Method to Purify Phase-Separated p62 Bodies Using Fluorescence-Activated Particle Sorting.

p62 bodies are ubiquitin-positive cytoplasmic condensates formed by liquid-liquid phase separation. They are targeted by selective autophagy and play important roles in intracellular quality control and stress responses. However, little is known about their constituents.

p62 bodies: cytosolic zoning by phase separation.

Cellular zoning or partitioning is critical in preventing macromolecules from random diffusion and orchestrating the spatiotemporal dynamics of biochemical reactions. Along with membranous organelles, membraneless organelles contribute to the precise regulation of biochemical reactions inside cells. In response to environmental cues, membraneless organelles rapidly form through liquid-liquid phase separation, sequester certain proteins and RNAs, mediate specific reactions and dissociate.

Vault-phagy: a phase-separation-mediated selective autophagy of vault, a non-membranous organelle.

SQSTM1/p62 bodies are phase-separated condensates that play a fundamental role in intracellular quality control and stress responses. Despite extensive studies investigating the mechanism of formation and degradation of SQSTM1/p62 bodies, the constituents of SQSTM1/p62 bodies remain elusive. We recently developed a purification method for intracellular SQSTM1/p62 bodies using a cell sorter and identified their constituents by mass spectrometry.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

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.

Regulation of ER-derived membrane dynamics by the DedA domain-containing proteins VMP1 and TMEM41B.

The endoplasmic reticulum (ER) is a central hub for the biogenesis of various organelles and lipid-containing structures. Recent studies suggest that vacuole membrane protein 1 (VMP1) and transmembrane protein 41B (TMEM41B), multispanning ER membrane proteins, regulate the formation of many of these ER-derived structures, including autophagosomes, lipid droplets, lipoproteins, and double-membrane structures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. VMP1 and TMEM41B possess a DedA domain that is widely distributed not only in eukaryotes but also in prokaryotes and predicted to adopt a characteristic structure containing two reentrant loops.

A new insight into the lens: cytosolic PLAAT phospholipases degrade organelles to make the lens transparent.

All membrane-bound organelles are degraded during the terminal differentiation of lens fiber cells. How these organelles are degraded has been a long-standing question in biology. We recently revealed that PLAAT (phospholipase A and acyltransferase)-family phospholipases degrade organelles in the lens independently of macroautophagy.

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy.

Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood.

Organelle degradation in the lens by PLAAT phospholipases.

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 zone. The mechanism that underlies this large-scale organelle degradation remains largely unknown, although it has previously been shown to be independent of macroautophagy. 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 mice-are essential for the degradation of lens organelles such as mitochondria, the endoplasmic reticulum and lysosomes.

Evolution and insights into the structure and function of the DedA superfamily containing TMEM41B and VMP1.

TMEM41B and VMP1 are endoplasmic reticulum (ER)-localizing multi-spanning membrane proteins required for ER-related cellular processes such as autophagosome formation, lipid droplet homeostasis and lipoprotein secretion in eukaryotes. Both proteins have a VTT domain, which is similar to the DedA domain found in bacterial DedA family proteins. However, the molecular function and structure of the DedA and VTT domains (collectively referred to as DedA domains) and the evolutionary relationships among the DedA domain-containing proteins are largely unknown.

No air without autophagy: autophagy is important for lung and swim bladder inflation.

Macroautophagy is a catabolic process critical for the degradation of intracellular material, but its physiological functions in vertebrates are not fully understood. Here, we discuss our recent finding that macroautophagy plays a role in lamellar body maturation. The lamellar body is a lysosome-related organelle and stores phospholipid-containing surfactant complexes that reduce the surface tension of the air-water interface in order to inflate the airspace in lungs and swim bladders.

Autophagy Is Required for Maturation of Surfactant-Containing Lamellar Bodies in the Lung and Swim Bladder.

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.

A critical role of VMP1 in lipoprotein secretion.

Lipoproteins are lipid-protein complexes that are primarily generated and secreted from the intestine, liver, and visceral endoderm and delivered to peripheral tissues. Lipoproteins, which are assembled in the endoplasmic reticulum (ER) membrane, are released into the ER lumen for secretion, but its mechanism remains largely unknown. Here, we show that the release of lipoproteins from the ER membrane requires VMP1, an ER transmembrane protein essential for autophagy and certain types of secretion.

Diverse Cellular Roles of Autophagy.

Macroautophagy is an intracellular degradation system that delivers diverse cytoplasmic materials to lysosomes via autophagosomes. Recent advances have enabled identification of several selective autophagy substrates and receptors, greatly expanding our understanding of the cellular functions of autophagy. In this review, we describe the diverse cellular functions of macroautophagy, including its essential contribution to metabolic adaptation and cellular homeostasis.

Genome-wide CRISPR screen identifies as a gene required for autophagosome formation.

Macroautophagy is an intracellular degradation process that requires multiple autophagy-related () genes. In this study, we performed a genome-wide screen using the autophagic flux reporter GFP-LC3-RFP and identified as a novel gene. TMEM41B is a multispanning membrane protein localized in the endoplasmic reticulum (ER).

Autophagy is essential for hearing in mice.

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.

Prognosis of infantile food protein-induced enterocolitis syndrome in Japan.

Background: Although serum C-reactive protein (CRP) and the percentage of eosinophils in peripheral blood (Eo) are increased at onset in infants with food protein-induced enterocolitis syndrome (FPIES), the relationship of these laboratory findings to prognosis is presently unknown.

Methods: Correlation of serum CRP and Eo at onset with prognosis was analyzed in 32 patients with FPIES caused by cow's milk (CM).

Results: The rate of tolerance acquisition was 18.

A new probe to measure autophagic flux in vitro and in vivo.

Macroautophagy is a catabolic process that delivers cytoplasmic components via the autophagosome to lysosomes for degradation. Measuring autophagic activity is critical to dissect molecular mechanisms and functions of autophagy but remains challenging due to the lack of a definitive method. We have recently developed a new fluorescent probe, GFP-LC3-RFP-LC3ΔG, to assess autophagic flux.

Cytokine profile after oral food challenge in infants with food protein-induced enterocolitis syndrome.

Background: Although food protein-induced enterocolitis syndrome (FPIES) is supposed to be caused by inflammation, the role of cytokines has not yet been clarified.

Methods: To elucidate the role of cytokines in the development of symptoms and abnormal laboratory findings at an oral food challenge (OFC), changes in serum cytokine levels were analyzed for 6 OFCs in 4 patients with FPIES. The result of OFC was judged positive if any gastrointestinal (GI) symptoms (vomiting, diarrhea, or bloody stool) were induced.

The ATG conjugation systems are important for degradation of the inner autophagosomal membrane.

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.

Targeted use of prednisolone with the second IVIG dose for refractory Kawasaki disease.

Background: Prednisolone (PSL) has been suggested to be useful for the treatment of Kawasaki disease (KD) resistant to i.v. immunoglobulin (IVIG), but much remains to be elucidated regarding its use.

Eosinophilia in infants with food protein-induced enterocolitis syndrome in Japan.

Background: Many Japanese infants with food protein-induced enterocolitis syndrome (FPIES) show eosinophilia, which has been thought to be a characteristic of food protein-induced proctocolitis (FPIP).

Methods: To elucidate the characteristics of eosinophilia in Japanese FPIES patients, 113 infants with non-IgE-mediated gastrointestinal food allergy due to cow's milk were enrolled and classified into FPIES (n = 94) and FPIP (n = 19).

Results: The percentage of peripheral blood eosinophils (Eo) was increased in most FPIES patients (median, 7.