Hideaki Morishita

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

Open sesame! The autophagosome is a double-membraned intracellular structure involved in the disposal of damaged or defunct organelles. Autophagosome formation requires a number of autophagy-related (ATG) proteins. Among them, the key conjugation systems ATG8 and ATG12 are widely exploited in the detection of autophagy in many organisms. However, their precise function in autophagy remains unknown. Tsuboyama et al. identified an unexpected role of ATG3, an important enzyme in the ATG conjugation systems, in efficient degradation and opening of the inner autophagosomal membrane after fusion with lysosomes (see the Perspective by Levine). Their live-imaging system revealed the entire life of an autophagosome in mammalian cells. Science, this issue p. 1036; see also p. 968 The requirements for autophagosome maturation and efficient autophagy within mammalian cells are dissected. 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.

Deletion of Autophagy-related 5 (Atg5) and Pik3c3 Genes in the Lens Causes Cataract Independent of Programmed Organelle Degradation

Background: The role of autophagy-dependent quality control in the lens remains unclear. Results: Deletion of Atg5 and Pik3c3/Vps34 in the lens does not affect programmed organelle degradation but causes cataract and a developmental defect, respectively. Conclusion: These genes are important for quality control and development of the lens. Significance: This study provides new insights into biology and age-related pathology of the lens. 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.

Autophagy in the lens.

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.

Fra-1 negatively regulates lipopolysaccharide-mediated inflammatory responses

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.

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. 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.

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

ABSTRACT 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. Upon intracellular expression, the probe is cleaved by ATG4 family proteases into equimolar amounts of GFP-LC3 and RFP-LC3ΔG. The former is degraded by autophagy while the latter persists as an internal control in the cytosol. Autophagic flux can thus be quantified by obtaining the ratio of GFP:RFP signals. Using this method, we have identified several autophagy-modulating drugs by screening an approved drug library. We have also demonstrated that induced and basal autophagic flux can be monitored in zebrafish and mice.

Genome-wide CRISPR screen identifies TMEM41B as a gene required for autophagosome formation

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.