| 1. |
Reo Kurusu, Hideaki Morishita, Masaaki Komatsu, p62 bodies: cytosolic zoning by phase separation, J Biochem, 10.1093/jb/mvad089, 2023.11, Summary
Cellular zoning or partitioning is critical in preventing macromolecules from random diffusion and in orchestrating the spatiotemporal dynamics of biochemical reactions. Along with membranous organelles, membrane-less organelles contribute to the precise regulation of biochemical reactions inside cells. In response to environmental cues, membrane-less organelles rapidly form through liquid-liquid phase separation, sequester certain proteins and RNAs, mediate specific reactions, and dissociate. Among membrane-less organelles, ubiquitin-positive condensates, namely, p62 bodies, maintain cellular homeostasis through selective autophagy of themselves to contribute to intracellular quality control. p62 bodies also activate the anti-oxidative stress response regulated by the KEAP1-NRF2 system. In this review, we present an overview of recent advancements in cellular and molecular biology related to p62 bodies, highlighting their dynamic nature and functions.. |
| 2. |
Vault-phagy: a phase-separation-mediated selective autophagy of vault, a non-membranous organelle. |
| 3. |
H Kuwata, M Matsumoto, K Atarashi, H Morishita, T Hirotani, R Koga, K Takeda, I kappa BNS inhibits induction of a subset of toll-like receptor-dependent genes and limits inflammation, Immunity, 10.1016/j.immuni.2005.11.004, 24, 1, 41-51, 2006.01, Toll-like receptor (TLR)-mediated immune responses are downregulated by several mechanisms that affect signaling pathways. However, it remains elusive how TLR-mediated gene expression is differentially modulated. Here, we show that I kappa BNS, a TLR-inducible nuclear I kappa B protein, negatively regulates induction of a subset of TLR-dependent genes through inhibition of NF-kappa B activity. I kappa BNS-deficient macrophages and dendritic cells show increased TLR-mediated expression of genes such as IL-6 and IL-12p40, which are induced late after TLR stimulation. In contrast, I kappa BNS-deficient cells showed normal induction of genes that are induced early or induced via IRF-3 activation. LPS stimulation of I kappa BNS-deficient macrophages prolonged NF-kappa B activity at the specific promoters, indicating that I kappa BNS mediates termination of NF-kappa B activity at selective gene promoters. Moreover, I kappa BNS-deficient mice are highly susceptible to LPS-induced endotoxin shock and intestinal inflammation. Thus, I kappa BNS regulates inflammatory responses by inhibiting the induction of a subset of TLR-dependent genes through modulation of NF-kappa B activity.. |
| 4. |
Hideaki Morishita, Fumiji Saito, Hisako Kayama, Koji Atarashi, Hirotaka Kuwata, Masahiro Yamamoto, Kiyoshi Takeda, Fra-1 negatively regulates lipopolysaccharide-mediated inflammatory responses., Int Immunol., 10.1093/intimm/dxp015, 21, 4, 457-65, 2009.04, Stimulation of macrophages with a Toll-like receptor ligand, LPS, facilitates gene expression. The activator protein-1 (AP-1) family of transcription factors mediates these responses. However, c-Fos, a member of the AP-1 family, has been shown to inhibit LPS-induced gene expression in macrophages. In this study, we analyzed the role of Fos-related antigen-1 (Fra-1), another member of the AP-1 family of transcription factors, in LPS-induced responses in RAW264.7 macrophages. Fra-1 was induced in LPS-stimulated macrophages with delayed time kinetics compared with c-Fos. Lentiviral introduction of Fra-1 blocked LPS-induced expression of pro-inflammatory mediators at the protein and mRNA levels. A Fra-1 mutant, which lacks the basic leucine zipper domain required for heterodimer formation and DNA binding, did not inhibit LPS-induced responses. c-Fos bound to the AP-1-binding site early, but afterward it was replaced by Fra-1 in LPS-stimulated macrophages. Over-expression of Fra-1 induced its association with Jun proteins and stable DNA binding from an early time point following LPS stimulation. These findings indicate that Fra-1 suppresses LPS-induced mRNA expression by binding to the AP-1-binding site. RNAi-mediated knockdown of Fra-1 in RAW264.7 macrophages resulted in enhanced LPS-induced expression of a subset of genes. Thus, like c-Fos, Fra-1 negatively regulates LPS-induced responses in RAW264.7 macrophages.. |
