including apoptosis, a cell suicide event, and necrosis, pathological cell deaths caused by environmental insults or genetic alterations. C. elegans has also been established as a model for understanding how dying cells are cleared from animal bodies. In particular, the transparent nature of worm bodies and eggshells make C. elegans particularly amenable for live-cell microscopy. Here we describe methods for identifying apoptotic and necrotic cells in living C. elegans embryos, larvae, and adults and for monitoring their clearance during development. We further discuss specific methods to distinguish engulfed from unengulfed apoptotic cells, and methods to monitor cellular and molecular events occurring during phagosome maturation. These methods are based on Differential Interference Contrast (DIC) microscopy or fluorescence microscopy using GFP-based reporters.

Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L) also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.

Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a signaling molecule important for many membrane trafficking events, including phagosome maturation. The level of PtdIns(3)P on phagosomes oscillates in two waves during phagosome maturation. However, the physiological significance of such oscillation remains unknown. Currently, the Class III PI 3-kinase (PI3K) Vps34 is regarded as the only kinase that produces PtdIns(3)P in phagosomal membranes. We report here that, in the nematode C. elegans, the Class II PI3K PIKI-1 plays a novel and crucial role in producing phagosomal PtdIns(3)P. PIKI-1 is recruited to extending pseudopods and nascent phagosomes prior to the appearance of PtdIns(3)P in a manner dependent on the large GTPase dynamin (DYN-1). PIKI-1 and VPS-34 act in sequence to provide overlapping pools of PtdIns(3)P on phagosomes. Inactivating both piki-1 and vps-34 completely abolishes the production of phagosomal PtdIns(3)P and disables phagosomes from recruiting multiple essential maturation factors, resulting in a complete arrest of apoptotic-cell degradation. We have further identified MTM-1, a PI 3-phosphatase that antagonizes the activities of PIKI-1 and VPS-34 by down-regulating PtdIns(3)P on phagosomes. Remarkably, persistent appearance of phagosomal PtdIns(3)P, as a result of inactivating mtm-1, blocks phagosome maturation. Our findings demonstrate that the proper oscillation pattern of PtdIns(3)P on phagosomes, programmed by the coordinated activities of two PI3Ks and one PI 3-phosphatase, is critical for phagosome maturation. They further shed light on how the temporally controlled reversible phosphorylation of phosphoinositides regulates the progression of multi-step cellular events.

Apoptosis is a cellular suicide process that quietly and efficiently eliminates unwanted or damaged cells. In metazoans, cells that undergo apoptosis are swiftly internalized by phagocytes and subsequently degraded inside phagosomes through phagosome maturation, a process that involves the fusion between phagosomes and multiple kinds of intracellular organelles and the gradual acidification of phagosomal lumen. In recent years, rapid progress has been made, in particular, through studies conducted in the model organism, the nematode Caenorhabditis elegans, in understanding the membrane trafficking events and molecular mechanisms that govern the degradation of apoptotic cells through phagosome maturation. These studies revealed the novel and essential functions of a large number of proteins, including the large GTPase dynamin, multiple Rab small GTPases and their regulatory proteins, the lipid second messenger PtdIns(3)P and its effectors, and unexpectedly, the phagosomal receptors for apoptotic cells, in promoting phagosome maturation. Further, novel signaling pathways essential for phagosome maturation have been delineated. Here, we discuss these exciting new findings, which have significantly deepened and broadened our understanding of the mechanisms that regulate the interaction between intracellular organelles and phagosomes.

Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genomewide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans , we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific protein-protein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1's association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells.

Dynamins are large GTPases that oligomerize along membranes. Dynamin's membrane fission activity is believed to underlie many of its physiological functions in membrane trafficking. Previously, we reported that DYN-1 (Caenorhabditis elegans dynamin) drove the engulfment and degradation of apoptotic cells through promoting the recruitment and fusion of intracellular vesicles to phagocytic cups and phagosomes, an activity distinct from dynamin's well-known membrane fission activity. Here, we have detected the oligomerization of DYN-1 in living C. elegans embryos and identified DYN-1 mutations that abolish DYN-1's oligomerization or GTPase activities. Specifically, abolishing self-assembly destroys DYN-1's association with the surfaces of extending pseudopods and maturing phagosomes, whereas inactivating guanosine triphosphate (GTP) binding blocks the dissociation of DYN-1 from these membranes. Abolishing the self-assembly or GTPase activities of DYN-1 leads to common as well as differential phagosomal maturation defects. Whereas both types of mutations cause delays in the transient enrichment of the RAB-5 GTPase to phagosomal surfaces, only the self-assembly mutation but not GTP binding mutation causes failure in recruiting the RAB-7 GTPase to phagosomal surfaces. We propose that during cell corpse removal, dynamin's self-assembly and GTP hydrolysis activities establish a precise dynamic control of DYN-1's transient association to its target membranes and that this control mechanism underlies the dynamic recruitment of downstream effectors to target membranes.

