The trigger to cell death determines the efficiency with which dying cells are cleared by neighbours.

Phagocytosis of apoptotic cells is required to prevent tissue injury. Professional phagocytes, such as monocyte-derived macrophages, are highly efficient scavengers of apoptotic cells but their presence cannot always be relied on; in that case, removal of effete cells is accomplished by helpful neighbours. This study describes differences in the efficiency with which apoptotic cells of the same type, but dying in response to different triggers, are engulfed; this varies from engulfment that is so proficient few or no unengulfed apoptotic cells are found, to engulfment that is so delayed apoptotic cells have become secondarily necrotic at the point of engulfment. In all cases the efficiency of engulfment is determined at least in part by the dying cells themselves. p53- and Bax-transfected kidney epithelial (293) cells (transiently transfected using a non-toxic method) were engulfed so proficiently by homotypic neighbours that cells did not show evidence of engagement of the apoptotic programme (chromatin condensation and TUNEL positivity) until engulfment had taken place. Engulfment nonetheless required activation of at least initiator caspases. 293 cells induced to apoptose by other means (etoposide and staurosporine treatment) were not so efficiently ingested: unengulfed apoptotic cells were consistently revealed at all doses and time points, even when treated cells were mixed with healthy, non-treated 293 cells. These data make it extremely unlikely that the fraction of viable, unaffected neighbours determines the efficiency with which engulfment proceeds. Furthermore, 293 cells treated with etoposide or staurosporine were differentially appealing both to homotypic neighbours and to cells in the professional phagocyte lineage (THP-1 cells). If different apoptotic stimuli programme cells to be recognised with different efficiencies, pathways to apoptosis may be injury limiting to greater or lesser degrees.     

Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos

Programmed cell death plays an essential role during Drosophila embryonic development. A stereotypic series of cellular changes occur during apoptosis, most of which are initiated by a caspase cascade that is triggered by a trio of proteins, RPR, HID and GRIM. The final step in apoptosis is engulfment of the cell corpse. To monitor cell engulfment in vivo, we developed a fluorogenic beta-galactosidase substrate that is cleaved by an endogenous, lysosomal beta-galactosidase activity. The pattern of cell engulfment in wild-type embryos correlated well with the known pattern of apoptosis. Surprisingly, the pattern of cell engulfment persisted in apoptosis-deficient embryos. We provide evidence for a caspase-independent engulfment process that affects the majority of cells expected to die in developing Drosophila embryos.     

Induction of entosis by epithelial cadherin expression

Cell engulfment typically targets dead or dying cells for clearance from metazoan tissues. However, recent evidence demonstrates that live cells can also be targeted and that engulfment can cause cell death. Entosis is one mechanism proposed to mediate the engulfment and killing of live tumor cells by their neighbors, an activity often referred to as cell cannibalism. Here we report that the expression of exogenous epithelial cadherin proteins (E- or P-cadherin) in human breast tumor cells lacking endogenous expression of epithelial cadherins induces entosis and inhibits transformed growth. Entosis induced by cadherin expression is associated with the polarized distribution of Rho and Rho-kinase (ROCK) activity within entotic cells, which is dependent on p190A RhoGAP activity. ROCK inhibition or downregulation of p190A RhoGAP expression reduces entosis and increases the transformed growth of epithelial cadherin-expressing tumor cells. These data define new cell systems for the study of entosis, and identify entosis as a mechanism of cell cannibalism that is induced by the establishment of epithelial adhesion and inhibits transformed growth.     