| 5. |
Hideaki Morishita, Satoshi Eguchi, Hirotaka Kimura, Junko Sasaki, Yuriko Sakamaki, Michael L. Robinson, Takehiko Sasaki, Noboru Mizushima, Deletion of Autophagy-related 5 (Atg5) and Pik3c3 Genes in the Lens Causes Cataract Independent of Programmed Organelle Degradation, J Biol Chem, 10.1074/jbc.M112.437103, 288, 16, 11436-11447, 2013.04, The lens of the eye is composed of fiber cells, which differentiate from epithelial cells and undergo programmed organelle degradation during terminal differentiation. Although autophagy, a major intracellular degradation system, is constitutively active in these cells, its physiological role has remained unclear. We have previously shown that Atg5-dependent macroautophagy is not necessary for lens organelle degradation, at least during the embryonic period. Here, we generated lens-specific Atg5 knock-out mice and showed that Atg5 is not required for lens organelle degradation at any period of life. However, deletion of Atg5 in the lens results in age-related cataract, which is accompanied by accumulation of polyubiquitinated and oxidized proteins, p62, and insoluble crystallins, suggesting a defect in intracellular quality control. We also produced lens-specific Pik3c3 knock-out mice to elucidate the possible involvement of Atg5-independent alternative autophagy, which is proposed to be dependent on Pik3c3 (also known as Vps34), in lens organelle degradation. Deletion of Pik3c3 in the lens does not affect lens organelle degradation, but it leads to congenital cataract and a defect in lens development after birth likely due to an impairment of the endocytic pathway. Taken together, these results suggest that clearance of lens organelles is independent of macroautophagy. These findings also clarify the physiological role of Atg5 and Pik3c3 in quality control and development of the lens, respectively.. |
| 6. |
Kotaro Tsuboyama, Ikuko Koyama-Honda, Yuriko Sakamaki, Masato Koike, Hideaki Morishita, Noboru Mizushima, The ATG conjugation systems are important for degradation of the inner autophagosomal membrane, Science, 10.1126/science.aaf6136, 354, 6315, 1036-1041, 2016.11, In macroautophagy, cytoplasmic contents are sequestered into the double-membrane autophagosome, which fuses with the lysosome to become the autolysosome. It has been thought that the autophagy-related (ATG) conjugation systems are required for autophagosome formation. Here, we found that autophagosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) syntaxin 17-positive autophagosome-like structures could be generated even in the absence of the ATG conjugation systems, although at a reduced rate. These syntaxin 17-positive structures could further fuse with lysosomes, but degradation of the inner autophagosomal membrane was significantly delayed. Accordingly, autophagic activity in ATG conjugation-deficient cells was strongly suppressed. We suggest that the ATG conjugation systems, which are likely required for the closure (i.e., fission) of the autophagosomal edge, are not absolutely essential for autolysosome formation but are important for efficient degradation of the inner autophagosomal membrane.. |
| 7. |
