Autophagy genes coordinate with the class II PI/PtdIns 3-kinase PIKI-1 to regulate apoptotic cell clearance in C. elegans

Phagocytosis and autophagy are two lysosome-mediated cellular degradation pathways designed to eliminate extracellular and intracellular constituents, respectively. Recent studies suggest that these two processes intersect. Several autophagy proteins have been shown to participate in clearance of apoptotic cells, but whether and how the autophagy pathway is involved is unclear. Here we showed that loss of function mutations in 19 genes acting at overlapping or distinct stages of autophagy caused increased numbers of cell corpses in C. elegans embryos. In contrast, genes that mediate specific clearance of P granules or protein aggregates through autophagy are dispensable for cell corpse removal. We showed that defective autophagy impairs phagosome maturation and that autophagy genes act in parallel to the class II phosphoinositide (PI)/phosphatidylinositol (PtdIns) 3-kinase PIKI-1 to regulate phagosomal PtdIns3P in a similar manner as VPS-34. Our data indicate that autophagy may coordinate with PIKI-1 to promote phagosome maturation, thus ensuring efficient clearance of apoptotic cells.     

Autophagy genes function sequentially to promote apoptotic cell corpse degradation in the engulfing cell

Apoptotic cell degradation is a fundamental process for organism development, and impaired clearance causes inflammatory or autoimmune disease. Although autophagy genes were reported to be essential for exposing the engulfment signal on apoptotic cells, their roles in phagocytes for apoptotic cell removal are not well understood. In this paper, we develop live-cell imaging techniques to study apoptotic cell clearance in the Caenorhabditis elegans Q neuroblast lineage. We show that the autophagy proteins LGG-1/LC3, ATG-18, and EPG-5 were sequentially recruited to internalized apoptotic Q cells in the phagocyte. In atg-18 or epg-5 mutants, apoptotic Q cells were internalized but not properly degraded; this phenotype was fully rescued by the expression of autophagy genes in the phagocyte. Time-lapse analysis of autophagy mutants revealed that recruitment of the small guanosine triphosphatases RAB-5 and RAB-7 to the phagosome and the formation of phagolysosome were all significantly delayed. Thus, autophagy genes act within the phagocyte to promote apoptotic cell degradation.     

V-ATPase V1 sector is required for corpse clearance and neurotransmission in Caenorhabditis elegans

The vacuolar-type ATPase (V-ATPase) is a proton pump composed of two sectors, the cytoplasmic V(1) sector that catalyzes ATP hydrolysis and the transmembrane V(o) sector responsible for proton translocation. The transmembrane V(o) complex directs the complex to different membranes, but also has been proposed to have roles independent of the V(1) sector. However, the roles of the V(1) sector have not been well characterized. In the nematode Caenorhabditis elegans there are two V(1) B-subunit genes; one of them, vha-12, is on the X chromosome, whereas spe-5 is on an autosome. vha-12 is broadly expressed in adults, and homozygotes for a weak allele in vha-12 are viable but are uncoordinated due to decreased neurotransmission. Analysis of a null mutation demonstrates that vha-12 is not required for oogenesis or spermatogenesis in the adult germ line, but it is required maternally for early embryonic development. Zygotic expression begins during embryonic morphogenesis, and homozygous null mutants arrest at the twofold stage. These mutant embryos exhibit a defect in the clearance of apoptotic cell corpses in vha-12 null mutants. These observations indicate that the V(1) sector, in addition to the V(o) sector, is required in exocytic and endocytic pathways.     

p62/SQSTM1: A Missing Link between Protein Aggregates and the Autophagy Machinery

In eukaryotic cells short-lived proteins are degraded in a specific process by the ubiquitin-proteasome system (UPS), whereas long-lived proteins and damaged organelles are degraded by macroautophagy (hereafter referred to as autophagy). A growing body of evidence now suggests that autophagy is important for clearance of protein aggregates that form in cells as a consequence of ageing, oxidative stress, alterations that elevate the amounts of certain aggregation-prone proteins or expression of aggregating mutant variants of specific proteins. Autophagy is generally considered to be a non-specific, bulk degradation process. However, a recent study suggests that p62/SQSTM1 may link the recognition of polyubiquitinated protein aggregates to the autophagy machinery.1 This protein is able to polymerize via its N-terminal PB1 domain and to recognize polyubiquitin via its C-terminal UBA domain. It can also recruit the autophagosomal protein LC3 and co-localizes with many types of polyubiquitinated protein aggregates.1 Here we discuss possible implications of these findings and raise some questions for further investigation.     

Crystal structure of the cell corpse engulfment protein CED-2 in Caenorhabditis elegans

In the nematode Caenorhabditis elegans, the cell corpse engulfment proteins CED-2, CED-5, and CED-12 act in the same pathway to regulate the activation of the Rac small GTPase, CED-10, leading to the rearrangement of the actin cytoskeleton for engulfing apoptotic cells. Nevertheless, it is not well understood how these proteins act together. Here we report the crystal structures of the CED-2 protein as determined by X-ray crystallography. The full-length CED-2 protein and its truncated form containing the N-terminal SH2 domain and the first SH3 domain show similar three-dimensional structures. A CED-2 point mutation (F125G) disrupting its interaction with the PXXP motif of CED-5 did not affect its rescuing activity. However, CED-2 was found to interact with the N-terminal region of CED-5. Our findings suggest that CED-2 may regulate cell corpse engulfment by interacting with CED-5 through the N-terminal region rather than the PXXP motif.     

