Engulfment Of Apoptotic Cells Research Articles

Pyroptotic cells externalize eat-me and release find-me signals and are efficiently engulfed by macrophages

Pathogenic intracellular bacteria often hijack macrophages for their propagation. The infected macrophages release IL-1β and IL-18 and simultaneously commit suicide, which is called pyroptosis; both responses require caspase-1. Here, we found that pyroptotic cells induced by microbial infection were efficiently engulfed by human monocytic THP-1-cell-derived macrophages or mouse peritoneal macrophages. This engulfment was inhibited by the D89E mutant of milk fat globule (MFG) epidermal growth factor (EGF) factor 8 (MFG-E8; a phosphatidylserine-binding protein) that has been shown previously to inhibit phosphatidylserine-dependent engulfment of apoptotic cells by macrophages, suggesting that the engulfment of pyroptotic cells by macrophages was also phosphatidylserine dependent. Using a pair of cell lines that respectively exhibited pyroptosis or apoptosis after muramyl dipeptide treatment, we showed that both pyroptotic and apoptotic cells bound to a T-cell immunoglobulin and mucin domain-containing 4 (Tim4; another phosphatidylserine-binding protein)-coated plate, whereas heat-killed necrotic cells did not, indicating that phosphatidylserine was externalized in pyroptosis and apoptosis but not in accidental necrosis. Macrophages engulfed apoptotic cells most efficiently, followed by pyroptotic and then heat-killed necrotic cells. Pyroptotic cells also released a macrophage attractant(s), 'find-me' signal, whose activity was diminished by apyrase that degrades nucleoside triphosphate to nucleoside monophosphate. Heat-killed necrotic cells and pyroptotic cells released ATP much more efficiently than apoptotic cells. These results suggest that pyroptotic cells, like apoptotic cells, actively induce phagocytosis by macrophages using 'eat-me' and find-me signals. Based on these results, a possible role of coordinated induction of pyroptosis and inflammatory cytokine production is discussed.

GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance

Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize ‘eat me’ signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80+ macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.

Measurement of Phagocytic Engulfment of Apoptotic Cells by Macrophages Using pHrodo Succinimidyl Ester

Considerable interest has emerged towards phagocytosis of apoptotic cells, due to its intricate molecular mechanisms and important regulatory functions in development, homoeostasis, and immune tolerance. Impaired clearance of apoptotic cells leads to immune-mediated disorders. Current quantification methods of the engulfment of apoptotic cells by macrophages are potentially flawed by several limitations. Adherent macrophage populations are overlaid with apoptotic targets in suspension and then co-cultured for a definite period, which may give rise to two different features: (1) engulfed and (2) non-engulfed macrophages that are surface-bound cell populations. Rigorous washing to dislodge surface-bound apoptotic cells before assessment of phagocytosis may lead to loss of phagocytes, thereby skewing the apparent magnitude of the overall phagocytic response. There is a need for simple and reliable methods to clearly determine the internalization of apoptotic cells. In this unit, we demonstrate the use of pHrodo-succinimidyl ester (SE), a pH-sensitive fluorescent dye, to label the apoptotic cells for monitoring the phagocytosis. After engulfment, the intensity of pHrodo light emission will be elevated due to the pH change inside of macrophages. The shift of pHrodo light emission can be detected by a flow cytometer or using a fluorescence microscope.

Reactive oxygen species H2O2 and [rad]OH, but not O2[rad]− promote oridonin-induced phagocytosis of apoptotic cells by human histocytic lymphoma U937 cells

We reported previously that phagocytosis of apoptotic cells by U937 cells was enhanced by the treatment with oridonin that showed high activity to induce the generation of reactive oxygen species (ROS) in many cells. ROS, important signaling molecules, are involved in the immune defenses, cell repair and proliferation. In this study, oridonin caused modest amount of ROS generation in U937 cells, with hydrogen peroxide (H2O2) and hydroxyl free radical (OH) as the major types. Meanwhile, H2O2 and OH were positive regulators involved in oridonin-enhanced engulfment of apoptotic cells through down-regulating mitochondrial membrane potential (MMP) and inducing autophagy. The ROS-mediated phagocytosis was independent of cellular adenosine triphosphate (ATP) levels. H2O2 and OH generation also activated phosphatidylinositol 3-kinases-Akt (PI3K-Akt) and phospholipase C γ-protein kinase C(PLC γ)-Ras-Raf-ERK signaling pathways, which were essential for oridonin-induced engulfment of apoptotic cells. Phagocytosis, the loss of MMP, autophagy and the activated signaling pathways were all suppressed by ROS scavenger N-acetyl-l-cysteine (NAC), H2O2 scavenger catalase or OH scavenger glutathione (GSH). However, superoxide anion (O2−) and its scavenger superoxide dismutase (SOD) did not significantly affect these oridonin-induced biological processes.