Hideaki Morishita, Noboru Mizushima, Autophagy in the lens, Exp Eye Res, 10.1016/j.exer.2015.08.019, 144, 22-28, 2016.03, The lens of the eye is a transparent tissue composed of lens fiber cells that differentiate from lens epithelial cells and degrade all cytoplasmic organelles during terminal differentiation. Autophagy is a major intracellular degradation system in which cytoplasmic proteins and organelles are degraded in the lysosome. Although autophagy is constitutively activated in the lens and has been proposed to be involved in lens organelle degradation, its precise role is not well understood. Recent genetic studies in mice have demonstrated that autophagy is critically important for intracellular quality control in the lens but can be dispensable for lens organelle degradation. Here, we review recent findings on the roles of autophagy and lysosomes in organelle degradation and intracellular quality control in the lens, and discuss their possible involvement in the development of human cataract. (C) 2015 Elsevier Ltd. All rights reserved.. |
| 8. |
Kaizuka T*, Morishita H*(*co-first authors), Hama Y, Tsukamoto S, Matsui T, Toyota Y, Kodama A, Ishihara T, Mizushima T, Mizushima N, An Autophagic Flux Probe that Releases an Internal Control, Mol Cell., 10.1016/j.molcel.2016.09.037, 64, 4, 835-849, 2016.11, Macroautophagy is an intracellular degradation system that utilizes the autophagosome to deliver cytoplasmic components to the lysosome. Measuring autophagic activity is critically important but remains complicated and challenging. Here, we have developed GFP-LC3-RFP-LC3 Delta G, a fluorescent probe to evaluate autophagic flux. This probe is cleaved by endogenous ATG4 proteases into equimolar amounts of GFP-LC3 and RFP-LC3 Delta G. GFP-LC3 is degraded by autophagy, while RFP-LC3 Delta G remains in the cytosol, serving as an internal control. Thus, autophagic flux can be estimated by calculating the GFP/RFP signal ratio. Using this probe, we re-evaluated previously reported autophagy-modulating compounds, performed a high-throughput screen of an approved drug library, and identified autophagy modulators. Furthermore, we succeeded in measuring both induced and basal autophagic flux in embryos and tissues of zebrafish and mice. The GFP-LC3-RFP-LC3 Delta G probe is a simple and quantitative method to evaluate autophagic flux in cultured cells and whole organisms.. |
| 9. |
Chisato Fujimoto, Shinichi Iwasaki, Shinji Urata, Hideaki Morishita, Yuriko Sakamaki, Masato Fujioka, Kenji Kondo, Noboru Mizushima, Tatsuya Yamasoba, Autophagy is essential for hearing in mice, Cell Death Dis., 10.1038/cddis.2017.194, 8, 5, e2780, 2017.05, Hearing loss is the most frequent sensory disorder in humans. Auditory hair cells (HCs) are postmitotic at late-embryonic differentiation and postnatal stages, and their damage is the major cause of hearing loss. There is no measurable HC regeneration in the mammalian cochlea, and the maintenance of cell function is crucial for preservation of hearing. Here we generated mice deficient in autophagy-related 5 (Atg5), a gene essential for autophagy, in the HCs to investigate the effect of basal autophagy on hearing acuity. Deletion of Atg5 resulted in HC degeneration and profound congenital hearing loss. In autophagy-deficient HCs, polyubiquitinated proteins and p62/SQSTM1, an autophagy substrate, accumulated as inclusion bodies during the first postnatal week, and these aggregates increased in number. These findings revealed that basal autophagy has an important role in maintenance of HC morphology and hearing acuity.. |
| 10. |