During the development of peripheral ganglia, 50% of the neurons that are generated undergo apoptosis. How the massive numbers of corpses are removed is unknown. We found that satellite glial cell precursors are the primary phagocytic cells for apoptotic corpse removal in developing mouse dorsal root ganglia (DRG). Confocal and electron microscopic analysis revealed that glial precursors, rather than macrophages, were responsible for clearing most of the dead DRG neurons. Moreover, we identified Jedi-1, an engulfment receptor, and MEGF10, a purported engulfment receptor, as homologs of the invertebrate engulfment receptors Draper and CED-1 expressed in the glial precursor cells. Expression of Jedi-1 or MEGF10 in fibroblasts facilitated binding to dead neurons, and knocking down either protein in glial cells or overexpressing truncated forms lacking the intracellular domain inhibited engulfment of apoptotic neurons. Together, these results suggest a cellular and molecular mechanism by which neuronal corpses are culled during DRG development.

Apoptosis ensures quick death and quiet clearance of unwanted or damaged cells, without inducing much, if any, immunological responses from the organism. In metazoan organisms, apoptotic cells are swiftly engulfed by other cells. The degradation of cellular content is initiated in apoptotic cells and completed within engulfing cells. In apoptotic cells, caspase-mediated proteolysis cleaves protein substrates into fragments; nuclear DNA is partially degraded into nucleosomal units; and autophagy potentially contributes to apoptotic cell removal. In engulfing cells, specific signaling pathways promote the sequential fusion of intracellular vesicles with phagosomes and lead to the complete degradation of apoptotic cells in an acidic environment. Phagocytic receptors that initiate the engulfment of apoptotic cells play an additional and crucial role in initiating phagosome maturation through activating these signaling pathways. Here we highlight recent discoveries made in invertebrate models and mammalian systems, focusing on the molecular mechanisms that regulate the efficient degradation of apoptotic cells.

Apoptosis is a genetically controlled process of cell suicide that plays an important role in animal development and in maintaining homeostasis. The nematode Caenorhabditis elegans has proven to be an excellent model organism for studying the mechanisms controlling apoptosis and the subsequent clearance of apoptotic cells, aided with cell-biological and genetic tools. In particular, the transparent nature of worm bodies and eggshells makes C. elegans particularly amiable for live cell microscopy. Here we describe a few methods for identifying apoptotic cells in living C. elegans embryos and adults and for monitoring their clearance during embryonic development. These methods are based on Differential Interference Contrast microscopy and on fluorescence microscopy using GFP-based reporters.

In metazoan organisms, cells undergoing apoptosis are rapidly engulfed and degraded by phagocytes. Defects in apoptotic-cell clearance result in inflammatory and autoimmune responses. However, little is known about how apoptotic-cell degradation is initiated and regulated and how different phagocytic targets induce different immune responses from their phagocytes. Recent studies in mammalian systems and invertebrate model organisms have led to major progress in identifying new factors involved in the maturation of phagosomes containing apoptotic cells. These studies have delineated signaling pathways that promote the sequential incorporation of intracellular organelles to phagosomes and have also discovered that phagocytic receptors produce the signals that initiate phagosome maturation. Here, we discuss these exciting new findings, focusing on the mechanisms that regulate the interactions between intracellular organelles and phagosomes.

