Publications by authors named "Maho Hamasaki"

34 Publications

Degradation of the NOTCH intracellular domain by elevated autophagy in osteoblasts promotes osteoblast differentiation and alleviates osteoporosis.

Autophagy 2022 Jan 13:1-10. Epub 2022 Jan 13.

Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.

Maintenance of bone integrity is mediated by the balanced actions of osteoblasts and osteoclasts. Because macroautophagy/autophagy regulates osteoblast mineralization, osteoclast differentiation, and their secretion from osteoclast cells, autophagy deficiency in osteoblasts or osteoclasts can disrupt this balance. However, it remains unclear whether upregulation of autophagy becomes beneficial for suppression of bone-associated diseases. In this study, we found that genetic upregulation of autophagy in osteoblasts facilitated bone formation. We generated mice in which autophagy was specifically upregulated in osteoblasts by deleting the gene encoding RUBCN/Rubicon, a negative regulator of autophagy. The mice showed progressive skeletal abnormalities in femur bones. Consistent with this, RUBCN deficiency in osteoblasts resulted in elevated differentiation and mineralization, as well as an increase in the elevated expression of key transcription factors involved in osteoblast function such as and . Furthermore, RUBCN deficiency in osteoblasts accelerated autophagic degradation of NOTCH intracellular domain (NICD) and downregulated the NOTCH signaling pathway, which negatively regulates osteoblast differentiation. Notably, osteoblast-specific deletion of RUBCN alleviated the phenotype in a mouse model of osteoporosis. We conclude that RUBCN is a key regulator of bone homeostasis. On the basis of these findings, we propose that medications targeting RUBCN or autophagic degradation of NICD could be used to treat age-related osteoporosis and bone fracture.: ALPL: alkaline phosphatase, liver/bone/kidney; BCIP/NBT: 5-bromo-4-chloro-3'-indolyl phosphate/nitro blue tetrazolium; BMD: bone mineral density; BV/TV: bone volume/total bone volume; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NICD: NOTCH intracellular domain; RB1CC1/FIP200: RB1-inducible coiled-coil 1; RUBCN/Rubicon: RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein; SERM: selective estrogen receptor modulator; TNFRSF11B/OCIF: tumor necrosis factor receptor superfamily, member 11b (osteoprotegerin).
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http://dx.doi.org/10.1080/15548627.2021.2017587DOI Listing
January 2022

Rubicon prevents autophagic degradation of GATA4 to promote Sertoli cell function.

PLoS Genet 2021 08 5;17(8):e1009688. Epub 2021 Aug 5.

Department of Genetics, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.

Autophagy degrades unnecessary proteins or damaged organelles to maintain cellular function. Therefore, autophagy has a preventive role against various diseases including hepatic disorders, neurodegenerative diseases, and cancer. Although autophagy in germ cells or Sertoli cells is known to be required for spermatogenesis and male fertility, it remains poorly understood how autophagy participates in spermatogenesis. We found that systemic knockout mice of Rubicon, a negative regulator of autophagy, exhibited a substantial reduction in testicular weight, spermatogenesis, and male fertility, associated with upregulation of autophagy. Rubicon-null mice also had lower levels of mRNAs of Sertoli cell-related genes in testis. Importantly, Rubicon knockout in Sertoli cells, but not in germ cells, caused a defect in spermatogenesis and germline stem cell maintenance in mice, indicating a critical role of Rubicon in Sertoli cells. In mechanistic terms, genetic loss of Rubicon promoted autophagic degradation of GATA4, a transcription factor that is essential for Sertoli cell function. Furthermore, androgen antagonists caused a significant decrease in the levels of Rubicon and GATA4 in testis, accompanied by elevated autophagy. Collectively, we propose that Rubicon promotes Sertoli cell function by preventing autophagic degradation of GATA4, and that this mechanism could be regulated by androgens.
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http://dx.doi.org/10.1371/journal.pgen.1009688DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8341604PMC
August 2021

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).

Autophagy 2021 Jan 8;17(1):1-382. Epub 2021 Feb 8.

