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Background: γ1-Adaptin is a subunit of adaptor protein complex-1 (AP-1), which regulates intracellular transport between the trans-Golgi network (TGN) and endosomes. Since expression levels of AP-1 subunits have been reported to be associated with cell proliferation and cancer malignancy, we investigated the relationships between the immunohistochemical expression of γ1-adaptin and both clinicopathological factors and relapse-free survival (RFS) in breast cancer tissue., Materials and Methods: SK-BR-3 cell line depleted of γ1-adaptin was used for cell proliferation, migration, and invasion assay. Intracellular localization of γ1-adaptin was examined with immunohistochemistry (IHC) using an antibody against γ1-adaptin, and with double immunohistofluorescence (IHF) microscopy using markers for the TGN and endosome. γ1-Adaptin intensities in IHC samples from 199 primary breast cancer patients were quantified and assessed in relation to clinicopathological factors and RFS., Results: Cell growth, migration, and invasion of SK-BR-3 cells were significantly suppressed by the depletion of γ1-adaptin. Although the staining patterns in the cancer tissues varied among cases by IHC, double IHF demonstrated that γ1-adaptin was mainly localized in EEA1-positive endosomes, but not in the TGN. γ1-Adaptin intensity was significantly higher in the tumor regions than in non-tumor regions. It was also higher in patients with Ki-67 (high), ER (-), PgR (-), and HER2 (+). Among subtypes of breast cancer, γ1-adaptin intensity was higher in HER2 than in luminal A or luminal B. The results of the survival analysis indicated that high γ1-adaptin intensity was significantly associated with worse RFS, and this association was also observed in group with ER (+), PgR (+), HER2 (-), Ki-67 (high), or luminal B. In addition, the Cox proportional hazards model showed that high γ1-adaptin intensity was an independent prognostic factor., Conclusion: These results suggest that the endosomal expression of γ1-adaptin is positively correlated with breast cancer malignancy and could be a novel prognostic marker., (© 2024. The Author(s).)

Chlamydia trachomatis infection can be regulated by autophagy-related ( ATG ) genes. Here, we found that the depletion of ATG9A , one of the core ATG genes, in HeLa cells suppressed C. trachomatis growth in the inclusion. The growth was restored by re-expressing ATG9A or an ATG9A mutant impairing lipid scramblase activity in ATG9A -knockout (KO) cells. Moreover, the depletion of lipid transfer proteins ATG2A/B, responsible for isolation membrane expansion together with ATG9A, did not significantly alter the growth, suggesting that the non-autophagic function of ATG9A supports C. trachomatis infection. ATG9A -KO cells showed no infection-induced redistribution of the Golgi from the perinuclear region to inclusion, which was restored by re-expressing the mutant but not the ATG9A mutant lacking an N-terminal adapter protein-binding domain. Re-expression of the N-terminal deletion mutant in ATG9A -KO cells did not rescue C. trachomatis growth, suggesting the importance of this domain for its growth. Although ATG9A -KO cells showed enhanced TBK1 activation, interferon (IFN)-β was not significantly increased, excluding the possibility that upregulation of stimulator of IFN genes (STING) signaling suppressed bacterial growth. Taken together, these findings suggest that the proper trafficking, rather than the isolation membrane expansion function, of ATG9A assists C. trachomatis growth in the inclusion. IMPORTANCE ATG9A is an autophagy-related gene that functions during the isolation membrane expansion process to form autophagosomes, but it also has other functions independent of autophagy. In this study, we employed ATG9A -deficient HeLa cells and found that the absence of ATG9A negatively impacted proliferation of Chlamydia trachomatis in inclusions. Furthermore, rescue experiments using ATG9A mutants revealed that this action was mediated not by its autophagic function but by its binding ability to clathrin adapter proteins. These findings suggest that the proper trafficking of ATG9A assists C. trachomatis growth in the inclusion.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62 S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62 S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The ability to engineer biologically viable hepatocytes and tissue matrices with long-term functional maintenance has attracted considerable interest in the fields of hepatocyte transplantation and liver tissue engineering. Here, newly developed hepatocyte sheets supplemented with adipose-derived stem cells (ADSCs) were evaluated to assess the effects of ADSCs on hepatocyte function and engraftment into the subcutaneous space. Eight-week-old male C57BL/6J mice were used as donors, and 6-week-old male C.B-17/Icr-scid/scid mice were used as recipients. Hepatocyte-ADSC composite sheets were developed using temperature-responsive culture dishes. Hepatocyte viability in the hepatocyte-ADSC composite sheets was evaluated in an in vitro assay, and the outcome of subcutaneous transplantation of the sheet was evaluated. Hepatocyte viability was sustained in the hepatocyte-ADSC composite sheets in vitro . Albumin secretion was significantly higher ( p  = 0.015) in the hepatocytes of the hepatocyte-ADSC composite sheets (70.5 μg/mL) than in hepatocyte-only sheets (24.0 μg/mL). Cytokine assays showed that hepatocyte growth factor and interleukin-6 were contributed by ADSCs and not hepatocytes, which were not capable of constitutively secreting them. Immunohistochemically, phosphorylated STAT3 and c-MET expression in hepatocytes in the hepatocyte-ADSC composite sheets was significantly higher than that in the hepatocyte-only sheets. Engraftment of the transplanted hepatocyte-ADSC composite sheets was significantly enhanced without pretreatment of the subcutaneous tissue to induce a vascular network. In the hepatocyte-ADSC composite sheets, the viability of the hepatocytes was significantly maintained as the co-cultured ADSCs provided cytokines, enhancing pivotal cell signaling necessary for hepatocyte activity. Impact statement Hepatocyte transplantation is a safe, less invasive bridge treatment for liver transplantation, but its effectiveness is low and transitory. Herein, we introduce newly developed hepatocyte-adipose-derived stem cell composite sheets with improved strength, easier transplantation, and increased hepatocyte viability in the subcutaneous transplantation compared with hepatocyte-only sheets.