Keigo Morita, Yutaro Hama, Tamaki Izume, Norito Tamura, Toshihide Ueno, Yoshihiro Yamashita, Yuriko Sakamaki, Kaito Mimura, Hideaki Morishita, Wataru Shihoya, Osamu Nureki, Hiroyuki Mano, Noboru Mizushima, Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation., J Cell Biol., 10.1083/jcb.201804132, 217, 11, 3817-3828, 2018.11, Macroautophagy is an intracellular degradation process that requires multiple autophagy-related (ATG) genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified TMEM41B as a novel ATG gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER). It has a conserved domain also found in vacuole membrane protein 1 (VMP1), another ER multispanning membrane protein essential for autophagy, yeast Tvp38, and the bacterial DedA family of putative half-transporters. Deletion of TMEM41B blocked the formation of autophagosomes at an early step, causing accumulation of ATG proteins and small vesicles but not elongating autophagosome-like structures. Furthermore, lipid droplets accumulated in TMEM41B-knockout (KO) cells. The phenotype of TMEM41B-KO cells resembled those of VMP1-KO cells. Indeed, TMEM41B and VMP1 formed a complex in vivo and in vitro, and overexpression of VMP1 restored autophagic flux in TMEM41B-KO cells. These results suggest that TMEM41B and VMP1 function together at an early step of autophagosome formation.. |
| 11. |
Morishita H*(* co-corresponding authors), Mizushima N*, Diverse Cellular Roles of Autophagy., Annu Rev Cell Dev Biol., 10.1146/annurev-cellbio-100818-125300, 35, 453-475, 2019.10. |
| 12. |
Morishita H, Zhao YG*, Tamura N*(*co-second authors), Nishimura T, Kanda Y, Sakamaki Y, Okazaki M, Li D, Mizushima N, A critical role of VMP1 in lipoprotein secretion, eLife, 10.7554/eLife.48834, 8:e48834, 2019.09. |
| 13. |
Morishita H, Kanda Y, Kaizuka T, Chino H, Nakao K, Miki Y, Taketomi Y, Guan J-L, Murakami M, Aiba A, Mizushima N, Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder, Cell Rep., 10.1016/j.celrep.2020.108477, 33, 10, 108477-108477, 2020.12, Autophagy is an intracellular degradation system, but its physiological functions in vertebrates are not yet fully understood. Here, we show that autophagy is required for inflation of air-filled organs: zebrafish swim bladder and mouse lung. In wild-type zebrafish swim bladder and mouse lung type II pulmonary epithelial cells, autophagosomes are formed and frequently fuse with lamellar bodies. The lamellar body is a lysosome-related organelle that stores a phospholipid-containing surfactant complex that lines the air-liquid interface and reduces surface tension. We find that autophagy is critical for maturation of the lamellar body. Accordingly, atg-deficient zebrafish fail to maintain their position in the water, and type-II-pneumocyte-specific Fip200-deficient mice show neonatal lethality with respiratory failure. Autophagy suppression does not affect synthesis of the surfactant phospholipid, suggesting that autophagy supplies lipids and membranes to lamellar bodies. These results demonstrate an evolutionarily conserved role of autophagy in lamellar body maturation.. |
| 14. |
Hideaki Morishita* (*co-corresponding authors), Tomoya Eguchi, Satoshi Tsukamoto, Yuriko Sakamaki, Satoru Takahashi, Chieko Saito, Ikuko Koyama-Honda, Noboru Mizushima*, Organelle degradation in the lens by PLAAT phospholipases., Nature, 10.1038/s41586-021-03439-w, 592, 7855, 634-638, 2021.04, The eye lens of vertebrates is composed of fibre cells in which all membrane-bound organelles undergo degradation during terminal differentiation to form an organelle-free zone1. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy2,3. Here we report that phospholipases in the PLAAT (phospholipase A/acyltransferase, also known as HRASLS) family-Plaat1 (also known as Hrasls) in zebrafish and PLAAT3 (also known as HRASLS3, PLA2G16, H-rev107 or AdPLA) in mice4-6-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes. Plaat1 and PLAAT3 translocate from the cytosol to various organelles immediately before organelle degradation, in a process that requires their C-terminal transmembrane domain. The translocation of Plaat1 to organelles depends on the differentiation of fibre cells and damage to organelle membranes, both of which are mediated by Hsf4. After the translocation of Plaat1 or PLAAT3 to membranes, the phospholipase induces extensive organelle rupture that is followed by complete degradation. Organelle degradation by PLAAT-family phospholipases is essential for achieving an optimal transparency and refractive function of the lens. These findings expand our understanding of intracellular organelle degradation and provide insights into the mechanism by which vertebrates acquired transparent lenses.. |
