Title: The Role of Autophagy in Mammalian Development: Cell Makeover Rather than Cell Death
Abstract: Autophagy is important for the degradation of bulk cytoplasm, long-lived proteins, and entire organelles. In lower eukaryotes, autophagy functions as a cell death mechanism or as a stress response during development. However, autophagy's significance in vertebrate development, and the role (if any) of vertebrate-specific factors in its regulation, remains unexplained. Through careful analysis of the current autophagy gene mutant mouse models, we propose that in mammals, autophagy may be involved in specific cytosolic rearrangements needed for proliferation, death, and differentiation during embryogenesis and postnatal development. Thus, autophagy is a process of cytosolic “renovation,” crucial in cell fate decisions. Autophagy is important for the degradation of bulk cytoplasm, long-lived proteins, and entire organelles. In lower eukaryotes, autophagy functions as a cell death mechanism or as a stress response during development. However, autophagy's significance in vertebrate development, and the role (if any) of vertebrate-specific factors in its regulation, remains unexplained. Through careful analysis of the current autophagy gene mutant mouse models, we propose that in mammals, autophagy may be involved in specific cytosolic rearrangements needed for proliferation, death, and differentiation during embryogenesis and postnatal development. Thus, autophagy is a process of cytosolic “renovation,” crucial in cell fate decisions. Autophagy is a lysosomal pathway used by eukaryotes for degrading and recycling various cellular constituents, such as long-lived proteins and entire organelles. There are three main forms of autophagy: microautophagy, macroautophagy, and chaperone-mediated autophagy (Klionsky, 2005Klionsky D.J. The molecular machinery of autophagy: unanswered questions.J. Cell Sci. 2005; 118: 7-18Crossref PubMed Scopus (716) Google Scholar, Massey et al., 2006Massey A.C. Zhang C. Cuervo A.M. Chaperone-mediated autophagy in aging and disease.Curr. Top. Dev. Biol. 2006; 73: 205-235Crossref PubMed Scopus (242) Google Scholar). Microautophagy and the mammalian-specific chaperone-mediated autophagy directly involve the lysosome, which either engulfs small portions of cytosol or receives chaperone-associated cargoes, respectively. Macroautophagy is responsible for the turnover of organelles and portions of cytosol sequestered in a double-membrane-bound vesicle, the autophagosome. Autophagosomes originate from a precursor structure, the phagophore, which starts growing at both ends and finally closes and wraps the bulk cytoplasm and organelles (Figure 1A). Autophagosomes slide along cytoskeletal structures and fuse with lysosomes, forming a single large and membrane-surrounded vesicle called the autophagolysosome, where both their membrane and contents are degraded by lytic enzymes (reviewed in Xie and Klionsky, 2007Xie Z. Klionsky D.J. Autophagosome formation: core machinery and adaptations.Nat. Cell Biol. 2007; 9: 1102-1109Crossref PubMed Scopus (1541) Google Scholar). Most of the genes involved in macroautophagy (hereafter autophagy), the so-called Autophagy-related (Atg) genes, have been discovered in S. cerevisiae (Ohsumi, 2001Ohsumi Y. Molecular dissection of autophagy: two ubiquitin-like systems.Nat. Rev. Mol. Cell Biol. 2001; 2: 211-216Crossref PubMed Scopus (971) Google Scholar). These include genes that regulate autophagosome formation, which requires two evolutionarily conserved ubiquitin-like conjugation systems—the Atg12-Atg5 and the Atg8(LC3)-PE (phosphatidylethanolamine) systems (Figure 1) (Ohsumi, 2001Ohsumi Y. Molecular dissection of autophagy: two ubiquitin-like systems.Nat. Rev. Mol. Cell Biol. 2001; 2: 211-216Crossref PubMed Scopus (971) Google Scholar, Suzuki and Ohsumi, 2007Suzuki K. Ohsumi Y. Molecular machinery of autophagosome formation in yeast, Saccharomyces cerevisiae.FEBS Lett. 2007; 581: 2156-2161Crossref PubMed Scopus (306) Google Scholar)—as well as genes that function in other stages of autophagy. Atg genes were originally described in yeast, and in some cases their orthologs have been isolated and functionally characterized in mammals (Table 1) (Ferraro and Cecconi, 2007Ferraro E. Cecconi F. Autophagic and apoptotic response to stress signals in mammalian cells.Arch. Biochem. Biophys. 