Autophagy Captures the Nobel Prize.

This year's Nobel Prize in Physiology or Medicine has been awarded to Yoshinori Ohsumi for the discovery of the molecular principles governing autophagy, an intracellular degradation pathway routed via lysosomes or vacuoles. It is a story of a simple yet insightful yeast genetic screen that revealed the inner circuitry of one of the most powerful quality-control pathways in cells.

Autophagy modulates endothelial junctions to restrain neutrophil diapedesis during inflammation.

The migration of neutrophils from the blood circulation to sites of infection or injury is a key immune response and requires the breaching of endothelial cells (ECs) that line the inner aspect of blood vessels. Unregulated neutrophil transendothelial cell migration (TEM) is pathogenic, but the molecular basis of its physiological termination remains unknown. Here, we demonstrated that ECs of venules in inflamed tissues exhibited a robust autophagic response that was aligned temporally with the peak of neutrophil trafficking and was strictly localized to EC contacts. Genetic ablation of EC autophagy led to excessive neutrophil TEM and uncontrolled leukocyte migration in murine inflammatory models, while pharmacological induction of autophagy suppressed neutrophil infiltration into tissues. Mechanistically, autophagy regulated the remodeling of EC junctions and expression of key EC adhesion molecules, facilitating their intracellular trafficking and degradation. Collectively, we have identified autophagy as a modulator of EC leukocyte trafficking machinery aimed at terminating physiological inflammation.

Quality Control: Maintaining molecular order and preventing cellular chaos.

We asked experts from different fields-from genome maintenance and proteostasis to organelle degradation via ubiquitin and autophagy-"What does quality control mean to you?" Despite their diverse backgrounds, they converge on and discuss the importance of continuous quality control at all levels, context, communication, timing, decisions on whether to repair or remove, and the significance of dysregulated quality control in disease.

ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation.

ATG9A and ATG2A are essential core members of the autophagy machinery. ATG9A is a lipid scramblase that allows equilibration of lipids across a membrane bilayer, whereas ATG2A facilitates lipid flow between tethered membranes. Although both have been functionally linked during the formation of autophagosomes, the molecular details and consequences of their interaction remain unclear. By combining data from peptide arrays, crosslinking, and hydrogen-deuterium exchange mass spectrometry together with cryoelectron microscopy, we propose a molecular model of the ATG9A-2A complex. Using this integrative structure modeling approach, we identify several interfaces mediating ATG9A-2A interaction that would allow a direct transfer of lipids from ATG2A into the lipid-binding perpendicular branch of ATG9A. Mutational analyses combined with functional activity assays demonstrate their importance for autophagy, thereby shedding light on this protein complex at the heart of autophagy.

Exploring the ATG9A interactome uncovers interaction with VPS13A.

ATG9A, a transmembrane protein of the core autophagy pathway, cycles between the Golgi, endosomes and a vesicular compartment. ATG9A was recently shown to act as a lipid scramblase, and this function is thought to require its interaction with another core autophagy protein, ATG2A, which acts as a lipid transfer protein. Together, ATG9A and ATG2A are proposed to function to expand the growing autophagosome. However, ATG9A is implicated in other pathways including membrane repair and lipid droplet homeostasis. To elucidate other ATG9A interactors within the autophagy pathway, or interactors beyond autophagy, we performed an interactome analysis through mass spectrometry. This analysis revealed a host of proteins involved in lipid synthesis and trafficking, including ACSL3, VPS13A and VPS13C. Furthermore, we show that ATG9A directly interacts with VPS13A and forms a complex that is distinct from the ATG9A-ATG2A complex.

Phosphoproteomic identification of ULK substrates reveals VPS15-dependent ULK/VPS34 interplay in the regulation of autophagy.

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.

The autophagosome: origins unknown, biogenesis complex.