| 15. |
Daniel J Klionsky, Amal Kamal Abdel-Aziz, Sara Abdelfatah, Mahmoud Abdellatif, Asghar Abdoli, Steffen Abel, Hagai Abeliovich, Marie H Abildgaard, ---, Hideaki Morishita, ---, Shigeru Oshima, Yueguang Rong, Judith C Sluimer, Christina L Stallings, Chun-Kit Tong, Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)., Autophagy, 10.1080/15548627.2020.1797280, 17, 1, 1-382, 2021.02, 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 bona fide 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.. |
| 16. |
Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy. |
| 17. |
Regulation of ER-derived membrane dynamics by the DedA domain-containing proteins VMP1 and TMEM41B. |
| 18. |
Kurusu R, Fujimoto Y, Morishita H,*(co-first authors,*co-corresponding authors), Noshiro D, Takada S, Yamano K, Tanaka H, Arai R, Kageyama S, Funakoshi T, Komatsu-Hirota S, Taka H, Kazuno S, Miura Y, Koike M, Wakai T, Waguri S, Noda NN, Komatsu M*, Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex, Dev Cell, 10.1016/j.devcel.2023.04.015, 2023.05. |
| 19. |
Autophagy in the eye: from physiology to pathophysology. |
| 20. |
Ikeda R, Noshiro D, Morishita H, Takada S, Kageyama S, Fujioka Y, Funakoshi T, Komatsu-Hirota S, Arai R, Ryzhii E, Abe M, Koga T, Motohashi H, Nakao M, Sakimura K, Horii A, Waguri S, Ichimura Y, Noda NN, Komatsu M, Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response, EMBO J, 2023.06. |
| 21. |
Kurusu R, Fujimoto Y, Morishita H,*(co-first authors,*co-corresponding authors), Noshiro D, Takada S, Yamano K, Tanaka H, Arai R, Kageyama S, Funakoshi T, Komatsu-Hirota S, Taka H, Kazuno S, Miura Y, Koike M, Wakai T, Waguri S, Noda NN, Komatsu M*, Integrated proteomics identifies p62-dependent selective autophagy of the supramolecular vault complex, Dev Cell, 2023.05. |
| 22. |
Hideaki Morishita* (*co-corresponding authors), Tomoya Eguchi, Satoshi Tsukamoto, Yuriko Sakamaki, Satoru Takahashi, Chieko Saito, Ikuko Koyama-Honda, Noboru Mizushima*, Organelle degradation in the lens by PLAAT phospholipases, Nature, 2021.04. |
| 23. |
Morishita H, Kanda Y, Kaizuka T, Chino H, Nakao K, Miki Y, Taketomi Y, Guan J-L, Murakami M, Aiba A, Mizushima N, Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder, Cell Rep, 2020.12. |
| 24. |
Morishita H, Zhao YG*, Tamura N*(*co-second authors), Nishimura T, Kanda Y, Sakamaki Y, Okazaki M, Li D, Mizushima N, A critical role of VMP1 in lipoprotein secretion, eLife, 2019.09. |
| 25. |
Kaizuka T*, Morishita H*(*co-first authors), Hama Y, Tsukamoto S, Matsui T, Toyota Y, Kodama A, Ishihara T, Mizushima T, Mizushima N, An Autophagic Flux Probe that Releases an Internal Control, Mol Cell, 2016.11. |
| 26. |
Tsuboyama K, Koyama-Honda I, Sakamaki Y, Koike M, Morishita H, Mizushima N, The ATG conjugation systems are important for degradation of the inner autophagosomal membrane, Science, 2016.11. |
| 27. |
Morishita H, Eguchi S, Kimura H, Sasaki J, Sakamaki Y, Robinson ML, Sasaki T, Mizushima N, Deletion of Autophagy-related 5 (Atg5) and Pik3c3 Genes in the Lens Causes Cataract Independent of Programmed Organelle Degradation, J Biol Chem, 2013.04. |
| 28. |
Kuwata H, Matsumoto M, Atarashi K, Morishita H, Hirotani T, Koga R, Takeda K, I kappa BNS inhibits induction of a subset of toll-like receptor-dependent genes and limits inflammation, Immunity, 10.1016/j.immuni.2005.11.004, 2016.01. |