University of Crete, School of Medicine, Laboratory of Clinical Microbiology and Microbial Pathogenesis, Voutes, Heraklion, Crete, Greece; Foundation for Research and Technology, Institute of Molecular Biology and Biotechnology (IMBB), Heraklion, Crete, Greece.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
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http://dx.doi.org/10.1080/15548627.2020.1797280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7996087PMC
January 2021

THOC4 regulates energy homeostasis by stabilizing mRNA during prolonged starvation.

J Cell Sci 2021 03 22;134(6). Epub 2021 Mar 22.

Department of Genetics, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan

TFEB, a basic helix-loop-helix transcription factor, is a master regulator of autophagy, lysosome biogenesis and lipid catabolism. Compared to posttranslational regulation of TFEB, the regulation of mRNA stability remains relatively uncharacterized. In this study, we identified the mRNA-binding protein THOC4 as a novel regulator of TFEB. In mammalian cells, siRNA-mediated knockdown of THOC4 decreased the level of TFEB protein to a greater extent than other bHLH transcription factors. THOC4 bound to mRNA and stabilized it after transcription by maintaining poly(A) tail length. We further found that this mode of regulation was conserved in and was essential for TFEB-mediated lipid breakdown, which becomes over-represented during prolonged starvation. Taken together, our findings reveal the presence of an additional layer of TFEB regulation by THOC4 and provide novel insights into the function of TFEB in mediating autophagy and lipid metabolism.
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http://dx.doi.org/10.1242/jcs.248203DOI Listing
March 2021

Amyloid Β-Peptide Increases Mitochondria-Endoplasmic Reticulum Contact Altering Mitochondrial Function and Autophagosome Formation in Alzheimer's Disease-Related Models.

Cells 2020 11 28;9(12). Epub 2020 Nov 28.

Division of Neurogeriatrics, Department of Neurobiology, Care Science and Society, Karolinska Institutet, BioClinicum J9:20, Visionsgatan 4, 171 64 Solna, Sweden.

Recent findings have shown that the connectivity and crosstalk between mitochondria and the endoplasmic reticulum (ER) at mitochondria-ER contact sites (MERCS) are altered in Alzheimer's disease (AD) and in AD-related models. MERCS have been related to the initial steps of autophagosome formation as well as regulation of mitochondrial function. Here, the interplay between MERCS, mitochondria ultrastructure and function and autophagy were evaluated in different AD animal models with increased levels of Aβ as well as in primary neurons derived from these animals. We start by showing that the levels of Mitofusin 1, Mitofusin 2 and mitochondrial import receptor subunit TOM70 are decreased in post-mortem brain tissue derived from familial AD. We also show that Aβ increases the juxtaposition between ER and mitochondria both in adult brain of different AD mouse models as well as in primary cultures derived from these animals. In addition, the connectivity between ER and mitochondria are also increased in wild-type neurons exposed to Aβ. This alteration in MERCS affects autophagosome formation, mitochondrial function and ATP formation during starvation. Interestingly, the increment in ER-mitochondria connectivity occurs simultaneously with an increase in mitochondrial activity and is followed by upregulation of autophagosome formation in a clear chronological sequence of events. In summary, we report that Aβ can affect cell homeostasis by modulating MERCS and, consequently, altering mitochondrial activity and autophagosome formation. Our data suggests that MERCS is a potential target for drug discovery in AD.
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http://dx.doi.org/10.3390/cells9122552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760163PMC
November 2020

LC3 lipidation is essential for TFEB activation during the lysosomal damage response to kidney injury.

Nat Cell Biol 2020 10 28;22(10):1252-1263. Epub 2020 Sep 28.

Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.

Sensing and clearance of dysfunctional lysosomes is critical for cellular homeostasis. Here we show that transcription factor EB (TFEB)-a master transcriptional regulator of lysosomal biogenesis and autophagy-is activated during the lysosomal damage response, and its activation is dependent on the function of the ATG conjugation system, which mediates LC3 lipidation. In addition, lysosomal damage triggers LC3 recruitment on lysosomes, where lipidated LC3 interacts with the lysosomal calcium channel TRPML1, facilitating calcium efflux essential for TFEB activation. Furthermore, we demonstrate the presence and importance of this TFEB activation mechanism in kidneys in a mouse model of oxalate nephropathy accompanying lysosomal damage. A proximal tubule-specific TFEB-knockout mouse exhibited progression of kidney injury induced by oxalate crystals. Together, our results reveal unexpected mechanisms of TFEB activation by LC3 lipidation and their physiological relevance during the lysosomal damage response.
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http://dx.doi.org/10.1038/s41556-020-00583-9DOI Listing
October 2020

Age-dependent loss of adipose Rubicon promotes metabolic disorders via excess autophagy.