Healthy cells use autophagy as a general 'housekeeping' mechanism and to survive stress, including stress induced by nutrient deprivation. Autophagy is initiated at the isolation membrane (originally termed the phagophore), and the coordinated action of ATG (autophagy-related) proteins results in the expansion of this membrane to form the autophagosome. Although the biogenesis of the isolation membrane and the autophagosome is complex and incompletely understood, insight has been gained into the molecular processes involved in initiating the isolation membrane, the source from which this originates (for example, it was recently proposed that the isolation membrane forms from the mitochondria-associated endoplasmic reticulum (ER) membrane (MAM)) and the role of ATG proteins and the vesicular trafficking machinery in autophagosome formation.

Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function.

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.

The Golgi as an Assembly Line to the Autophagosome.

Autophagy is traditionally depicted as a signaling cascade that culminates in the formation of an autophagosome that degrades cellular cargo. However, recent studies have identified myriad pathways and cellular organelles underlying the autophagy process, be it as signaling platforms or through the contribution of proteins and lipids. The Golgi complex is recognized as being a central transport hub in the cell, with a critical role in endocytic trafficking and endoplasmic reticulum (ER) to plasma membrane (PM) transport. However, the Golgi is also an important site of key autophagy regulators, including the protein autophagy-related (ATG)-9A and the lipid, phosphatidylinositol-4-phosphate [PI(4)P]. In this review, we highlight the central function of this organelle in autophagy as a transport hub supplying various components of autophagosome formation.

The phosphatidylinositol 3-phosphate-binding protein SNX4 controls ATG9A recycling and autophagy.

Late endosomes and lysosomes (endolysosomes) receive proteins and cargo from the secretory, endocytic and autophagic pathways. Although these pathways and the degradative processes of endolysosomes are well characterized, less is understood about protein traffic from these organelles. In this study, we demonstrate the direct involvement of the phosphatidylinositol 3-phosphate (PI3P)-binding SNX4 protein in membrane protein recycling from endolysosomes, and show that SNX4 is required for proper autophagic flux. We show that SNX4 mediates recycling of the lipid scramblase ATG9A, which drives expansion of nascent autophagosome membranes, from endolysosomes to early endosomes, from where ATG9A is recycled to the trans-Golgi network in a retromer-dependent manner. Upon siRNA-mediated depletion of SNX4 or the retromer component VPS35, we observed accumulation of ATG9A on endolysosomes and early endosomes, respectively. Moreover, starvation-induced autophagosome biogenesis and autophagic flux were inhibited when SNX4 was downregulated. We propose that proper ATG9A recycling by SNX4 sustains autophagy by preventing exhaustion of the available ATG9A pool.This article has an associated First Person interview with the first author of the paper.

Activation of ULK Kinase and Autophagy by GABARAP Trafficking from the Centrosome Is Regulated by WAC and GM130.

Starvation-induced autophagy requires activation of the ULK complex at the phagophore. Two Golgi proteins, WAC and GM130, regulate autophagy, however their mechanism of regulation is unknown. In search of novel interaction partners of WAC, we found that GM130 directly interacts with WAC, and this interaction is required for autophagy. WAC is bound to the Golgi by GM130. WAC and GM130 interact with the Atg8 homolog GABARAP and regulate its subcellular localization. GABARAP is on the pericentriolar matrix, and this dynamic pool contributes to autophagosome formation. Tethering of GABARAP to the Golgi by GM130 inhibits autophagy, demonstrating an unexpected role for a golgin. WAC suppresses GM130 binding to GABARAP, regulating starvation-induced centrosomal GABARAP delivery to the phagophore. GABARAP, unlipidated and lipidated, but not LC3B, GABARAPL1, and GATE-16, specifically promotes ULK kinase activation dependent on the ULK1 LIR motif, elucidating a unique non-hierarchical role for GABARAP in starvation-induced activation of autophagy.

Autophagy coordinates chondrocyte development and early joint formation in zebrafish.