Tim4- and MerTK-Mediated Engulfment of Apoptotic Cells by Mouse Resident Peritoneal Macrophages

Apoptotic cells are swiftly engulfed by macrophages to prevent the release of noxious materials from dying cells. Apoptotic cells expose phosphatidylserine (PtdSer) on their surface, and macrophages engulf them by recognizing PtdSer using specific receptors and opsonins. Here, we found that mouse resident peritoneal macrophages expressing Tim4 and MerTK are highly efficient at engulfing apoptotic cells. Neutralizing antibodies against either Tim4 or MerTK inhibited the macrophage engulfment of apoptotic cells. Tim4-null macrophages exhibited reduced binding and engulfment of apoptotic cells, whereas MerTK-null macrophages retained the ability to bind apoptotic cells but failed to engulf them. The incubation of wild-type peritoneal macrophages with apoptotic cells induced the rapid tyrosine phosphorylation of MerTK, which was not observed with Tim4-null macrophages. When mouse Ba/F3 cells were transformed with Tim4, apoptotic cells bound to the transformants but were not engulfed. Transformation of Ba/F3 cells with MerTK had no effect on the binding or engulfment of apoptotic cells; however, Tim4/MerTK transformants exhibited strong engulfment activity. Taken together, these results indicate that the engulfment of apoptotic cells by resident peritoneal macrophages proceeds in two steps: binding to Tim4, a PtdSer receptor, followed by MerTK-mediated cell engulfment.     

Evidence that the SpoIIIE DNA translocase participates in membrane fusion during cytokinesis and engulfment

During Bacillus subtilis sporulation, SpoIIIE is required for translocation of the trapped forespore chromosome across the sporulation septum, for compartmentalization of cell-specific gene expression, and for membrane fusion after engulfment. We isolated mutations within the SpoIIIE membrane domain that block localization and function. One mutant protein initially localizes normally and completes DNA translocation, but shows reduced membrane fusion after engulfment. Fluorescence recovery after photobleaching experiments demonstrate that in this mutant the sporulation septum remains open, allowing cytoplasmic contents to diffuse between daughter cells, suggesting that it blocks membrane fusion after cytokinesis as well as after engulfment. We propose that SpoIIIE catalyses these topologically opposite fusion events by assembling or disassembling a proteinaceous fusion pore. Mutants defective in SpoIIIE assembly also demonstrate that the ability of SpoIIIE to provide a diffusion barrier is directly proportional to its ability to assemble a focus at the septal midpoint during DNA translocation. Thus, SpoIIIE mediates compartmentalization by two distinct mechanisms: the SpoIIIE focus first provides a temporary diffusion barrier during DNA translocation, and then mediates the completion of membrane fusion after division to provide a permanent diffusion barrier. SpoIIIE-like proteins might therefore serve to couple the final step in cytokinesis, septal membrane fusion, to the completion of chromosome segregation.     

The scoop on the fly brain: glial engulfment functions in Drosophila

Glial cells provide support and protection for neurons in the embryonic and adult brain, mediated in part through the phagocytic activity of glia. Glial cells engulf apoptotic cells and pruned neurites from the developing nervous system, and also clear degenerating neuronal debris from the adult brain after neural trauma. Studies indicate that Drosophila melanogaster is an ideal model system to elucidate the mechanisms of engulfment by glia. The recent studies reviewed here show that many features of glial engulfment are conserved across species and argue that work in Drosophila will provide valuable cellular and molecular insight into glial engulfment activity in mammals.     

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.     

Membrane cell fusion activity of the vaccinia virus A17-A27 protein complex

Vaccinia virus enters cells by endocytosis and via a membrane fusion mechanism mediated by viral envelope protein complexes. While several proteins have been implicated in the entry/fusion event, there is no direct proof for fusogenic activity of any viral protein in heterologous systems. Transient coexpression of A17 and A27 in mammalian cells led to syncytia formation in a pH-dependent manner, as ascertained by confocal fluorescent immunomicroscopy. The pH-dependent fusion activity was identified to reside in A17 amino-terminal ectodomain after overexpression in insect cells using recombinant baculoviruses. Through the use of A17 ectodomain deletion mutants, it was found that the domain important for fusion spanned between residues 18 and 34. To further characterize A17–A27 fusion activity in mammalian cells, 293T cell lines stably expressing A17, A27 or coexpressing both proteins were generated using lentivectors. A27 was exposed on the cell surface only when A17 was coexpressed. In addition, pH-dependent fusion activity was functionally demonstrated in mammalian cells by cytoplasmic transfer of fluorescent proteins, only when A17 and A27 were coexpressed. Bioinformatic tools were used to compare the putative A17–A27 protein complex with well-characterized fusion proteins. Finally, all experimental evidence was integrated into a working model for A17–A27-induced pH-dependent cell-to-cell fusion.     