Signaling unmasked: Autophagy and catalase promote programmed cell death

Autophagy contributes to the removal of harmful cellular refuse, whereas catalase plays an important protective role by detoxifying reactive oxygen species. We recently found that autophagy and catalase are also required for promoting programmed cell death induced during plant immune responses. Here we discuss the difficulties in identifying cell death effectors, which are also required to maintain cellular homeostasis, and how their prodeath roles were unmasked using an unbiased forward genetics approach.     

Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap

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Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids

The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8^−/− mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.     

A BORC-dependent molecular pathway for vesiculation of cell corpse phagolysosomes

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Autophagy and cell death

Autophagy exerts dual functions in cancer, acting as both a tumor suppressor, for example, by preventing the accumulation of damaged proteins and organelles, and as a tumor promoter that supports tumor growth. Many anticancer therapies engage autophagy as part of a cellular response. However, the question of whether or not autophagic activity in cells undergoing cell death is the cause of death or whether it is actually an attempt to support survival in response to cellular stress conditions has been discussed with great controversy.     

Autophagy in stem cell aging

Aging is responsible for changes in mammalian tissues that result in an imbalance to tissue homeostasis and a decline in the regeneration capacity of organs due to stem cell exhaustion. Autophagy is a constitutive pathway necessary to degrade damaged organelles and protein aggregates. Autophagy is one of the hallmarks of aging, which involves a decline in the number and functionality of stem cells. Recent studies show that stem cells require autophagy to get rid of cellular waste produced during the quiescent stage. In particular, two independent studies in muscle and hematopoietic stem cells demonstrate the relevance of the autophagy impairment for stem cell exhaustion and aging. In this review, we summarize the main results of these works, which helped to elucidate the impact of autophagy in stem cell activity as well as in age‐associated diseases.     

Control of non-apoptotic nurse cell death by engulfment genes in Drosophila

Programmed cell death occurs as a normal part of oocyte development in Drosophila. For each egg that is formed, 15 germline-derived nurse cells transfer their cytoplasmic contents into the oocyte and die. Disruption of apoptosis or autophagy only partially inhibits the death of the nurse cells, indicating that other mechanisms significantly contribute to nurse cell death. Recently, we demonstrated that the surrounding stretch follicle cells non-autonomously promote nurse cell death during late oogenesis and that phagocytosis genes including draper, ced-12, and the JNK pathway are crucial for this process. When phagocytosis genes are inhibited in the follicle cells, events specifically associated with death of the nurse cells are impaired. Death of the nurse cells is not completely blocked in draper mutants, suggesting that other engulfment receptors are involved. Indeed, we found that the integrin subunit, αPS3, is enriched on stretch follicle cells during late oogenesis and is required for elimination of the nurse cells. Moreover, double mutant analysis revealed that integrins act in parallel to draper. Death of nurse cells in the Drosophila ovary is a unique example of programmed cell death that is both non-apoptotic and non-cell autonomously controlled.     

Anion Transport or Nucleotide Binding by Ucp2 Is Indispensable for Ucp2-Mediated Efferocytosis

Rapid and efficient engulfment of apoptotic cells is an essential property of phagocytes for removal of the large number of apoptotic cells generated in multicellular organisms. To achieve this, phagocytes need to be able to continuously uptake apoptotic cells. It was recently reported that uncoupling protein 2 (Ucp2) promotes engulfment of apoptotic cells by increasing the phagocytic capacity, thereby allowing cells to continuously ingest apoptotic cells. However, the functions of Ucp2, beyond its possible role in dissipating the mitochondrial membrane potential, that contribute to elevation of the phagocytic capacity have not been determined. Here, we report that the anion transfer or nucleotide binding activity of Ucp2, as well as its dissipation of the mitochondrial membrane potential, is necessary for Ucp2-mediated engulfment of apoptotic cells. To study these properties, we generated Ucp2 mutations that affected three different functions of Ucp2, namely, dissipation of the mitochondrial membrane potential, transfer of anions, and binding of purine nucleotides. Mutations of Ucp2 that affected the proton leak did not enhance the engulfment of apoptotic cells. Although anion transfer and nucleotide binding mutations did not affect the mitochondrial membrane potential, they exerted a dominant-negative effect on Ucp2-mediated engulfment. Furthermore, none of our Ucp2 mutations increased the phagocytic capacity. We conclude that dissipation of the proton gradient by Ucp2 is not the only determinant of the phagocytic capacity and that anion transfer or nucleotide binding by Ucp2 is also essential for Ucp2-mediated engulfment of apoptotic cells.     

Autophagy promotes cell motility by driving focal adhesion turnover

In eukaryotic cells, cell migration is a dynamic and complex process that involves finely tuned orchestration of a multitude of proteins including, for example, those involved in focal adhesions (FAs). Cell migration plays an indispensable role in particular stages of development and its proper regulation is crucial in various biological processes, from wound healing to the immune response. FAs are transmembrane protein complexes that traverse cytoskeletal infrastructures all the way to the extracellular matrix, producing traction at the leading edge of the cell, thus allowing for motility. The assembly of FAs has been extensively studied, whereas disassembly remains poorly understood. Here, we highlight 2 recent studies (see the corresponding puncta in the previous and current issues of the journal) that demonstrate a requirement for macroautophagy/autophagy in FA disassembly. These studies also provide a deeper understanding of how autophagy can contribute to cell migration among multiple cell types.