Nat Commun 2020 08 18;11(1):4150. Epub 2020 Aug 18.

Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.

The systemic decline in autophagic activity with age impairs homeostasis in several tissues, leading to age-related diseases. A mechanistic understanding of adipocyte dysfunction with age could help to prevent age-related metabolic disorders, but the role of autophagy in aged adipocytes remains unclear. Here we show that, in contrast to other tissues, aged adipocytes upregulate autophagy due to a decline in the levels of Rubicon, a negative regulator of autophagy. Rubicon knockout in adipocytes causes fat atrophy and hepatic lipid accumulation due to reductions in the expression of adipogenic genes, which can be recovered by activation of PPARγ. SRC-1 and TIF2, coactivators of PPARγ, are degraded by autophagy in a manner that depends on their binding to GABARAP family proteins, and are significantly downregulated in Rubicon-ablated or aged adipocytes. Hence, we propose that age-dependent decline in adipose Rubicon exacerbates metabolic disorders by promoting excess autophagic degradation of SRC-1 and TIF2.
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http://dx.doi.org/10.1038/s41467-020-17985-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7434891PMC
August 2020

Structural basis for autophagy inhibition by the human Rubicon-Rab7 complex.

Proc Natl Acad Sci U S A 2020 07 6;117(29):17003-17010. Epub 2020 Jul 6.

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720;

Rubicon is a potent negative regulator of autophagy and a potential target for autophagy-inducing therapeutics. Rubicon-mediated inhibition of autophagy requires the interaction of the C-terminal Rubicon homology (RH) domain of Rubicon with Rab7-GTP. Here we report the 2.8-Å crystal structure of the Rubicon RH domain in complex with Rab7-GTP. Our structure reveals a fold for the RH domain built around four zinc clusters. The switch regions of Rab7 insert into pockets on the surface of the RH domain in a mode that is distinct from those of other Rab-effector complexes. Rubicon residues at the dimer interface are required for Rubicon and Rab7 to colocalize in living cells. Mutation of Rubicon RH residues in the Rab7-binding site restores efficient autophagic flux in the presence of overexpressed Rubicon, validating the Rubicon RH domain as a promising therapeutic target.
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http://dx.doi.org/10.1073/pnas.2008030117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382272PMC
July 2020

ERdj8 governs the size of autophagosomes during the formation process.

J Cell Biol 2020 08;219(8)

Institute for Protein Dynamics, Kyoto Sangyo University, Kyoto, Japan.

In macroautophagy, membrane structures called autophagosomes engulf substrates and deliver them for lysosomal degradation. Autophagosomes enwrap a variety of targets with diverse sizes, from portions of cytosol to larger organelles. However, the mechanism by which autophagosome size is controlled remains elusive. We characterized a novel ER membrane protein, ERdj8, in mammalian cells. ERdj8 localizes to a meshwork-like ER subdomain along with phosphatidylinositol synthase (PIS) and autophagy-related (Atg) proteins. ERdj8 overexpression extended the size of the autophagosome through its DnaJ and TRX domains. ERdj8 ablation resulted in a defect in engulfing larger targets. C. elegans, in which the ERdj8 orthologue dnj-8 was knocked down, could perform autophagy on smaller mitochondria derived from the paternal lineage but not the somatic mitochondria. Thus, ERdj8 may play a critical role in autophagosome formation by providing the capacity to target substrates of diverse sizes for degradation.
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http://dx.doi.org/10.1083/jcb.201903127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401821PMC
August 2020

Suppression of autophagic activity by Rubicon is a signature of aging.

Nat Commun 2019 02 19;10(1):847. Epub 2019 Feb 19.

Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.