The membrane domain of SpoIIIE is required for membrane fusion during Bacillus subtilis sporulation

During Bacillus subtilis sporulation, SpoIIIE is required for both postseptational chromosome segregation and membrane fusion after engulfment. Here we demonstrate that SpoIIIE must be present in the mother cell to promote membrane fusion and that the N-terminal membrane-spanning segments constitute a minimal membrane fusion domain, as well as direct septal localization.     

Forespore engulfment mediated by a ratchet-like mechanism

A key step in bacterial endospore formation is engulfment, during which one bacterial cell engulfs another in a phagocytosis-like process that normally requires SpoIID, SpoIIM, and SpoIIP (DMP). We here describe a second mechanism involving the zipper-like interaction between the forespore protein SpoIIQ and its mother cell ligand SpoIIIAH, which are essential for engulfment when DMP activity is reduced or SpoIIB is absent. They are also required for the rapid engulfment observed during the enzymatic removal of peptidoglycan, a process that does not require DMP. These results suggest the existence of two separate engulfment machineries that compensate for one another in intact cells, thereby rendering engulfment robust. Photobleaching analysis demonstrates that SpoIIQ assembles a stationary structure, suggesting that SpoIIQ and SpoIIIAH function as a ratchet that renders forward membrane movement irreversible. We suggest that ratchet-mediated engulfment minimizes the utilization of chemical energy during this dramatic cellular reorganization, which occurs during starvation.     

Cell wall synthesis is necessary for membrane dynamics during sporulation of Bacillus subtilis

During Bacillus subtilis sporulation, an endocytic‐like process called engulfment results in one cell being entirely encased in the cytoplasm of another cell. The driving force underlying this process of membrane movement has remained unclear, although components of the machinery have been characterized. Here we provide evidence that synthesis of peptidoglycan, the rigid, strength bearing extracellular polymer of bacteria, is a key part of the missing force‐generating mechanism for engulfment. We observed that sites of peptidoglycan synthesis initially coincide with the engulfing membrane and later with the site of engulfment membrane fission. Furthermore, compounds that block muropeptide synthesis or polymerization prevented membrane migration in cells lacking a component of the engulfment machinery (SpoIIQ), and blocked the membrane fission event at the completion of engulfment in all cells. In addition, these compounds inhibited bulge and vesicle formation that occur in spoIID mutant cells unable to initiate engulfment, as did genetic ablation of a protein that polymerizes muropeptides. This is the first report to our knowledge that peptidoglycan synthesis is necessary for membrane movements in bacterial cells and has implications for the mechanism of force generation during cytokinesis.     

Recent advances in understanding the mechanisms of osteoclast precursor fusion

Bone marrow macrophages fuse on the bone surface to form multinucleated osteoclasts that then organize to efficiently resorb bone. Many, if not all, of the stages of macrophage fusion involve cytoskeletal components that reorganize the cells. Recruitment may involve chemotactic responses to bone matrix protein and calcium ion gradients and/or chemokine production by bone forming osteoblasts. The roles of integrins vary, depending on the particular subunits with some interfering with fusion and others having a participatory role. RANKL is essential for fusion and many identified modulators of fusion influence RANKL signaling pathways. Tetraspanins have been implicated in fusion of macrophages and myoblasts, but differences in impacts exist between these two cell types. Macrophage recruitment to apoptotic cells prior to their engulfment is driven by the exposed phospholipids on the external surface of the apoptotic cells and there is evidence that this same identification mechanism is employed in macrophage fusion. Because loss of cadherin or ADAM family members suppresses macrophage fusion, a crucial role for these membrane glycoproteins is evident. The Ig membrane glycoprotein superfamily members CD200 and MFR/SIRPα are involved in macrophage fusion, although their influences are unresolved. Differential screenings have identified the structurally related membrane proteins DC‐STAMP and OC‐STAMP as required components for fusion and the contributions to fusion remain active areas of investigation. While many of the key components involved in these processes have been identified, a great deal of work remains in resolving the precise processes involved and the interactions between key contributors to multinucleated osteoclast formation. J. Cell. Biochem. 110: 1058–1062, 2010. Published 2010 Wiley‐Liss, Inc.     