Background: The Great East Japan Earthquake and the resulting tsunami and nuclear disaster on March 11, 2011 have had a profound and lasting effect on residents of Japan's Fukushima Prefecture, particularly among evacuees. While there continues to be extensive news coverage and academic study of Fukushima Prefecture's recovery, there has been little exploration of individual narratives. This study aims to illuminate some individual stories of medical students at Fukushima Medical University (FMU) who lived in the Prefecture at the time of the Earthquake., Methods: A qualitative approach was taken in order to investigate individuals' experiences with the goal of adding a personal dimension to quantitative studies on the subject. 10 open-ended ethnographic interviews were conducted with medical students at FMU in years 1-5 who lived in Fukushima Prefecture at the time of the Great East Japan Earthquake. All interviews were audio recorded and transcribed. Transcriptions were reviewed using inductive thematic analysis under the lens of ethnographic anthropology., Results: Three major themes emerged from these interviews: first, that the events following the Earthquake influenced not only these students' decisions to pursue careers in medicine, but the ways in which they hope to practice medicine in the future. Second, that these students were motivated to share their experiences by a want to change Fukushima Prefecture's public image. And lastly, that the students viewed the opportunity to discuss their experiences through these interviews as healing, both for themselves and for the future., Conclusions: While multiple factors undoubtably contributed these students' medical education, they cite the Earthquake as essential to their approach to their medical careers. Additionally, opportunities for the participants to discuss their experiences following the Earthquake appear to be rare but valued, as the students view their stories as their "legacies." The enduring, burdening effects of the Earthquake appear to have galvanized the participating students to act on behalf of their communities and their Prefecture. Further qualitative studies in more generalizable populations are needed to improve and deepen our understanding of the societal, cultural, and personal impacts of the Great East Japan Earthquake., (© 2023. The Author(s).)