2007; 462: 210-219Crossref PubMed Scopus (0) Google Scholar, Levine and Kroemer, 2008Levine B. Kroemer G. Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (4871) Google Scholar).Table 1Mouse Models of Autophagy Impairment with Clear Developmental PhenotypesMolecule and Role in AutophagyMouse Model Allele Symbol and TypeType of MutationKey Aspects of the PhenotypeReferenceAmbra1: WD40 protein promotes Vps34 complex formation (autophagosome nucleation)ambra1gt/gt (all tissues)gene trap allele: Ambra1Gt(pGT1.8geo)1Fcecgene trap insertion predicted to cause a 459 aa C-terminal truncation; fusion transcript, but no protein, detecteddecreased autophagy, increased apoptosis and cell proliferation in embryonic brain; neural tube defects and embryonic lethalityFimia et al., 2007Fimia G.M. Stoykova A. Romagnoli A. Giunta L. Di Bartolomeo S. Nardacci R. Corazzari M. Fuoco C. Ucar A. Schwartz P. et al.Ambra1 regulates autophagy and development of the nervous system.Nature. 2007; 447: 1121-1125Crossref PubMed Scopus (682) Google ScholarAtg5: ubiquitin-like protein covalently modifies Atg12 and binds Atg16L to promote LC3-PE recruitment to autophagosome membranes (autophagosome elongation)atg5−/− (all tissues)knockout allele: Atg5tm1Nmzneomycin-resistance (neo) gene replaces the transcription start site and first two exonsperinatal death due to nutrient and energy depletion; suckling defect; increased apoptosis in embryosKuma et al., 2004Kuma A. Hatano M. Matsui M. Yamamoto A. Nakaya H. Yoshimori T. Ohsumi Y. Tokuhisa T. Mizushima N. The role of autophagy during the early neonatal starvation period.Nature. 2004; 432: 1032-1036Crossref PubMed Scopus (2177) Google Scholar, Qu et al., 2007Qu X. Zou Z. Sun Q. Luby-Phelps K. Cheng P. Hogan R.N. Gilpin C. Levine B. Autophagy gene-dependent clearance of apoptotic cells during embryonic development.Cell. 2007; 128: 931-946Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholaratg5−/flox (tissue-specific)conditional allele: Atg5tm1Myok (floxed)loxP sites flank exon 3 and neo cassette; Cre removes both exon 3 and neoAtg5tm1Myok combined w/Tg(Nes-cre)1Kln transgenic (Cre driver)Cre expressed in neuronal and glial precursors by e11.0 (Nestin promoter)ubiquitin-positive inclusions in the brains of e15.5 embryos and postnatal mice, early onset neurodegenerationHara et al., 2006Hara T. Nakamura K. Matsui M. Yamamoto A. Nakahara Y. Suzuki-Migishima R. Yokoyama M. Mishima K. Saito I. Okano H. Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice.Nature. 2006; 441: 885-889Crossref PubMed Scopus (2787) Google ScholarAtg5tm1Myok combined w/Tg(Zp3-cre)93Knw transgenic (Cre driver)Cre expressed in female germline (Zp3 promoter)null embryos derived from Atg5-deficient eggs fail to develop beyond the eight-cell stage; Atg5-deficient eggs fertilized by wild-type sperm are normalTsukamoto et al., 2008Tsukamoto S. Kuma A. Murakami M. Kishi C. Yamamoto A. Mizushima N. Autophagy is essential for preimplantation development of mouse embryos.Science. 2008; 321: 117-120Crossref PubMed Scopus (382) Google ScholarAtg5tm1Myok combined w/Cd19tm1(cre)Cgn transgenic (Cre driver)Cre expressed throughout B lymphocyte development (Cd19 promoter)the pro-B to pre-B cell transition is defectiveMiller et al., 2008Miller B.C. Zhao Z. Stephenson L.M. Cadwell K. Pua H.H. Lee H.K. Mizushima N. Iwasaki A. He Y.W. Swat W. Virgin 4th, H.W. The autophagy gene ATG5 plays an essential role in B lymphocyte development.Autophagy. 2008; 4: 309-314Crossref PubMed Google ScholarAtg7: E1 ligase-like protein activates LC3-I and Atg12 (autophagosome elongation)atg7−/− (all tissues)conditional allele: Atg7tm1.1TchiloxP sites flank exon 14excised during oogenesis, this allele results in a similar phenotype to Atg5tm1Myok / Tg(Zp3-cre)93Knw as aboveKomatsu et al., 2005Komatsu M. Waguri S. Ueno T. Iwata J. Murata S. Tanida I. Ezaki J. Mizushima N. Ohsumi Y. Uchiyama Y. et al.Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.J. Cell Biol. 2005; 169: 425-434Crossref PubMed Scopus (1707) Google Scholaratg7−/flox (tissue-specific)combined w/Tg(Nes-cre)1Wme transgenic (Cre/Flp)this Nestin-Cre, in which Cre has a nuclear localization signal, drives variable/mosaic activity by e10.5, detectable in all adult tissues examined, including the germlineprogressive deficits in motor function and death a few months after birthKomatsu et al., 2006Komatsu M. Waguri S. Chiba T. Murata S. Iwata J. Tanida I. Ueno T. Koike M. Uchiyama Y. Kominami E. Tanaka K. Loss of autophagy in the central nervous system causes neurodegeneration in mice.Nature. 2006; 441: 880-884Crossref PubMed Scopus (2544) Google Scholarcombined w/Tg(Pcp2-cre)2Mpin transgenic (Cre/Flp)Cre expressed in Purkinje cells starting at P6 and complete 2–3 weeks postpartum (L7/Pcp2 promoter)degeneration of the axon terminals during postnatal brain developmentKomatsu et al., 2007Komatsu M. Wang Q.J. Holstein G.R. Friedrich Jr., V.L. Iwata J. Kominami E. Chait B.T. Tanaka K. Yue Z. Essential role for autophagy protein Atg7 in the maintenance of axonal homeostasis and the prevention of axonal degeneration.Proc. Natl. Acad. Sci. USA. 2007; 104: 14489-14494Crossref PubMed Scopus (435) Google ScholarBeclin 1/Atg6: subunit of the Vps34 complex (autophagosome nucleation)becn1−/− (all tissues)knockout allele: Becn1tm1Blevneo replaces exons 1 and 2abnormal ectodermal layer with reduced cavitation and early embryonic lethalityQu et al., 2003Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. et al.Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.J. Clin. Invest. 2003; 112: 1809-1820Crossref PubMed Scopus (1655) Google Scholar, Qu et al., 2007Qu X. Zou Z. Sun Q. Luby-Phelps K. Cheng P. Hogan R.N. Gilpin C. Levine B. Autophagy gene-dependent clearance of apoptotic cells during embryonic development.Cell. 2007; 128: 931-946Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholarknockout allele: Becn1tm1Htzneo replaces exons 1 and 2 and 1.6 kb of upstream sequencewidespread cell death, reduced embryo sizeYue et al., 2003Yue Z. Jin S. Yang C. Levine A.J. Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor.Proc. Natl. Acad. Sci. USA. 2003; 100: 15077-15082Crossref PubMed Scopus (1586) Google Scholar.Allele symbols are standard according to http://www.informatics.jax.org. Open table in a new tab Allele symbols are standard according to http://www.informatics.jax.org. A core molecule in autophagy regulation is the kinase mammalian Target Of Rapamycin (mTOR). By sensing signals that monitor nutrient levels, mTOR can trigger protein translation by specific phosphorylation of the ribosomal protein S6 kinase (pS6K) (Blommaart et al., 1995Blommaart E.F. Luiken J.J. Blommaart P.J. van Woerkom G.M. Meijer A.J. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes.J. Biol. Chem. 1995; 270: 2320-2326Abstract Full Text Full Text PDF PubMed Scopus (534) Google Scholar). When nutrients are lacking, mTOR repression shifts cellular metabolism toward autophagy and recycling of cytosolic constituents (Figure 1), although the precise targets of mTOR in autophagy remain unidentified. The discovery of the molecular basis of autophagy has enabled the search for links with human pathological conditions. Any genetic or pharmacological alteration in this process impairs cell survival rate or cell metabolism, thereby affecting tissue homeostasis (Levine and Kroemer, 2008Levine B. Kroemer G. Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (4871) Google Scholar, Mizushima et al., 2008Mizushima N. Levine B. Cuervo A.M. Klionsky D.J. Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (4562) Google Scholar). Many neurodegenerative conditions, for example, can be traced back to defective autophagy. The role of autophagy in Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, or Alzheimer's disease may reside in the failure to clear aggregates of mutated toxic proteins (reviewed in Rubinsztein, 2006Rubinsztein D.C. The roles of intracellular protein-degradation pathways in neurodegeneration.Nature. 