A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia

Following neuronal injury, microglia initiate repair by phagocytosing dead neurons without eliciting inflammation. Prior evidence indicates triggering receptor expressed by myeloid cells-2 (TREM2) promotes phagocytosis and retards inflammation. However, evidence that microglia and neurons directly interact through TREM2 to orchestrate microglial function is lacking. We here demonstrate that TREM2 interacts with endogenous ligands on neurons. Staining with TREM2-Fc identified TREM2 ligands (TREM2-L) on Neuro2A cells and on cultured cortical and dopamine neurons. Apoptosis greatly increased the expression of TREM2-L. Furthermore, apoptotic neurons stimulated TREM2 signaling, and an anti-TREM2 mAb blocked stimulation. To examine the interaction between TREM2 and TREM2-L in phagocytosis, we studied BV2 microglial cells and their engulfment of apoptotic Neuro2A. One of our anti-TREM2 mAb, but not others, reduced engulfment, suggesting the presence of a functional site on TREM2 interacting with neurons. Further, Chinese hamster ovary cells transfected with TREM2 conferred phagocytic activity of neuronal cells demonstrating that TREM2 is both required and sufficient for competent uptake of apoptotic neuronal cells. Finally, while TREM2-L are expressed on neurons, TREM2 is not; in the brain, it is found on microglia. TREM2 and TREM2-L form a receptor–ligand pair connecting microglia with apoptotic neurons, directing removal of damaged cells to allow repair.     

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.     

Integrin α PAT-2/CDC-42 signaling is required for muscle-mediated clearance of apoptotic cells in Caenorhabditis elegans

Clearance of apoptotic cells by engulfment plays an important role in the homeostasis and development of multicellular organisms. Despite the fact that the recognition of apoptotic cells by engulfment receptors is critical in inducing the engulfment process, the molecular mechanisms are still poorly understood. Here, we characterize a novel cell corpse engulfment pathway mediated by the integrin α subunit PAT-2 in Caenorhabditis elegans and show that it specifically functions in muscle-mediated engulfment during embryogenesis. Inactivation of pat-2 results in a defect in apoptotic cell internalization. The PAT-2 extracellular region binds to the surface of apoptotic cells in vivo, and the intracellular region may mediate signaling for engulfment. We identify essential roles of small GTPase CDC-42 and its activator UIG-1, a guanine-nucleotide exchange factor, in PAT-2-mediated cell corpse removal. PAT-2 and CDC-42 both function in muscle cells for apoptotic cell removal and are co-localized in growing muscle pseudopods around apoptotic cells. Our data suggest that PAT-2 functions through UIG-1 for CDC-42 activation, which in turn leads to cytoskeletal rearrangement and apoptotic cell internalization by muscle cells. Moreover, in contrast to PAT-2, the other integrin α subunit INA-1 and the engulfment receptor CED-1, which signal through the conserved signaling molecules CED-5 (DOCK180)/CED-12 (ELMO) or CED-6 (GULP) respectively, preferentially act in epithelial cells to mediate cell corpse removal during mid-embryogenesis. Our results show that different engulfing cells utilize distinct repertoires of receptors for engulfment at the whole organism level.     