Stimulator of interferon genes (STING) is essential for the type I interferon response against a variety of DNA pathogens. Upon emergence of cytosolic DNA, STING translocates from the endoplasmic reticulum to the Golgi where STING activates the downstream kinase TBK1, then to lysosome through recycling endosomes (REs) for its degradation. Although the molecular machinery of STING activation is extensively studied and defined, the one underlying STING degradation and inactivation has not yet been fully elucidated. Here we show that STING is degraded by the endosomal sorting complexes required for transport (ESCRT)-driven microautophagy. Airyscan super-resolution microscopy and correlative light/electron microscopy suggest that STING-positive vesicles of an RE origin are directly encapsulated into Lamp1-positive compartments. Screening of mammalian Vps genes, the yeast homologues of which regulate Golgi-to-vacuole transport, shows that ESCRT proteins are essential for the STING encapsulation into Lamp1-positive compartments. Knockdown of Tsg101 and Vps4, components of ESCRT, results in the accumulation of STING vesicles in the cytosol, leading to the sustained type I interferon response. Knockdown of Tsg101 in human primary T cells leads to an increase the expression of interferon-stimulated genes. STING undergoes K63-linked ubiquitination at lysine 288 during its transit through the Golgi/REs, and this ubiquitination is required for STING degradation. Our results reveal a molecular mechanism that prevents hyperactivation of innate immune signalling, which operates at REs., (© 2023. The Author(s).)

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions. Here we identify a UFM1 substrate, NADH-cytochrome b5 reductase 3 (CYB5R3), that localizes on the ER membrane. Ufmylation of CYB5R3 depends on the E3 components UFL1 and UFBP1 on the ER, and converts CYB5R3 into its inactive form. Ufmylated CYB5R3 is recognized by UFBP1 through the UFM1-interacting motif, which plays an important role in the further uyfmylation of CYB5R3. Ufmylated CYB5R3 is degraded in lysosomes, which depends on the autophagy-related protein Atg7- and the autophagy-adaptor protein CDK5RAP3. Mutations of CYB5R3 and genes involved in the UFM1 system cause hereditary developmental disorders, and ufmylation-defective Cyb5r3 knock-in mice exhibit microcephaly. Our results indicate that CYB5R3 ufmylation induces ER-phagy, which is indispensable for brain development., (© 2022. The Author(s).)

A germ line copy number duplication of chromosome 14q32, which contains ATG2B and GSKIP , was identified in families with myeloproliferative neoplasm (MPN). Here, we show that mice lacking both Atg2b and Gskip , but not either alone, exhibited decreased hematopoiesis, resulting in death in utero accompanied by anemia. In marked contrast to MPN patients with duplication of ATG2B and GSKIP , the number of hematopoietic stem cells (HSCs), in particular long-term HSCs, in double-knockout fetal livers was significantly decreased due to increased cell death. Although the remaining HSCs still had the ability to differentiate into hematopoietic progenitor cells, the differentiation efficiency was quite low. Remarkably, mice with knockout of Atg2b or Gskip alone did not show any hematopoietic abnormality. Mechanistically, while loss of both genes had no effect on autophagy, it increased the expression of genes encoding enzymes involved in oxidative phosphorylation. Taken together, our results indicate that Atg2b and Gskip play a synergistic effect in maintaining the pool size of HSCs.

The role of Golgi/endosome-localized clathrin adapters in the maintenance of steady-state cell surface epidermal growth factor receptor (EGFR) is not well known. Here, we show that EGFR associates preferentially with both AP-1 and GGA2 in vitro. AP-1 depletion caused a reduction in the EGFR protein by promoting its lysosomal degradation. Triple immunofluorescence microscopy and proximity ligation assays demonstrated that the interaction of EGFR with AP-1 or GGA2 occurred more frequently in Rab11-positive recycling endosomes than in Rab5-positive early endosomes. Biochemical recycling assay revealed that the depletion of AP-1 or GGA2 significantly suppressed EGFR recycling to the plasma membrane regardless of the EGF stimulation. Depletion of AP-1 or GGA2 also reduced cell contents of other tyrosine kinases, MET and ErbB4, and therefore, suppressed the growth of H1975 cancer cells in culture and xenograft model. Moreover, AP-1 was expressed in endosomes at higher levels in some cancer tissues. Collectively, these results suggest that AP-1 and GGA2 function in recycling endosomes to retrieve endocytosed EGFR, thereby sustaining its cell surface expression and, consequently, cancer cell growth., (© 2021. The Author(s).)