2006; 443: 780-786Crossref PubMed Scopus (1215) Google Scholar). Autophagy has also been identified as a crucial process in oncogenesis and cancer progression (Gozuacik and Kimchi, 2004Gozuacik D. Kimchi A. 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Bray K. Degenhardt K. Chen G. Jin S. White E. Autophagy suppresses tumor progression by limiting chromosomal instability.Genes Dev. 2007; 21: 1367-1381Crossref PubMed Scopus (675) Google Scholar). In cancer progression, autophagy may, on the other hand, provide tumor cells with a survival strategy, thus suggesting a therapeutic use for autophagy downregulation in solid tumors (reviewed in Levine and Kroemer, 2008Levine B. Kroemer G. Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (4871) Google Scholar, Mizushima et al., 2008Mizushima N. Levine B. Cuervo A.M. Klionsky D.J. Autophagy fights disease through cellular self-digestion.Nature. 2008; 451: 1069-1075Crossref PubMed Scopus (4562) Google Scholar). 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Meanwhile, other studies focused on the peculiar morphology that some cells with autophagic activity show during embryogenesis in lower eukaryotes. In 1977, Jacques Beaulaton and Richard Lockshin elegantly demonstrated that in Lepidoptera the normal degeneration of intersegmental muscles, a crucial step in insect metamorphosis, was accompanied by the presence of autophagic vacuoles containing mitochondria (Beaulaton and Lockshin, 1977Beaulaton J. Lockshin R.A. Ultrastructural study of the normal degeneration of the intersegmental muscles of Anthereae polyphemus and Manduca sexta (Insecta, Lepidoptera) with particular reference of cellular autophagy.J. Morphol. 1977; 154: 39-57Crossref PubMed Google Scholar). Similar evidence was found in the regressing prothoracic and salivary glands of other insects, including Drosophila (Scharrer, 1966Scharrer B. Ultrastructural study of the regressing prothoracic glands of blattarian insects.Z. Zellforsch. Mikrosk. 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Growth arrest and autophagy are required for salivary gland cell degradation in Drosophila.Cell. 2007; 131: 1137-1148Abstract Full Text Full Text PDF PubMed Scopus (458) Google Scholar). In only a few cases, most notably in the developing nervous system, was such a death pathway found in higher eukaryotes (Clarke, 1990Clarke P.G. Developmental cell death: morphological diversity and multiple mechanisms.Anat. Embryol. (Berl.). 1990; 181: 195-213Crossref PubMed Google Scholar, Hornung et al., 1989Hornung J.P. Koppel H. Clarke P.G. Endocytosis and autophagy in dying neurons: an ultrastructural study in chick embryos.J. Comp. Neurol. 1989; 283: 425-437Crossref PubMed Google Scholar). These studies suggested that autophagy has an important role in development, although its specific functions remained unclear when using morphologic analysis alone. The era of Atg gene discovery in lower eukaryotes was followed by in-depth investigation of the same process in vertebrates. In 1998, Beth Levine's and Noboru Mizushima's laboratories isolated, in mammals, Beclin 1, and Atg5 and Atg12, respectively (Liang et al., 1998Liang X.H. Kleeman L.K. Jiang H.H. Gordon G. Goldman J.E. Berry G. Herman B. Levine B. Protection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein.J. Virol. 1998; 72: 8586-8596Crossref PubMed Google Scholar, Mizushima et al., 1998bMizushima N. Sugita H. Yoshimori T. Ohsumi Y. A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy.J. Biol. Chem. 1998; 273: 33889-33892Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Several mouse models for Atg gene inactivation revealed interesting embryonic phenotypes (Table 1, and listed in Table 1 of Levine and Kroemer, 2008Levine B. Kroemer G. Autophagy in the pathogenesis of disease.Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (4871) Google Scholar). In theory, there could be a link between apoptosis and autophagy in these cases, suggesting that autophagy may trigger cell demise upon strong environmental or extrinsic stress during development, thereby matching the scenario believed to occur in lower eukaryotes. However, the mechanisms and functions of autophagy need further investigation. The question remains as to whether autophagy constitutes an understudied form of cell destruction or, alternatively or in parallel, whether it is a regulated process capable of rapidly changing the cell in response to environmental stimuli or intrinsic metabolic needs. In order to function during embryogenesis (in proliferation, migration, communication, differentiation, or death), cells undergo phases of quiescence or enhanced metabolism. Thus, they require dynamic tools to suddenly modify their organelle or protein content so as to adapt rapidly and respond to adverse conditions. Since development time is so rapid, proteasome-mediated turnover of individual proteins is probably not sufficient to effect the necessary cellular architectural changes. Autophagy, which can mediate bulk protein and organelle turnover, could therefore help to profoundly renovate cells or modify their external appearance within a few hours. Therefore, a fuller knowledge of autophagy in development might not only reveal the evolutionary significance of this process, it may also explain its roles in human pathologies. To establish the role of autophagy in mammalian development or adult tissue homeostasis, various mouse and cellular models have been generated by using multiple genetic approaches, such as gene targeting, gene trapping, dysregulation in cell or tissue culture of embryonic origin, tissue chimerism, and tissue-specific conditional mutagenesis. The resulting developmental phenotypes and the genetic features of the most relevant mouse models are summarized in Table 1. Note that the complex phenotypes we include here may not be uniquely related to autophagy defects, but could also be due to the inactivation of nonautophagic functions of these genes. The mouse ortholog of yeast Atg1 is Ulk1/Unc51.1 (Figure 1), a serine/threonine kinase involved in the early steps of autophagosome generation (Tomoda et al., 1999Tomoda T. Bhatt R.S. Kuroyanagi H. Shirasawa T. Hatten M.E. A mouse serine/threonine kinase homologous to C. elegans UNC51 functions in parallel fiber formation of cerebellar granule neurons.Neuron. 1999; 24: 833-846Abstract Full Text Full Text PDF PubMed Google Scholar). Atg1 forms a complex with other Atg regulatory proteins such as Atg13 and Atg17 (Kamada et al., 2000Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. Tor-mediated induction of autophagy via an Apg1 protein kinase complex.J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (849) Google Scholar, Tsukada and Ohsumi, 1993Tsukada M. Ohsumi Y. Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae.FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1228) Google Scholar). Under nutrient-rich conditions, i.e., upon autophagy inhibition, yeast Atg13 is hyperphosphorylated, blocking its association with Atg1. However, under nutrient starvation, i.e., when autophagy activation is required, Atg13 becomes partially dephosphorylated, with subsequent Atg1-Atg13 interaction and the generation of the autophagosome (Kamada et al., 2000Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. Tor-mediated induction of autophagy via an Apg1 protein kinase complex.J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (849) Google Scholar). Ulk1 was the first autophagy-related gene to be manipulated in a mouse system, where it is expressed in granule cells in the cerebellar cortex. Retroviral infection of immature granule cells with a dominant-negative Ulk1 resulted in inhibition of neurite outgrowth in vitro and in vivo (Tomoda et al., 1999Tomoda T. Bhatt R.S. Kuroyanagi H. Shirasawa T. Hatten M.E. A mouse serine/threonine kinase homologous to C. elegans UNC51 functions in parallel fiber formation of cerebellar granule neurons.Neuron. 