Apoptotic cells in animals are engulfed by phagocytic cells and subsequently degraded inside phagosomes. To study the mechanisms controlling the degradation of apoptotic cells, we developed time-lapse imaging protocols in developing Caenorhabditis elegans embryos and established the temporal order of multiple events during engulfment and phagosome maturation. These include sequential enrichment on phagocytic membranes of phagocytic receptor cell death abnormal 1 (CED-1), large GTPase dynamin (DYN-1), phosphatidylinositol 3-phosphate (PI(3)P), and the small GTPase RAB-7, as well as the incorporation of endosomes and lysosomes to phagosomes. Two parallel genetic pathways are known to control the engulfment of apoptotic cells in C. elegans. We found that null mutations in each pathway not only delay or block engulfment, but also delay the degradation of engulfed apoptotic cells. One of the pathways, composed of CED-1, the adaptor protein CED-6, and DYN-1, controls the rate of enrichment of PI(3)P and RAB-7 on phagosomal surfaces and the formation of phagolysosomes. We further identified an essential role of RAB-7 in promoting the recruitment and fusion of lysosomes to phagosomes. We propose that RAB-7 functions as a downstream effector of the CED-1 pathway to mediate phagolysosome formation. Our work suggests that phagocytic receptors, which were thought to act specifically in initiating engulfment, also control phagosome maturation through the sequential activation of multiple effectors such as dynamin, PI(3)P, and Rab GTPases.

We identify here a novel class of loss-of-function alleles of uncoordinated locomotion(unc)-108, which encodes the Caenorhabditis elegans homologue of the mammalian small guanosine triphosphatase Rab2. Like the previously isolated dominant-negative mutants, unc-108 loss-of-function mutant animals are defective in locomotion. In addition, they display unique defects in the removal of apoptotic cells, revealing a previously uncharacterized function for Rab2. unc-108 acts in neurons and engulfing cells to control locomotion and cell corpse removal, respectively, indicating that unc-108 has distinct functions in different cell types. Using time-lapse microscopy, we find that unc-108 promotes the degradation of engulfed cell corpses. It is required for the efficient recruitment and fusion of lysosomes to phagosomes and the acidification of the phagosomal lumen. In engulfing cells, UNC-108 is enriched on the surface of phagosomes. We propose that UNC-108 acts on phagosomal surfaces to promote phagosome maturation and suggest that mammalian Rab2 may have a similar function in the degradation of apoptotic cells.

Phagocytes recognize apoptotic cells using cell surface receptors, and subsequently engulf these cells. In a recent issue of Nature, two papers reported the identification of novel phagocytic receptors that directly interact with apoptotic cell surface phosphatidylserine (PS). The studies provide new insights into the apoptotic cell clearance process and implicate PS receptors in additional signaling events.

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 engulfment of dying cells is a specialized form of phagocytosis that is extremely conserved across evolution. In the worm, it is genetically controlled by two parallel pathways, which are only partially reconstituted in mammals. We focused on the recapitulation of the CED-1 defined pathway in mammalian systems. We first explored and validated MEGF10, a novel receptor bearing striking structural similarities to CED-1, as a bona fide functional ortholog in mammals and hence progressed toward the analysis of molecular interactions along the corresponding pathway. We ascertained that, in a system of forced expression by transfection, MEGF10 function can be modulated by the ATP binding cassette transporter ABCA1, ortholog to CED-7. Indeed, the coexpression of either a functional or a mutant ABCA1 exerted a transdominant positive or negative modulation on the MEGF10-dependent engulfment. The combined use of biochemical and biophysical approaches indicated that this functional cooperation relies on the alternate association of these receptors with a common partner, endogenously expressed in our cell system. We provide the first working model structuring in mammals the CED-1 dependent pathway.

Dynamins are large GTPases that act in multiple vesicular trafficking events. We identified 14 loss-of-function alleles of the C. elegans dynamin gene, dyn-1, that are defective in the removal of apoptotic cells. dyn-1 functions in engulfing cells to control the internalization and degradation of apoptotic cells. dyn-1 acts in the genetic pathway composed of ced-7 (ABC transporter), ced-1 (phagocytic receptor), and ced-6 (CED-1's adaptor). DYN-1 transiently accumulates to the surface of pseudopods in a manner dependent on ced-1, ced-6, and ced-7, but not on ced-5, ced-10, or ced-12. Abnormal vesicle structures accumulate in engulfing cells upon dyn-1 inactivation. dyn-1 and ced-1 mutations block the recruitment of intracellular vesicles to pseudopods and phagosomes. We propose that DYN-1 mediates the signaling of the CED-1 pathway by organizing an intracellular vesicle pool and promoting vesicle delivery to phagocytic cups and phagosomes to support pseudopod extension and apoptotic cell degradation.