Clearing the Dead: Apoptotic Cell Sensing, Recognition, Engulfment, and Digestion

Clearance of apoptotic cells is the final stage of programmed cell death. Uncleared corpses can become secondarily necrotic, promoting inflammation and autoimmunity. Remarkably, even in tissues with high cellular turnover, apoptotic cells are rarely seen because of efficient clearance mechanisms in healthy individuals. Recently, significant progress has been made in understanding the steps involved in prompt cell clearance in vivo. These include the sensing of corpses via "find me" signals, the recognition of corpses via "eat me" signals and their cognate receptors, the signaling pathways that regulate cytoskeletal rearrangement necessary for engulfment, and the responses of the phagocyte that keep cell clearance events "immunologically silent." This study focuses on our understanding of these steps.

Apoptotic regulators promote cytokinetic midbody degradation in C. elegans

Cell death genes are essential for apoptosis and other cellular events, but their nonapoptotic functions are not well understood. The midbody is an important cytokinetic structure required for daughter cell abscission, but its fate after cell division remains elusive in metazoans. In this paper, we show through live-imaging analysis that midbodies generated by Q cell divisions in Caenorhabditis elegans were released to the extracellular space after abscission and subsequently internalized and degraded by the phagocyte that digests apoptotic Q cell corpses. We further show that midbody degradation is defective in apoptotic cell engulfment mutants. Externalized phosphatidylserine (PS), an engulfment signal for corpse phagocytosis, exists on the outer surface of the midbody, and inhibiting PS signaling delayed midbody clearance. Thus, our findings uncover a novel function of cell death genes in midbody internalization and degradation after cell division.

SLI-1 Cbl Inhibits the Engulfment of Apoptotic Cells in C. elegans through a Ligase-Independent Function

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway, which includes the small GTPase CED-10 Rac and the cytoskeletal regulator ABI-1, acts to rearrange the cytoskeleton of the engulfing cell. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The second pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Cbl, the mammalian homolog of the C. elegans E3 ubiquitin ligase and adaptor protein SLI-1, interacts with Rac and Abi2 and modulates the actin cytoskeleton, suggesting it might act in engulfment. Our genetic studies indicate that SLI-1 inhibits apoptotic cell engulfment and DTC migration independently of the CED-10 Rac and CED-1 pathways. We found that the RING finger domain of SLI-1 is not essential to rescue the effects of SLI-1 deletion on cell migration, suggesting that its role in this process is ubiquitin ligase-independent. We propose that SLI-1 opposes the engulfment of apoptotic cells via a previously unidentified pathway.

Interactions of Apoptotic Cells with Macrophages in Radiation-Induced Bystander Signaling

Nontargeted effects that result in ongoing cellular and tissue damage show genotype-dependency in murine models with CBA/Ca, but not C57BL/6, exhibiting sensitivity to induced genomic instability. In vivo, radiation exposure is associated with genotype-dependent macrophage activation, and these cells are a source of bystander signaling involving cytokines and reactive oxygen and nitrogen species. The mechanisms responsible for macrophage activation and production of damaging bystander signals after irradiation are unclear. Macrophages from CBA/Ca exhibit an M1 (proinflammatory) phenotype compared to the M2 (anti-inflammatory) phenotype of C57BL/6 macrophages. Using the murine RAW264.7 macrophage-like cell line, we show that the ability of macrophages to interact with apoptotic cells and their responses to interaction varies significantly according to macrophage phenotype. Nonstimulated and M2 macrophages induce anti-inflammatory markers arginase and TGFβ after engulfment of apoptotic cells. In contrast, M1 macrophages do not induce anti-inflammatory responses, but express the proinflammatory markers NOS2, IL-6, TNFα, superoxide and NO, able to contribute to a damaging microenvironment. Macrophages stimulated with both inflammatory and anti-inflammatory agents prior to exposure to apoptotic cells induce a mixed response. The results indicate a complex cross-talk between macrophages and apoptotic cells and demonstrate that phagocytic clearance of apoptotic cells induced by genotoxic stress can produce microenvironmental responses consistent with the induction of a chromosomal instability phenotype in sensitive CBA/Ca mice with M1 macrophage activation, but not in resistant C57BL/6 mice with M2 macrophage activation. Modulation of macrophage phenotypes may represent a novel approach for reducing the nontargeted effects of radiation.