Autophagy, an evolutionarily conserved cytoplasmic degradation system, has been implicated as a convergent mechanism in various longevity pathways. Autophagic activity decreases with age in several organisms, but the underlying mechanism is unclear. Here, we show that the expression of Rubicon, a negative regulator of autophagy, increases in aged worm, fly and mouse tissues at transcript and/or protein levels, suggesting that an age-dependent increase in Rubicon impairs autophagy over time, and thereby curtails animal healthspan. Consistent with this idea, knockdown of Rubicon extends worm and fly lifespan and ameliorates several age-associated phenotypes. Tissue-specific experiments reveal that Rubicon knockdown in neurons has the greatest effect on lifespan. Rubicon knockout mice exhibits reductions in interstitial fibrosis in kidney and reduced α-synuclein accumulation in the brain. Rubicon is suppressed in several long-lived worms and calorie restricted mice. Taken together, our results suggest that suppression of autophagic activity by Rubicon is one of signatures of aging.
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http://dx.doi.org/10.1038/s41467-019-08729-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6381146PMC
February 2019

Three-Color Simultaneous Live Imaging of Autophagy-Related Structures.

Methods Mol Biol 2019 ;1880:223-230

Andor Technology Ltd., Newark, DE, USA.

Simultaneous live cell imaging of multiple proteins helps to analyze mobility and interactions among proteins over time. Since autophagosomes depend on other organelles for their formation, it is necessary to observe this process with multiple fluorsphores to mark multiple organelles and the autophagosomes. To do so, we set up three cameras on one microscope to be able to acquire three colors at the same time. Here we describe the setup using a Yokogawa spinning disk confocal microscope (CSU-W1) with Andor TuCam system attaching 3 × Zyla 4.2 CMOS cameras (Andor) and detail the method for acquiring live images.
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http://dx.doi.org/10.1007/978-1-4939-8873-0_14DOI Listing
June 2019

Seeking and embracing change.

Authors:
Maho Hamasaki

Nat Cell Biol 2018 Sep;20(9):1002

Osaka University, Osaka, Japan.

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http://dx.doi.org/10.1038/s41556-018-0180-6DOI Listing
September 2018

Ubiquitination of exposed glycoproteins by SCF directs damaged lysosomes for autophagy.

Proc Natl Acad Sci U S A 2017 08 25;114(32):8574-8579. Epub 2017 Jul 25.

Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan;

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCF ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCF ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy.
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http://dx.doi.org/10.1073/pnas.1702615114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5559013PMC
August 2017

Endothelial cells are intrinsically defective in xenophagy of Streptococcus pyogenes.

PLoS Pathog 2017 Jul 6;13(7):e1006444. Epub 2017 Jul 6.

Department of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

Group A Streptococcus (GAS) is deleterious pathogenic bacteria whose interaction with blood vessels leads to life-threatening bacteremia. Although xenophagy, a special form of autophagy, eliminates invading GAS in epithelial cells, we found that GAS could survive and multiply in endothelial cells. Endothelial cells were competent in starvation-induced autophagy, but failed to form double-membrane structures surrounding GAS, an essential step in xenophagy. This deficiency stemmed from reduced recruitment of ubiquitin and several core autophagy proteins in endothelial cells, as demonstrated by the fact that it could be rescued by exogenous coating of GAS with ubiquitin. The defect was associated with reduced NO-mediated ubiquitin signaling. Therefore, we propose that the lack of efficient clearance of GAS in endothelial cells is caused by their intrinsic inability to target GAS with ubiquitin to promote autophagosome biogenesis for xenophagy.
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http://dx.doi.org/10.1371/journal.ppat.1006444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500369PMC
July 2017

Mitochondrial division occurs concurrently with autophagosome formation but independently of Drp1 during mitophagy.

J Cell Biol 2016 Dec 30;215(5):649-665. Epub 2016 Nov 30.

Department of Cellular Physiology, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8510, Japan

Mitophagy is thought to play an important role in mitochondrial quality control. Mitochondrial division is believed to occur first, and autophagosome formation subsequently occurs to enwrap mitochondria as a process of mitophagy. However, there has not been any temporal analysis of mitochondrial division and autophagosome formation in mitophagy. Therefore, the relationships among these processes remain unclear. We show that the mitochondrial division factor Dnm1 in yeast or Drp1 in mammalian cells is dispensable for mitophagy. Autophagosome formation factors, such as FIP200, ATG14, and WIPIs, were essential for the mitochondrial division for mitophagy. Live-cell imaging showed that isolation membranes formed on the mitochondria. A small portion of the mitochondria then divided from parental mitochondria simultaneously with the extension of isolation membranes and autophagosome formation. These findings suggest the presence of a mitophagy process in which mitochondrial division for mitophagy is accomplished together with autophagosome formation.
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http://dx.doi.org/10.1083/jcb.201605093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5147001PMC
December 2016

Rubicon inhibits autophagy and accelerates hepatocyte apoptosis and lipid accumulation in nonalcoholic fatty liver disease in mice.