Arf GTPase-Activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors (Arfs), which are critical to form transport intermediates. ArfGAPs have been thought to be negative regulators of Arfs; however, accumulating evidence indicates that ArfGAPs are important for cargo sorting and promote membrane traffic. Weibel-Palade bodies (WPBs) are cigar-shaped secretory granules in endothelial cells that contain von Willebrand factor (vWF) as their main cargo. WPB biogenesis at the Golgi was reported to be regulated by Arf and their regulators, but the role of ArfGAPs has been unknown. In this study, we performed siRNA screening of ArfGAPs to investigate the role of ArfGAPs in the biogenesis of WPBs. We found two ArfGAPs, SMAP1 and AGFG2, to be involved in WPB size and vWF exocytosis, respectively. SMAP1 depletion resulted in small-sized WPBs, and the lysosomal inhibitor leupeptin recovered the size of WPBs. The results indicate that SMAP1 functions in preventing the degradation of cigar-shaped WPBs. On the other hand, AGFG2 downregulation resulted in the inhibition of vWF secretion upon Phorbol 12-myristate 13-acetate (PMA) or histamine stimulation, suggesting that AGFG2 plays a role in vWF exocytosis. Our study revealed unexpected roles of ArfGAPs in vWF transport., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Coat protein complex I (COP-I) mediates the retrograde transport from the Golgi apparatus to the endoplasmic reticulum (ER). Mutation of the COPA gene, encoding one of the COP-I subunits (α-COP), causes an immune dysregulatory disease known as COPA syndrome. The molecular mechanism by which the impaired retrograde transport results in autoinflammation remains poorly understood. Here we report that STING, an innate immunity protein, is a cargo of the retrograde membrane transport. In the presence of the disease-causative α-COP variants, STING cannot be retrieved back to the ER from the Golgi. The forced Golgi residency of STING results in the cGAS-independent and palmitoylation-dependent activation of the STING downstream signaling pathway. Surf4, a protein that circulates between the ER/ ER-Golgi intermediate compartment/ Golgi, binds STING and α-COP, and mediates the retrograde transport of STING to the ER. The STING/Surf4/α-COP complex is disrupted in the presence of the disease-causative α-COP variant. We also find that the STING ligand cGAMP impairs the formation of the STING/Surf4/α-COP complex. Our results suggest a homeostatic regulation of STING at the resting state by retrograde membrane traffic and provide insights into the pathogenesis of COPA syndrome.

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.

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., (© 2020 Yamamoto et al.)

Autophagy is an essential intracellular self-degradation system, and is known to maintain the homeostatic balance between the synthesis, degradation, and recycling of cellular proteins and organelles. Recent studies have suggested a possible role of autophagy in systemic sclerosis (SSc);however, differences in autophagy among pathological phases of SSc have not yet been examined. Therefore, in the current study we investigated the expression pattern of an autophagosome marker protein, microtubule-associated protein 1 light chain 3 (LC3) in the lesional skin of a murine model and human SSc. In bleomycin-induced mouse scleroderma skin, the number of LC3-positive puncta was significantly higher than that in phosphate buffered salts-injected control skin after 4 weeks of treatment. Such an increase, however, was not observed in the skin after 2 weeks of bleomycin treatment, in which few myofibroblasts were detected. In the sclerotic phase of SSc patients, the number of LC3-positive puncta in the lower dermis was significantly higher than in the upper dermis. It was also significantly higher than in the lower dermis of the control patients. No increase in LC3-positive puncta was observed in the skin from SSc patients in edematous phase, in which myofibroblasts were hardly detected. These results suggest that changes in the autophagic degradation system reflect a skin remodeling process that leads to fibrosis.

p62/SQSTM1 is a multivalent protein that has the ability to cause liquid-liquid phase separation and serves as a receptor protein that participates in cargo isolation during selective autophagy. This protein is also involved in the non-canonical activation of the Keap1-Nrf2 system, a major oxidative stress response pathway. Here, we show a role of neighbor of BRCA1 gene 1 (NBR1), an autophagy receptor structurally similar to p62/SQSTM1, in p62-liquid droplet formation and Keap1-Nrf2 pathway activation. Overexpression of NBR1 blocks selective degradation of p62/SQSTM1 through autophagy and promotes the accumulation and phosphorylation of p62/SQSTM1 in liquid-like bodies, which is required for the activation of Nrf2. NBR1 is induced in response to oxidative stress, which triggers p62-mediated Nrf2 activation. Conversely, loss of Nbr1 suppresses not only the formation of p62/SQSTM1-liquid droplets, but also of p62-dependent Nrf2 activation during oxidative stress. Taken together, our results show that NBR1 mediates p62/SQSTM1-liquid droplet formation to activate the Keap1-Nrf2 pathway., (© 2020 The Authors.)