1999; 24: 833-846Abstract Full Text Full Text PDF PubMed Google Scholar, Tomoda et al., 2004Tomoda T. Kim J.H. Zhan C. Hatten M.E. Role of Unc51.1 and its binding partners in CNS axon outgrowth.Genes Dev. 2004; 18: 541-558Crossref PubMed Scopus (143) Google Scholar). Moreover, infected neurons failed to express any specific markers of neuronal differentiation, suggesting that neurodevelopment was arrested early (Tomoda et al., 1999Tomoda T. Bhatt R.S. Kuroyanagi H. Shirasawa T. Hatten M.E. A mouse serine/threonine kinase homologous to C. elegans UNC51 functions in parallel fiber formation of cerebellar granule neurons.Neuron. 1999; 24: 833-846Abstract Full Text Full Text PDF PubMed Google Scholar). Ulk1 localizes to both axonal shafts and growth cones of extending axons and is essential for neurite extension and parallel fiber formation in cerebellar granule neurons (Tomoda et al., 2004Tomoda T. Kim J.H. Zhan C. Hatten M.E. Role of Unc51.1 and its binding partners in CNS axon outgrowth.Genes Dev. 2004; 18: 541-558Crossref PubMed Scopus (143) Google Scholar). An autophagy-independent role for Ulk1 has been suggested in various species, such as control of axonal elongation and branching in C. elegans (Ogura et al., 1994Ogura K. Wicky C. Magnenat L. Tobler H. Mori I. Müller F. Ohshima Y. Caenorhabditis elegans unc-51 gene required for axonal elongation encodes a novel serine/threonine kinase.Genes Dev. 1994; 8: 2389-2400Crossref PubMed Google Scholar) and mammals (Zhou et al., 2007Zhou X. Babu J.R. da Silva S. Shu Q. Graef I.A. Oliver T. Tomoda T. Tani T. Wooten M.W. Wang F. Unc-51-like kinase 1/2-mediated endocytic processes regulate filopodia extension and branching of sensory axons.Proc. Natl. Acad. Sci. USA. 2007; 104: 5842-5847Crossref PubMed Scopus (0) Google Scholar). What seems to be misregulated by Ulk1 manipulation is either the establishment of a proper constellation of surface factors necessary for cell migration and adhesion, or normal cell metabolism, essential for an energy-consumptive cellular task such as axonal growth. Interestingly, Ulk1 was found to localize to autophagic phagophores under starvation conditions, interacting with the Atg17 ortholog FIP200 (Hara et al., 2008Hara T. Takamura A. Kishi C. Iemura S. Natsume T. Guan J.L. Mizushima N. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells.J. Cell Biol. 2008; 181: 497-510Crossref PubMed Scopus (623) Google Scholar). FIP200 itself was relocalized upon starvation, switching from the cytoplasm to the phagophore (Hara et al., 2008Hara T. Takamura A. Kishi C. Iemura S. Natsume T. Guan J.L. Mizushima N. FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells.J. Cell Biol. 2008; 181: 497-510Crossref PubMed Scopus (623) Google Scholar). Given that FIP200 regulates diverse cellular processes such as cell size, proliferation, migration, and focal adhesion, the Ulk1-FIP200 interaction might have other vital functions aside from autophagy. The autophagy-related gene becn1 (encoding Beclin 1; coiled-coil, myosin-like BCL2-interacting protein) has been targeted and inactivated in mice (Qu et al., 2003Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. et al.Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.J. Clin. Invest. 2003; 112: 1809-1820Crossref PubMed Scopus (1655) Google Scholar, Yue et al., 2003Yue Z. Jin S. Yang C. Levine A.J. Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor.Proc. Natl. Acad. Sci. USA. 2003; 100: 15077-15082Crossref PubMed Scopus (1586) Google Scholar). Beclin 1 is the mammalian ortholog of yeast Atg6, a component of the class III phosphatidylinositol-3-OH kinase (also known as Vps34) complex regulating autophagosome formation in mammals (Liang et al., 1999Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Induction of autophagy and inhibition of tumorigenesis by beclin 1.Nature. 1999; 402: 672-676Crossref PubMed Scopus (2490) Google Scholar). 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Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. et al.Promotion of tumorigenesis by heterozygous disruption o