Release of lysophospholipid ‘find-me’ signals during apoptosis requires the ATP-binding cassette transporter A1

Efficient engulfment of apoptotic cells is essential in multi-cellular organisms in order to prevent inflammatory responses. Apoptotic cells secure this process by releasing ‘find-me’ signals for the attraction of phagocytes. A major ‘find-me’ signal liberated from apoptotic cells is lysophosphatidylcholine (LPC). So far, however, the mechanisms underlying LPC release are poorly understood. In this study, we demonstrate that pharmacological inhibition and RNAi-mediated knock-down of the lipid transporter ABCA1 in apoptotic cells completely abolished phagocyte attraction. Moreover, ectopic expression of ABCA1 significantly enhanced monocyte migration to supernatants of apoptotic cells. Hence, ABCA1 represents a novel regulator of LPC release during apoptosis.

Cell surface-expressed phosphatidylserine as therapeutic target to enhance phagocytosis of apoptotic cells

Impaired efferocytosis has been shown to be associated with, and even to contribute to progression of, chronic inflammatory diseases such as atherosclerosis. Enhancing efferocytosis has been proposed as strategy to treat diseases involving inflammation. Here we present the strategy to increase 'eat me' signals on the surface of apoptotic cells by targeting cell surface-expressed phosphatidylserine (PS) with a variant of annexin A5 (Arg-Gly-Asp-annexin A5, RGD-anxA5) that has gained the function to interact with α(v)β(3) receptors of the phagocyte. We describe design and characterization of RGD-anxA5 and show that introduction of RGD transforms anxA5 from an inhibitor into a stimulator of efferocytosis. RGD-anxA5 enhances engulfment of apoptotic cells by phorbol-12-myristate-13-acetate-stimulated THP-1 (human acute monocytic leukemia cell line) cells in vitro and resident peritoneal mouse macrophages in vivo. In addition, RGD-anxA5 augments secretion of interleukin-10 during efferocytosis in vivo, thereby possibly adding to an anti-inflammatory environment. We conclude that targeting cell surface-expressed PS is an attractive strategy for treatment of inflammatory diseases and that the rationally designed RGD-anxA5 is a promising therapeutic agent.

Efferocytosis Is an Innate Antibacterial Mechanism

Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M.tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M.tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M.tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M.tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism.

Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding.

BackgroundActivation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer’s disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells.MethodsPrimary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events.ResultsThe direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB-EGF neutralizing antibody abrogated the phenotype of activated microglia induced by the sPLA2-IIA.ConclusionThese results support the hypothesis that sPLA2-IIA may act as a potent modulator of microglial functions through its ability to induce EGFR transactivation and HB-EGF release. Accordingly, pharmacological modulation of EGFR might be a useful tool for treating neuroinflammatory diseases characterized by sPLA2-IIA accumulation.

Synergistic effect of Tim4 and MFG-E8 null mutations on the development of autoimmunity

Phagocytes, including macrophages, recognize phosphatidylserine exposed on apoptotic cells as an "eat me" signal. Milk Fat Globule EGF Factor VIII (MFG-E8) is secreted by one subset of macrophages, whereas Tim4, a type I membrane protein, is expressed by another. These proteins bind tightly to phosphatidylserine on apoptotic cells and enhance their engulfment by macrophages. To study the contribution of these proteins to the engulfment of apoptotic cells, we established a mouse line that was deficient in the genes encoding MFG-E8 and Tim4. The null mutation of Tim4 impaired the ability of resident peritoneal macrophages, but not thioglycollate-elicited macrophages, to engulf apoptotic cells. Mice deficient in either MFG-E8 or Tim4 on the C57BL/6 background developed hardly any autoantibodies, but aged female mice deficient in both MFG-E8 and Tim4 developed autoantibodies in an age-dependent manner. Tumour necrosis factor (TNF) α is known to protect against systemic lupus erythematosus-type autoimmunity, whereas type I IFN accelerates the disease. Indeed, the administration of an anti-TNFα antibody or a reagent that stimulates the IFN-α production [2,6,10,14-tetramethylpentadecane (TMPD; also known as pristane)] enhanced the production of autoantibodies in the MFG-E8- and Tim4-double-deficient mice. These results suggest that the double deficiency of Tim4 and MFG-E8, phosphatidylserine-binding proteins, can trigger autoimmunity and that TNFα and type I IFN regulate reciprocally the development of autoimmune disease.

Identification of TLT2 as an Engulfment Receptor for Apoptotic Cells

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

Structural study of TTR-52 reveals the mechanism by which a bridging molecule mediates apoptotic cell engulfment.