Chondrogenesis is accompanied by not only cellular renovation, but also metabolic stress. Therefore, macroautophagy/autophagy is postulated to be involved in this process. Previous reports have shown that suppression of autophagy during chondrogenesis causes mild growth retardation. However, the role of autophagy in chondrocyte differentiation still largely remains unclear. Here, we show the important role of autophagy on chondrogenesis. The transition of mesenchymal cells to chondrocytes was severely impaired by ablation of Atg7 , a gene essential for autophagy. Mice lacking Atg7 after the transition exhibited phenotypes severer than mutant mice in which Atg7 was removed before the transition. Atg7 -deficient chondrocytes accumulated large numbers of glycogen granules, hardly proliferate and died specifically in the proliferative zone without any ER-stress signal. Our results suggest that the suppression of autophagy in prechondrogenic cells drives compensatory mechanism(s) that mitigate defective chondrogenesis, and that autophagy participates in glycogenolysis to supply glucose in avascular growth plates. Abbreviations: DDIT3/CHOP: DNA damage inducible transcript 3; ER: endoplasmic reticulum; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; SQSTM1/p62: sequestosome 1; STBD1: starch-binding domain-containing protein 1.

GGAs (Golgi-localized, γ-adaptin ear-containing, ADP ribosylation factor [Arf]-binding proteins) are a family of ubiquitously expressed, Arf-dependent monomeric clathrin adaptor proteins, and are conserved from yeast to humans. Mammals have three GGAs (GGA1-3) that work not only at the trans-Golgi network, but also in endosomes to sort transmembrane cargo proteins such as mannose 6-phosphate receptors, sortilin, β-site amyloid precursor protein cleaving enzyme 1, and epidermal growth factor receptor. The cytoplasmic regions of these cargoes possess motifs of acidic amino acid cluster-dileucine and/or ubiquitination sites, which can be recognized by GGAs. Despite seminal investigations of the three molecules, their in vivo roles and functional redundancies, as well as relationships with a heterotetrameric adaptor protein, AP-1, which is functionally similar to GGAs were still poorly understood. Studies over the past two decades, however, discovered several new GGA cargoes, their interaction modes, and accessory proteins. These findings collectively suggest distinct and more fundamental roles of each GGA in regulating neuronal survival, lipid metabolism, and cell proliferation. This review aims to provide an update to the GGA research focusing on how GGAs became considered not only as players in the context of the TGN-endosome transport, but also as key regulators for physiologically and pathologically important phenomena.

Lipid droplets (LDs) are dynamic organelles that store neutral lipids during times of energy excess, such as after a meal. LDs serve as an energy reservoir during fasting and have a buffering capacity that prevents lipotoxicity. Autophagy and the autophagic machinery have been proposed to play a role in LD biogenesis, but the underlying molecular mechanism remains unclear. Here, we show that when nuclear receptor co-repressor 1 (NCoR1), which inhibits the transactivation of nuclear receptors, accumulates because of autophagy suppression, LDs decrease in size and number. Ablation of ATG7 , a gene essential for autophagy, suppressed the expression of gene targets of liver X receptor α, a nuclear receptor responsible for fatty acid and triglyceride synthesis in an NCoR1-dependent manner. LD accumulation in response to fasting and after hepatectomy was hampered by the suppression of autophagy. These results suggest that autophagy controls physiological hepatosteatosis by fine-tuning NCoR1 protein levels., (© 2019 Takahashi et al.)