Hepatology 2016 12 21;64(6):1994-2014. Epub 2016 Oct 21.

Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Osaka, Japan.

Nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease worldwide. It encompasses a spectrum ranging from simple steatosis to fatty liver with hepatocellular injury, termed nonalcoholic steatohepatitis. Recent studies have demonstrated hepatic autophagy being impaired in NAFLD. In the present study, we investigated the impact of Rubicon, a Beclin1-interacting negative regulator for autophagosome-lysosome fusion, in the pathogenesis of NAFLD. In HepG2 cells, BNL-CL2 cells, and murine primary hepatocytes, Rubicon was posttranscriptionally up-regulated by supplementation with saturated fatty acid palmitate. Up-regulation of Rubicon was associated with suppression of the late stage of autophagy, as evidenced by accumulation of both LC3-II and p62 expression levels as well as decreased autophagy flux. Its blockade by small interfering RNA attenuated autophagy impairment and reduced palmitate-induced endoplasmic reticulum stress, apoptosis, and lipid accumulation. Rubicon was also up-regulated in association with autophagy impairment in livers of mice fed a high-fat diet (HFD). Hepatocyte-specific Rubicon knockout mice generated by crossing Rubicon floxed mice with albumin-Cre transgenic mice did not produce any phenotypes on a normal diet. In contrast, on an HFD, they displayed significant improvement of both liver steatosis and injury as well as attenuation of both endoplasmic reticulum stress and autophagy impairment in the liver. In humans, liver tissues obtained from patients with NAFLD expressed significantly higher levels of Rubicon than those without steatosis.

Conclusion: Rubicon is overexpressed and plays a pathogenic role in NAFLD by accelerating hepatocellular lipoapoptosis and lipid accumulation, as well as inhibiting autophagy. Rubicon may be a novel therapeutic target for regulating NAFLD development and progression. (Hepatology 2016;64:1994-2014).
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http://dx.doi.org/10.1002/hep.28820DOI Listing
December 2016

Atg9A trafficking through the recycling endosomes is required for autophagosome formation.

J Cell Sci 2016 10 1;129(20):3781-3791. Epub 2016 Sep 1.

Graduate School of Frontier Bioscience, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Department of Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Autophagy is an intracellular degradation pathway conserved in eukaryotes. Among core autophagy-related (Atg) proteins, mammalian Atg9A is the sole multi-spanning transmembrane protein, and both of its N- and C-terminal domains are exposed to the cytoplasm. It is known that Atg9A travels through the trans-Golgi network (TGN) and the endosomal system under nutrient-rich conditions, and transiently localizes to the autophagosome upon autophagy induction. However, the significance of Atg9A trafficking for autophagosome formation remains elusive. Here, we identified sorting motifs in the N-terminal cytosolic stretch of Atg9A that interact with the adaptor protein AP-2. Atg9A with mutations in the sorting motifs could not execute autophagy and was abnormally accumulated at the recycling endosomes. The combination of defects in autophagy and Atg9A accumulation in the recycling endosomes was also found upon the knockdown of TRAPPC8, a specific subunit of the TRAPPIII complex. These results show directly that the trafficking of Atg9A through the recycling endosomes is an essential step for autophagosome formation.
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http://dx.doi.org/10.1242/jcs.196196DOI Listing
October 2016

Autophagosome-lysosome fusion in neurons requires INPP5E, a protein associated with Joubert syndrome.

EMBO J 2016 09 23;35(17):1853-67. Epub 2016 Jun 23.