Autophagy is a catabolic process responsible for the removal of waste and damaged cellular components by lysosomal degradation. It plays a key role in fundamental cell processes, including ER stress mitigation, control of cell metabolism, and cell differentiation and proliferation, all of which are essential for cartilage cell (chondrocyte) development and survival, and for the formation of cartilage. Correspondingly, autophagy dysregulation has been implicated in several skeletal disorders such as osteoarthritis and osteoporosis. To test the requirement for autophagy during skeletal development in zebrafish, we generated an atg13 CRISPR knockout zebrafish line. This line showed a complete loss of atg13 expression, and restricted autophagic activity in vivo. In the absence of autophagy, chondrocyte maturation was accelerated, with chondrocytes exhibiting signs of premature hypertrophy. Focussing on the jaw element, autophagy disruption affected joint articulation causing restricted mouth opening. This gross behavioural phenotype corresponded with a failure to thrive, and death in homozygote atg13 nulls within 17 days. Taken together, our results are consistent with autophagy contributing to the timely regulation of chondrocyte maturation and for extracellular matrix formation.

WIPI2 links LC3 conjugation with PI3P, autophagosome formation, and pathogen clearance by recruiting Atg12-5-16L1.

Mammalian cell homeostasis during starvation depends on initiation of autophagy by endoplasmic reticulum-localized phosphatidylinositol 3-phosphate (PtdIns(3)P) synthesis. Formation of double-membrane autophagosomes that engulf cytosolic components requires the LC3-conjugating Atg12-5-16L1 complex. The molecular mechanisms of Atg12-5-16L1 recruitment and significance of PtdIns(3)P synthesis at autophagosome formation sites are unknown. By identifying interacting partners of WIPIs, WD-repeat PtdIns(3)P effector proteins, we found that Atg16L1 directly binds WIPI2b. Mutation experiments and ectopic localization of WIPI2b to plasma membrane show that WIPI2b is a PtdIns(3)P effector upstream of Atg16L1 and is required for LC3 conjugation and starvation-induced autophagy through recruitment of the Atg12-5-16L1 complex. Atg16L1 mutants, which do not bind WIPI2b but bind FIP200, cannot rescue starvation-induced autophagy in Atg16L1-deficient MEFs. WIPI2b is also required for autophagic clearance of pathogenic bacteria. WIPI2b binds the membrane surrounding Salmonella and recruits the Atg12-5-16L1 complex, initiating LC3 conjugation, autophagosomal membrane formation, and engulfment of Salmonella.

Molecular definitions of autophagy and related processes.

Over the past two decades, the molecular machinery that underlies autophagic responses has been characterized with ever increasing precision in multiple model organisms. Moreover, it has become clear that autophagy and autophagy-related processes have profound implications for human pathophysiology. However, considerable confusion persists about the use of appropriate terms to indicate specific types of autophagy and some components of the autophagy machinery, which may have detrimental effects on the expansion of the field. Driven by the overt recognition of such a potential obstacle, a panel of leading experts in the field attempts here to define several autophagy-related terms based on specific biochemical features. The ultimate objective of this collaborative exchange is to formulate recommendations that facilitate the dissemination of knowledge within and outside the field of autophagy research.

TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic.

Macroautophagy requires membrane trafficking and remodelling to form the autophagosome and deliver its contents to lysosomes for degradation. We have previously identified the TBC domain-containing protein, TBC1D14, as a negative regulator of autophagy that controls delivery of membranes from RAB11-positive recycling endosomes to forming autophagosomes. In this study, we identify the TRAPP complex, a multi-subunit tethering complex and GEF for RAB1, as an interactor of TBC1D14. TBC1D14 binds to the TRAPP complex via an N-terminal 103 amino acid region, and overexpression of this region inhibits both autophagy and secretory traffic. TRAPPC8, the mammalian orthologue of a yeast autophagy-specific TRAPP subunit, forms part of a mammalian TRAPPIII-like complex and both this complex and TBC1D14 are needed for RAB1 activation. TRAPPC8 modulates autophagy and secretory trafficking and is required for TBC1D14 to bind TRAPPIII. Importantly, TBC1D14 and TRAPPIII regulate ATG9 trafficking independently of ULK1. We propose a model whereby TBC1D14 and TRAPPIII regulate a constitutive trafficking step from peripheral recycling endosomes to the early Golgi, maintaining the cycling pool of ATG9 required for initiation of autophagy.