Programmed cell death, or apoptosis, is a genetically controlled process of cell suicide that is a common fate during an animal's life. In metazoans, apoptotic cells are rapidly removed from the body through the process of phagocytosis. Genetic analyses probing the mechanisms controlling the engulfment of apoptotic cells were pioneered in the nematode Caenorhabditis elegans. So far, at least seven genes have been identified that are required for the recognition and engulfment of apoptotic cells and have been shown to function in two partially redundant signaling pathways. Molecular characterization of their gene products has lead to the finding that similar genes act to control the same processes in other organisms, including mammals. In this paper, we review these exciting findings in C. elegans and discuss their implications in understanding the clearance of apoptotic cells in mammals.

The success of engulfment and degradation relies on the proper coordination of multiple cellular and subcellular events, including cytoskeletal reorganization, membrane remodeling, membrane fusion, and the initiation of lysosome-phagosome fusion. This chapter reviews the logic and methods employed to study how apoptotic cells are recognized, internalized, and degraded by engulfing cells in C. elegans and Drosophila. New findings in these two systems resulting from a combination of genetic and molecular studies of gene function are also described. The implications of these results for the understanding of the clearance of apoptotic cells in mammals are discussed. Many components of the engulfment machinery are conserved during evolution. In Drosophila, the vast majority of apoptotic cells are cleared by hemocytes, although engulfment by nonprofessional phagocytes is also reported. Phagocytic Receptor in Drosophila is encoded by croquemort. The engulfment of apoptotic cells I required for proper patterning of the central nervous system. DNA degradation in drosophila proceeds in two steps: (1) cell-autonomous DNA degradation, and (2) cell nonautonomous DNA degradation. Although engulfment and subsequent degradation of apoptotic cells are considered as downstream events to cell death––there is an emerging consensus that there is some cross-talk among these pathways.

The C. elegans gene ced-12 functions in the engulfment of apoptotic cells and in cell migration, acting in a signaling pathway with ced-2 Crkll, ced-5 DOCK180, and ced-10 Rac GTPase and acting upstream of ced-10 Rac. ced-12 encodes a protein with a pleckstrin homology (PH) domain and an SH3 binding motif, both of which are important for ced-12 function. CED-12 acts in engulfing cells for cell corpse engulfment and interacts physically with CED-5, which contains an SH3 domain. CED-12 has Drosophila and human counterparts. Expression of CED-12 and its counterparts in murine Swiss 3T3 fibroblasts induced Rho GTPase-dependent formation of actin filament bundles. We propose that through interactions with membranes and with a CED-2/CED-5 protein complex, CED-12 regulates Rho/Rac GTPase signaling and leads to cytoskeletal reorganization by an evolutionarily conserved mechanism.

We cloned the C. elegans gene ced-1, which is required for the engulfment of cells undergoing programmed cell death. ced-1 encodes a transmembrane protein similar to human SREC (Scavenger Receptor from Endothelial Cells). We showed that ced-1 is expressed in and functions in engulfing cells. The CED-1 protein localizes to cell membranes and clusters around neighboring cell corpses. CED-1 failed to cluster around cell corpses in mutants defective in the engulfment gene ced-7. Motifs in the intracellular domain of CED-1 known to interact with PTB and SH2 domains were necessary for engulfment but not for clustering. Our results indicate that CED-1 is a cell surface phagocytic receptor that recognizes cell corpses. We suggest that the ABC transporter CED-7 promotes cell corpse recognition by CED-1, possibly by exposing a phospholipid ligand on the surfaces of cell corpses.

The Caenorhabditis elegans Bcl-2-like protein CED-9 prevents programmed cell death by antagonizing the Apaf-1-like cell-death activator CED-4. Endogenous CED-9 and CED-4 proteins localized to mitochondria in wild-type embryos, in which most cells survive. By contrast, in embryos in which cells had been induced to die, CED-4 assumed a perinuclear localization. CED-4 translocation induced by the cell-death activator EGL-1 was blocked by a gain-of-function mutation in ced-9 but was not dependent on ced-3 function, suggesting that CED-4 translocation precedes caspase activation and the execution phase of programmed cell death. Thus, a change in the subcellular localization of CED-4 may drive programmed cell death.