Laboratory of Intracellular Membrane Dynamics, Graduate School of Frontier Biosciences Osaka University, Osaka, Japan Department of Genetics, Graduate School of Medicine Osaka University, Osaka, Japan

Autophagy is a multistep membrane traffic pathway. In contrast to autophagosome formation, the mechanisms underlying autophagosome-lysosome fusion remain largely unknown. Here, we describe a novel autophagy regulator, inositol polyphosphate-5-phosphatase E (INPP5E), involved in autophagosome-lysosome fusion process. In neuronal cells, INPP5E knockdown strongly inhibited autophagy by impairing the fusion step. A fraction of INPP5E is localized to lysosomes, and its membrane anchoring and enzymatic activity are necessary for autophagy. INPP5E decreases lysosomal phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), one of the substrates of the phosphatase, that counteracts cortactin-mediated actin filament stabilization on lysosomes. Lysosomes require actin filaments on their surface for fusing with autophagosomes. INPP5E is one of the genes responsible for Joubert syndrome, a rare brain abnormality, and mutations found in patients with this disease caused defects in autophagy. Taken together, our data reveal a novel role of phosphoinositide on lysosomes and an association between autophagy and neuronal disease.
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http://dx.doi.org/10.15252/embj.201593148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007553PMC
September 2016

Autophagy sequesters damaged lysosomes to control lysosomal biogenesis and kidney injury.

EMBO J 2013 Aug 6;32(17):2336-47. Epub 2013 Aug 6.

Department of Genetics, Graduate School of Medicine, Osaka University, Osaka, Japan.

Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.
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http://dx.doi.org/10.1038/emboj.2013.171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3770333PMC
August 2013

Up-to-date membrane biogenesis in the autophagosome formation.

Curr Opin Cell Biol 2013 Aug 8;25(4):455-60. Epub 2013 Apr 8.

Department of Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

When cells are starved, are invaded by foreign bodies such as bacteria, and contain damaged organelles or aggregated proteins, double-membrane organelles called autophagosomes are formed within the cytoplasm to surround, isolate and deliver these materials to lysosomes for degradation. This pathway, called 'autophagy', is conserved from yeast to mammalian cells. Unlike other organelles, the autophagosome forms de novo, thus raising unique questions regarding its membrane biogenesis. Here we highlight a number of recent findings related to autophagosome formation and possible involvement of autophagy-specific vesicles originating from other organelles, but with particular attention on the formation sites and the relationship of the autophagosome to other organelles.
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http://dx.doi.org/10.1016/j.ceb.2013.03.004DOI Listing
August 2013

Autophagosomes form at ER-mitochondria contact sites.

Nature 2013 Mar 3;495(7441):389-93. Epub 2013 Mar 3.

Department of Genetics, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.

Autophagy is a tightly regulated intracellular bulk degradation/recycling system that has fundamental roles in cellular homeostasis. Autophagy is initiated by isolation membranes, which form and elongate as they engulf portions of the cytoplasm and organelles. Eventually isolation membranes close to form double membrane-bound autophagosomes and fuse with lysosomes to degrade their contents. The physiological role of autophagy has been determined since its discovery, but the origin of autophagosomal membranes has remained unclear. At present, there is much controversy about the organelle from which the membranes originate--the endoplasmic reticulum (ER), mitochondria and plasma membrane. Here we show that autophagosomes form at the ER-mitochondria contact site in mammalian cells. Imaging data reveal that the pre-autophagosome/autophagosome marker ATG14 (also known as ATG14L) relocalizes to the ER-mitochondria contact site after starvation, and the autophagosome-formation marker ATG5 also localizes at the site until formation is complete. Subcellular fractionation showed that ATG14 co-fractionates in the mitochondria-associated ER membrane fraction under starvation conditions. Disruption of the ER-mitochondria contact site prevents the formation of ATG14 puncta. The ER-resident SNARE protein syntaxin 17 (STX17) binds ATG14 and recruits it to the ER-mitochondria contact site. These results provide new insight into organelle biogenesis by demonstrating that the ER-mitochondria contact site is important in autophagosome formation.
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http://dx.doi.org/10.1038/nature11910DOI Listing
March 2013

Critical roles for lipomannan and lipoarabinomannan in cell wall integrity of mycobacteria and pathogenesis of tuberculosis.

mBio 2013 Feb 19;4(1):e00472-12. Epub 2013 Feb 19.

Research Institute for Microbial Diseases, WPI-Immunology Frontier Research Center, Osaka University, Osaka, Japan.