Autophagy is considered an adaptive mechanism against hyperosmotic stress. Although the process has been reported to be triggered by the inhibition of mTORC1, the precise downstream mechanisms remain elusive. Here, we demonstrate that hyperosmotic-stress-induced autophagy is different from conventional macroautophagy in mouse embryonic fibroblasts (MEFs) and human T24 cells. Our results indicated that cytoplasmic puncta for the isolation membrane markers WIPI2 and Atg16L increased after hyperosmotic stress. They were found to partially colocalize with puncta for a selective autophagy substrate, SQSTM1/p62, and were shown to be diminished by inhibitors of phosphatidylinositol 3-kinase (PI3K) or by knockdown of human Vps34 (hVps34), a component of PI3K. In addition, flux assays showed that SQSTM1/p62 and NcoA4 were degraded by the lysosomal pathway. Surprisingly, Ulk1, which is essential for starvation-induced macroautophagy, remained inactivated under hyperosmotic stress, which was partially caused by mTOR activity. Accordingly, the Ulk1 complex was not nucleated under hyperosmotic stress. Finally, autophagy proceeded even in MEFs deficient in RB1CC1/FIP200 or Atg13 , which encode components of the Ulk1 complex. These data suggest that hyperosmotic-stress-induced autophagy represents an unconventional type of autophagy that bypasses Ulk1 signaling., (Copyright © 2019 Tamura et al.)

Selective autophagy ensures the removal of specific soluble proteins, protein aggregates, damaged mitochondria, and invasive bacteria from cells. Defective autophagy has been directly linked to metabolic disorders. However how selective autophagy regulates metabolism remains largely uncharacterized. Here we show that a deficiency in selective autophagy is associated with suppression of lipid oxidation. Hepatic loss of Atg7 or Atg5 significantly impairs the production of ketone bodies upon fasting, due to decreased expression of enzymes involved in β-oxidation following suppression of transactivation by PPARα. Mechanistically, nuclear receptor co-repressor 1 (NCoR1), which interacts with PPARα to suppress its transactivation, binds to the autophagosomal GABARAP family proteins and is degraded by autophagy. Consequently, loss of autophagy causes accumulation of NCoR1, suppressing PPARα activity and resulting in impaired lipid oxidation. These results suggest that autophagy contributes to PPARα activation upon fasting by promoting degradation of NCoR1 and thus regulates β-oxidation and ketone bodies production.

Autophagy-related organelles, including omegasomes, isolation membranes (or phagophores), autophagosomes, and autolysosomes, are characterized by dynamic changes in lipid membranes including morphology as well as their associated proteins. Therefore, it is critical to define and track membranous elements for identification and detailed morphological analyses of these organelles. However, it is often difficult to clearly observe these organelles with good morphology in conventional electron microscopy (EM), thus hampering 3D analyses and correlative light-electron microscopy (CLEM). Here, we focus on describing fixation procedures using (1) ferrocyanide-reduced osmium for CLEM and (2) aldehyde/OsO 4 mixture for detecting omegasome structures and isolation membrane-associated tubules (IMATs). These methods can be easily applied to cultured mammalian cells for conventional and cutting-edge EM analyses, leading to a better understanding of ultrastructural details in autophagosome formation.

[This corrects the article DOI: 10.1371/journal.pone.0207533.].

During myogenesis, myogenic stem cells undergo several sequential events, including cell division, migration, and cell-cell fusion, leading to the formation of multinuclear myotubes, which are the precursors of myofibers. To understand the molecular mechanisms underlying these complex processes, an RNA interference-based gene depletion approach was used. Golgi associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1), a Golgi-resident monomeric clathrin adaptor, was found to be required for the process of myotube formation in C2C12 cells, a cultured murine myoblast cell line. Gga1 mRNA expression was upregulated during myogenesis, and Gga1 depletion prevented the formation of large multi-nucleated myotubes. Moreover, inhibition of lysosomal proteases in Gga1 knockdown myoblasts increased the amount of insulin receptor, suggesting that GGA1 is involved in the cell surface expression and sorting of the insulin receptor. These results suggested that GGA1 plays a significant role in the formation and maturation of myotubes by targeting the insulin receptor to the cell surface to establish functionally mature myofibers., Competing Interests: The authors have declared that no competing interests exist.