Peptidoglycan transformations during Bacillus subtilis sporulation

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross‐linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high‐resolution electron cryotomographic imaging. Three‐dimensional reconstructions of whole cells in near‐native states revealed a thin PG‐like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram‐negative‐like PG layers as well as its thick, archetypal Gram‐positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.     

PTEN negatively regulates engulfment of apoptotic cells by modulating activation of Rac GTPase

Efficient clearance of apoptotic cells by phagocytes (efferocytosis) is critical for normal tissue homeostasis and regulation of the immune system. Apoptotic cells are recognized by a vast repertoire of receptors on macrophage that lead to transient formation of phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] and subsequent cytoskeletal reorganization necessary for engulfment. Certain PI3K isoforms are required for engulfment of apoptotic cells, but relatively little is known about the role of lipid phosphatases in this process. In this study, we report that the activity of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a phosphatidylinositol 3-phosphatase, is elevated upon efferocytosis. Depletion of PTEN in macrophage results in elevated PtdIns(3,4,5)P(3) production and enhanced phagocytic ability both in vivo and in vitro, whereas overexpression of wild-type PTEN abrogates this process. Loss of PTEN in macrophage leads to activation of the pleckstrin homology domain-containing guanine-nucleotide exchange factor Vav1 and subsequent activation of Rac1 GTPase, resulting in increased amounts of F-actin upon engulfment of apoptotic cells. PTEN disruption also leads to increased production of anti-inflammatory cytokine IL-10 and decreased production of proinflammatory IL-6 and TNF-α upon engulfment of apoptotic cells. These data suggest that PTEN exerts control over efferocytosis potentially by regulating PtdIns(3,4,5)P(3) levels that modulate Rac GTPase and F-actin reorganization through Vav1 exchange factor and enhancing apoptotic cell-induced anti-inflammatory response.     

Function of GRIM-19, a Mitochondrial Respiratory Chain Complex I Protein, in Innate Immunity

Mitochondria respiratory chain (RC), consisting of five multisubunit complexes, is crucial for cellular energy production, reactive oxygen species generation, and regulation of apoptosis. Recently, a few mitochondrial proteins have been reported to be essential for innate immunity, but the function of mitochondrial RC in innate immunity is largely unknown. By knock-out of GRIM-19, a newly identified subunit protein of mitochondrial complex I, in mice, we found that heterogeneous mice (GRIM-19(+/-)) are prone to spontaneous urinary tract infection, mostly by Staphylococcus saprophyticus. Macrophages derived from these mice have compromised mitochondrial complex I activity and increased reactive oxygen species level. Bacterial infection induces a rapid up-regulation of GRIM-19 and complex I activity in the wild-type macrophages, but both are reduced in the macrophages from GRIM-19(+/-) mice. These cells also have decreased intracellular killing ability against S. saprophyticus. The defects for this probably occur in the fusion of bacteria to lysosome, but not in the bacterial engulfment and macrophage migration. In addition, production of proinflammatory cytokines, such as interleukin (IL)-1, IL-12, IL-6, and interferon (IFN)-γ, induced by both bacterial infection and lipopolysaccharide (LPS) and monodansylcadaverine treatment, is also decreased in the GRIM19(+/-) macrophages. Inhibition of mitochondrial RC activity by inhibitors shows a similar reduction on the cytokine production. Due to low cytokine production, the inflammatory response caused by in vivo bacterial challenge in the bladders of GRIM-19(+/-) mice is compromised. This study provides genetic evidence for a critical role of mitochondrial RC in innate immunity.     

The Role of Engulfment in the Dictyosteliaceae

SYNOPSIS. Experiments with mixtures of latex-tagged cultures of amoebae of strains, species and genera of the Dictyosteliaceae indicate little specificity in engulfment. This is in sharp contrast to the decided specificity for compatibility in completion of morphogenesis in the same organisms. Lack of specificity in engulfment is considered additional evidence favoring engulfment as a normal feeding activity rather than a process associated with syngamy in the cellular slime molds.