During apoptosis, apoptotic cells are removed by professional phagocytes or neighboring engulfing cells either directly through phagocytic receptors or indirectly through bridging molecules that cross-link dying cells to phagocytes. However, how bridging molecules recognize "eat me" signals and phagocytic receptors to mediate engulfment remains unclear. Here, we report the structural and functional studies of Caenorhabditis elegans TTR-52, a recently identified bridging molecule that cross-links surface-exposed phosphatidylserine (PtdSer) on apoptotic cells to the CED-1 receptor on phagocytes. Crystal structure studies show that TTR-52 has an open β-barrel-like structure with some similarities to the PKCα-C2 domain. TTR-52 is proposed to bind PtdSer via an "ion-mediating" PtdSer-binding mode. Intensive functional studies show that CED-1 binds TTR-52 through its N-terminal EMI domain and that the hydrophobic region of the TTR-52 C terminus is involved in this interaction. In addition, unlike other PtdSer-binding domains, TTR-52 forms dimers, and its dimerization is important for its function in vivo. Our results reveal the first full-length structure of a bridging molecule and the mechanism underlying bridging molecule-mediated apoptotic cell recognition.

MiR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching.

Complement component C1q regulates macrophage expression of Mer tyrosine kinase to promote clearance of apoptotic cells (171.31)

Abstract Failure to efficiently clear apoptotic cells is linked to defects in development and autoimmunity. Complement component C1q is required for efficient clearance of apoptotic cells, and C1q deficiency is the strongest known susceptibility factor for autoimmunity associated with lupus. Despite the importance of C1q in apoptotic cell clearance, the mechanism by which C1q enhances apoptotic cell clearance is poorly understood. Here we describe a novel C1q-dependent engulfment mechanism for apoptotic cells. We found that macrophage activation with C1q resulted in cycloheximide-sensitive enhanced engulfment, indicating a requirement for de novo protein synthesis. To investigate the cycloheximide-sensitive pathway, C1q-elicited macrophage transcripts were identified by microarray. C1q triggered the expression of Mer tyrosine kinase and Gas 6: a receptor-ligand pair that mediates clearance of apoptotic cells. This novel pathway is specific to C1q since MBL, a related collectin, failed to upregulate Mer expression and function. Soluble Mer-Fc fusion protein inhibited C1q-dependent engulfment of apoptotic cells. In addition, Mer-/- macrophages failed to respond to C1q with enhanced engulfment of apoptotic cells, indicating a requirement for Mer. Our results suggest that C1q elicits a macrophage phenotype specifically tailored for apoptotic cell clearance, and these data are consistent with the established requirement for C1q in prevention of autoimmunity.

Oxygenated Lipids: A Mode to WiPE Out Inflammation?

The immunologically silent clearance of apoptotic cells is crucial for maintaining self-tolerance. In this issue of Immunity, Uderhardt etal. (2012) reveal a mechanism by which lipid oxidation by tissue resident macrophages could inhibit the engulfment of apoptotic cells by inflammatory monocytes.

Complement Component C1q Regulates Macrophage Expression of Mer Tyrosine Kinase To Promote Clearance of Apoptotic Cells

Failure to efficiently clear apoptotic cells is linked to defects in development and the onset of autoimmunity. Complement component C1q is required for efficient engulfment of apoptotic cells in mice and humans; however, the molecular mechanisms leading to C1q-dependent engulfment are not fully understood. In this study, we used primary mouse macrophages to identify and characterize a novel molecular mechanism for macrophage-mediated C1q-dependent engulfment of apoptotic cells. We found that macrophage activation with C1q resulted in cycloheximide-sensitive enhanced engulfment, indicating a requirement for de novo protein synthesis. To investigate the cycloheximide-sensitive pathway, C1q-elicited macrophage transcripts were identified by microarray. C1q triggered the expression of Mer tyrosine kinase (Mer) and the Mer ligand growth arrest-specific 6: a receptor-ligand pair that mediates clearance of apoptotic cells. Full-length native C1q, and not the collagen-like tail or heat-denatured protein, stimulated Mer expression. This novel pathway is specific to C1q because mannose-binding lectin, a related collectin, failed to upregulate Mer expression and function. Soluble Mer-Fc fusion protein inhibited C1q-dependent engulfment of apoptotic cells, indicating a requirement for Mer. Moreover, Mer-deficient macrophages failed to respond to C1q with enhanced engulfment. Our results suggest that C1q elicits a macrophage phenotype specifically tailored for apoptotic cell clearance, and these data are consistent with the established requirement for C1q in prevention of autoimmunity.

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.