Lipomannan (LM) and lipoarabinomannan (LAM) are mycobacterial glycolipids containing a long mannose polymer. While they are implicated in immune modulations, the significance of LM and LAM as structural components of the mycobacterial cell wall remains unknown. We have previously reported that a branch-forming mannosyltransferase plays a critical role in controlling the sizes of LM and LAM and that deletion or overexpression of this enzyme results in gross changes in LM/LAM structures. Here, we show that such changes in LM/LAM structures have a significant impact on the cell wall integrity of mycobacteria. In Mycobacterium smegmatis, structural defects in LM and LAM resulted in loss of acid-fast staining, increased sensitivity to β-lactam antibiotics, and faster killing by THP-1 macrophages. Furthermore, equivalent Mycobacterium tuberculosis mutants became more sensitive to β-lactams, and one mutant showed attenuated virulence in mice. Our results revealed previously unknown structural roles for LM and LAM and further demonstrated that they are important for the pathogenesis of tuberculosis. IMPORTANCE Tuberculosis (TB) is a global burden, affecting millions of people worldwide. Mycobacterium tuberculosis is a causative agent of TB, and understanding the biology of M. tuberculosis is essential for tackling this devastating disease. The cell wall of M. tuberculosis is highly impermeable and plays a protective role in establishing infection. Among the cell wall components, LM and LAM are major glycolipids found in all Mycobacterium species, show various immunomodulatory activities, and have been thought to play roles in TB pathogenesis. However, the roles of LM and LAM as integral parts of the cell wall structure have not been elucidated. Here we show that LM and LAM play critical roles in the integrity of mycobacterial cell wall and the pathogenesis of TB. These findings will now allow us to seek the possibility that the LM/LAM biosynthetic pathway is a chemotherapeutic target.
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http://dx.doi.org/10.1128/mBio.00472-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573661PMC
February 2013

Guidelines for the use and interpretation of assays for monitoring autophagy.

Autophagy 2012 Apr;8(4):445-544

Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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http://dx.doi.org/10.4161/auto.19496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404883PMC
April 2012

Vaccinia virus lacking A17 induces complex membrane structures composed of open membrane sheets.

Arch Virol 2011 Sep 18;156(9):1647-53. Epub 2011 May 18.

European Molecular Biology Laboratory, Heidelberg, Meyerhofstraße 1, 69117 Heidelberg, Germany.

The vaccinia virus (VACV) precursor membrane, the crescent, consists of an open membrane sheet and is formed by rupture of a cellular compartment. Here, we asked whether A17, a viral membrane protein, plays a role in membrane rupture. Without A17 synthesis, crescents are not formed, and instead, tubular and vesicular membranes accumulate (Rodriguez et al. in J Virol 69:4640-4648, 1). We used electron tomography (ET) to analyze whether the viral membranes lacking A17 consist of open membrane sheets. Tubular, vesicular and so far not described onion-shaped membranes, which consisted of open membrane sheets, were seen. Thus, the data show that membrane rupture occurs independently of the A17 protein.
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http://dx.doi.org/10.1007/s00705-011-1012-1DOI Listing
September 2011

Where do they come from? Insights into autophagosome formation.

FEBS Lett 2010 Apr 25;584(7):1296-301. Epub 2010 Feb 25.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

Autophagosomes (APs) are unique organelles that enwrap cytoplasmic components when necessary. APs then fuse with lysosomes and enclosed materials are degraded. Although approximately 30 autophagy-related genes (ATG) required for AP formation have been identified, fundamental questions on the membrane source or dynamics during the formation remain unresolved. Here, we present a comprehensive overview of the putative membrane sources identified to date.
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http://dx.doi.org/10.1016/j.febslet.2010.02.061DOI Listing
April 2010

Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy.

Traffic 2010 Apr 6;11(4):468-78. Epub 2010 Jan 6.

Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-Oka, Suita, Osaka 565-0871, Japan.

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant-negative inactive mutant of Myotubularin-related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy-related PtdIns3P-binding proteins, GFP-DFCP1 and GFP-WIPI-1alpha (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock-down of MTMR3 increased autophagosome formation, and overexpression of wild-type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3-kinase and PI 3-phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.
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http://dx.doi.org/10.1111/j.1600-0854.2010.01034.xDOI Listing
April 2010

[Involvement of the secretory pathway in the autophagosome formation].

Tanpakushitsu Kakusan Koso 2006 Aug;51(10 Suppl):1469-73

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August 2006
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