SNX18 regulates ATG9A trafficking from recycling endosomes by recruiting Dynamin-2.

Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18-induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin-2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1- and WIPI2-positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin-2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.

A mutation in the major autophagy gene, WIPI2, associated with global developmental abnormalities.

We describe a large consanguineous pedigree from a remote area of Northern Pakistan, with a complex developmental disorder associated with wide-ranging symptoms, including mental retardation, speech and language impairment and other neurological, psychiatric, skeletal and cardiac abnormalities. We initially carried out a genetic study using the HumanCytoSNP-12 v2.1 Illumina gene chip on nine family members and identified a single region of homozygosity shared amongst four affected individuals on chromosome 7p22 (positions 3059377-5478971). We performed whole-exome sequencing on two affected individuals from two separate branches of the extended pedigree and identified a novel nonsynonymous homozygous mutation in exon 9 of the WIPI2 (WD-repeat protein interacting with phosphoinositide 2) gene at position 5265458 (c.G745A;pV249M). WIPI2 plays a critical role in autophagy, an evolutionary conserved cellular pathway implicated in a growing number of medical conditions. The mutation is situated in a highly conserved and critically important region of WIPI2, responsible for binding PI(3)P and PI(3,5)P2, an essential requirement for autophagy to proceed. The mutation is absent in all public databases, is predicted to be damaging and segregates with the disease phenotype. We performed functional studies in vitro to determine the potential effects of the mutation on downstream pathways leading to autophagosome assembly. Binding of the V231M mutant of WIPI2b to ATG16L1 (as well as ATG5-12) is significantly reduced in GFP pull-down experiments, and fibroblasts derived from the patients show reduced WIPI2 puncta, reduced LC3 lipidation and reduced autophagic flux.

A molecular perspective of mammalian autophagosome biogenesis.

Autophagy is a highly conserved process and is essential for the maintenance of cellular homeostasis. Autophagy occurs at a basal level in all cells, but it can be up-regulated during stress, starvation, or infection. Misregulation of autophagy has been linked to various disorders, including cancer, neurodegeneration, and immune diseases. Here, we discuss the essential proteins acting in the formation of an autophagosome, with a focus on the ULK and VPS34 kinase complexes, phosphatidylinositol 3-phosphate effector proteins, and the transmembrane autophagy-related protein ATG9. The function and regulation of these and other autophagy-related proteins acting during formation will be addressed, in particular during amino acid starvation.

Control of GABARAP-mediated autophagy by the Golgi complex, centrosome and centriolar satellites.

Within minutes of induction of autophagy by amino-acid starvation in mammalian cells, multiple autophagosomes form throughout the cell cytoplasm. During their formation, the autophagosomes sequester cytoplasmic material and deliver it to lysosomes for degradation. How these organelles can be so rapidly formed and how their formation is acutely regulated are major questions in the autophagy field. Protein and lipid trafficking from diverse cell compartments contribute membrane to, or regulate the formation of the autophagosome. In addition, recruitment of Atg8 (in yeast), and the ATG8-family members (in mammalian cells) to autophagosomes is required for efficient autophagy. Recently, it was discovered that the centrosome and centriolar satellites regulate autophagosome formation by delivery of an ATG8-family member, GABARAP, to the forming autophagosome membrane, the phagophore. We propose that GABARAP regulates phagophore expansion by activating the ULK complex, the amino-acid controlled initiator complex. This finding reveals a previously unknown link between the centrosome, centriolar satellites and autophagy.