Background/aim: Clear cell sarcoma (CCS) of soft tissue is exceedingly rare and frequently exhibits aggressive behavior. Toward the goals of improving the aggressive course and poor prognosis of CCS, and establish new therapeutic methods, molecular genetic and biological characterizations of CCS are required., Materials and Methods: A new human CCS cell line (designated RSAR001) was established from the pleural effusion of a 44-year-old man with multiple lung metastases and pleural dissemination. The cell line and its xenograft were characterized including their morphology, immunohistochemistry, cytogenetic analysis, reverse transcription-polymerase chain reaction, direct sequencing analysis, and fluorescence in situ hybridization analysis., Results: The cell line has been maintained for over 12 months with more than 50 passages. RSAR001 cells exhibited a fascicular or diffuse growth pattern of short spindle- or oval-shaped cells with clear cytoplasm in heterotransplanted tumor, that was similar to the primary tumor. Immunophenotypically, RSAR001 cells in vitro and in vivo exhibited almost the same characteristics as the primary tumor. Cytogenetic analyses revealed a translocation, t(12;22)(q13;q12). Reverse transcription-polymerase chain reaction and direct sequencing analysis detected transcripts of the Ewing sarcoma breakpoint region 1-activating transcription factor 1 (EWSR1-ATF1) type 1 fusion gene. Fluorescence in situ hybridization using a break-apart probe for the EWSR1 gene on 22q12 showed a rearrangement., Conclusion: These findings indicate that the RSAR001 cell line harbors EWSR1-ATF1 type 1 chimeric fusion gene, which is specific to CCS. RSAR001 cells might be useful for investigating biological behaviors and developing new treatments such as molecular-targeting antitumor drugs or immunological drugs for CCS., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Autophagy is important for liver homeostasis, and the deficiency leads to injury, inflammation, ductular reaction (DR), fibrosis, and tumorigenesis. It is not clear how these events are mechanistically linked to autophagy deficiency. Here, we reveal the role of high-mobility group box 1 (HMGB1) in two of these processes. First, HMGB1 was required for DR, which represents the expansion of hepatic progenitor cells (HPCs) implicated in liver repair and regeneration. DR caused by hepatotoxic diets (3,5-diethoxycarbonyl-1,4-dihydrocollidine [DDC] or choline-deficient, ethionine-supplemented [CDE]) also depended on HMGB1, indicating that HMGB1 may be generally required for DR in various injury scenarios. Second, HMGB1 promoted tumor progression in autophagy-deficient livers. Receptor for advanced glycation end product (RAGE), a receptor for HMGB1, was required in the same two processes and could mediate the proliferative effects of HMBG1 in isolated HPCs. HMGB1 was released from autophagy-deficient hepatocytes independently of cellular injury but depended on NRF2 and the inflammasome, which was activated by NRF2. Pharmacological or genetic activation of NRF2 alone, without disabling autophagy or causing injury, was sufficient to cause inflammasome-dependent HMGB1 release. In conclusion, HMGB1 release is a critical mechanism in hepatic pathogenesis under autophagy-deficient conditions and leads to HPC expansion as well as tumor progression.

To investigate the possible implications of autophagy, one of the degradation pathways induced by metabolic stress, in the dynamic reconstructive process of wound healing, the appearance and changes of punctate structures for microtubule-associated protein 1 light chain 3 (LC3), an autophagosome marker, were examined in a rat skin wound healing model. Although the ratio of LC3-II/LC3-I in Western blotting was not evidently changed during the wound healing process, LC3-positive dots were clearly observed in fibroblasts and myofibroblasts, and occasionally in macrophages, by immunohistofluorescence microscopy. Some of the LC3-positive dots were colocalized with Atg16L signal, an isolation membrane marker, and electron microscopy revealed the presence of typical autophagosomes in fibroblasts near the margin of the wound. The number of LC3-positive dots per fibroblast increased during the later period of the proliferation phase, and interestingly, it was higher in the margin than the center of the wound. It was also high in the periwound skin area. These results suggest that drastic functional changes in fibroblasts during wound healing process are accompanied by the alteration of the autophagy-lysosomal degradation system.