An mTORC1-to-CDK1 Switch Maintains Autophagy Suppression during Mitosis.

Since nuclear envelope breakdown occurs during mitosis in metazoan cells, it has been proposed that macroautophagy must be inhibited to maintain genome integrity. However, repression of macroautophagy during mitosis remains controversial and mechanistic detail limited to the suggestion that CDK1 phosphorylates VPS34. Here, we show that initiation of macroautophagy, measured by the translocation of the ULK complex to autophagic puncta, is repressed during mitosis, even when mTORC1 is inhibited. Indeed, mTORC1 is inactive during mitosis, reflecting its failure to localize to lysosomes due to CDK1-dependent RAPTOR phosphorylation. While mTORC1 normally represses autophagy via phosphorylation of ULK1, ATG13, ATG14, and TFEB, we show that the mitotic phosphorylation of these autophagy regulators, including at known repressive sites, is dependent on CDK1 but independent of mTOR. Thus, CDK1 substitutes for inhibited mTORC1 as the master regulator of macroautophagy during mitosis, uncoupling autophagy regulation from nutrient status to ensure repression of macroautophagy during mitosis.

Sorting nexin 5 mediates virus-induced autophagy and immunity.

Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.

TFG binds LC3C to regulate ULK1 localization and autophagosome formation.

The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

Autophagy in major human diseases.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

Functions and mechanisms of the GPCR adaptor protein Norbin.

Norbin (Neurochondrin, NCDN) is a highly conserved 79 kDa adaptor protein that was first identified more than a quarter of a century ago as a gene up-regulated in rat hippocampus upon induction of long-term potentiation. Most research has focussed on the role of Norbin in the nervous system, where the protein is highly expressed. Norbin regulates neuronal morphology and synaptic plasticity, and is essential for normal brain development and homeostasis. Dysregulation of Norbin is linked to a variety of neurological conditions. Recently, Norbin was shown to be expressed in myeloid cells as well as neurons. Myeloid-cell specific deletion revealed an important role of Norbin as a suppressor of neutrophil-derived innate immunity. Norbin limits the ability of neutrophils to clear bacterial infections by curbing the responsiveness of these cells to inflammatory and infectious stimuli. Mechanistically, Norbin regulates cell responses through binding to its interactors, in particular to a wide range of G protein-coupled receptors (GPCRs). Norbin association with GPCRs controls GPCR trafficking and signalling. Other important Norbin interactors are the Rac guanine-nucleotide exchange factor P-Rex1 and protein kinase A. Downstream signalling pathways regulated by Norbin include ERK, Ca2+ and the small GTPase Rac. Here, we review the current understanding of Norbin structure, expression and its roles in health and disease. We also explore Norbin signalling through its interactors, with a particular focus on GPCR trafficking and signalling. Finally, we discuss avenues that could be pursued in the future to increase our understanding of Norbin biology.

Ubiquitination Is a Novel Post-Translational Modification of VMP1 in Autophagy of Human Tumor Cells.

Autophagy is a tightly regulated catabolic process involved in the degradation and recycling of proteins and organelles. Ubiquitination plays an important role in the regulation of autophagy. Vacuole Membrane Protein 1 (VMP1) is an essential autophagy protein. The expression of VMP1 in pancreatic cancer stem cells carrying the activated Kirsten rat sarcoma viral oncogene homolog (KRAS) triggers autophagy and enables therapy resistance. Using biochemical and cellular approaches, we identified ubiquitination as a post-translational modification of VMP1 from the initial steps in autophagosome biogenesis. VMP1 remains ubiquitinated as part of the autophagosome membrane throughout autophagic flux until autolysosome formation. However, VMP1 is not degraded by autophagy, nor by the ubiquitin-proteasomal system. Mass spectrometry and immunoprecipitation showed that the cell division cycle protein cdt2 (Cdt2), the substrate recognition subunit of the E3 ligase complex associated with cancer, cullin-RING ubiquitin ligase complex 4 (CRL4), is a novel interactor of VMP1 and is involved in VMP1 ubiquitination. VMP1 ubiquitination decreases under the CRL inhibitor MLN4924 and increases with Cdt2 overexpression. Moreover, VMP1 recruitment and autophagosome formation is significantly affected by CRL inhibition. Our results indicate that ubiquitination is a novel post-translational modification of VMP1 during autophagy in human tumor cells. VMP1 ubiquitination may be of clinical relevance in tumor-cell-therapy resistance.

Ultrastructural insights into pathogen clearance by autophagy.

Autophagy defends cells against proliferation of bacteria such as Salmonella in the cytosol. After escape from a damaged Salmonella-containing vacuole (SCV) exposing luminal glycans that bind to Galectin-8, the host cell ubiquitination machinery deposits a dense layer of ubiquitin around the cytosolic bacteria. The nature and spatial distribution of this ubiquitin coat in relation to other autophagy-related membranes are unknown. Using transmission electron microscopy, we determined the exact localisation of ubiquitin, the ruptured SCV membrane and phagophores around cytosolic Salmonella. Ubiquitin was not predominantly present on the Salmonella surface, but enriched on the fragmented SCV. Cytosolic bacteria without SCVs were less efficiently targeted by phagophores. Single bacteria were contained in single phagophores but multiple bacteria could be within large autophagic vacuoles reaching 30 µm in circumference. These large phagophores followed the contour of the engulfed bacteria, they were frequently in close association with endoplasmic reticulum membranes and, within them, remnants of the SCV were seen associated with each engulfed particle. Our data suggest that the Salmonella SCV has a major role in the formation of autophagic phagophores and highlight evolutionary conserved parallel mechanisms between xenophagy and mitophagy with the fragmented SCV and the damaged outer mitochondrial membrane serving similar functions.

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.

The dynamics of mitochondrial autophagy at the initiation stage.

The pathway of mitochondrial-specific autophagy (mitophagy, defined here as the specific elimination of mitochondria following distinct mitochondrial injuries or developmental/metabolic alterations) is important in health and disease. This review will be focussed on the earliest steps of the pathway concerning the mechanisms and requirements for initiating autophagosome formation on a mitochondrial target. More specifically, and in view of the fact that we understand the basic mechanism of non-selective autophagy and are beginning to reshape this knowledge towards the pathways of selective autophagy, two aspects of mitophagy will be covered: (i) How does a machinery normally working in association with the endoplasmic reticulum (ER) to make an autophagosome can also do so at a site distinct from the ER such as on the surface of the targeted cargo? and (ii) how does the machinery deal with cargo of multiple sizes?

Targeting of early endosomes by autophagy facilitates EGFR recycling and signalling.

Despite recently uncovered connections between autophagy and the endocytic pathway, the role of autophagy in regulating endosomal function remains incompletely understood. Here, we find that the ablation of autophagy-essential players disrupts EGF-induced endocytic trafficking of EGFR. Cells lacking ATG7 or ATG16L1 exhibit increased levels of phosphatidylinositol-3-phosphate (PI(3)P), a key determinant of early endosome maturation. Increased PI(3)P levels are associated with an accumulation of EEA1-positive endosomes where EGFR trafficking is stalled. Aberrant early endosomes are recognised by the autophagy machinery in a TBK1- and Gal8-dependent manner and are delivered to LAMP2-positive lysosomes. Preventing this homeostatic regulation of early endosomes by autophagy reduces EGFR recycling to the plasma membrane and compromises downstream signalling and cell survival. Our findings uncover a novel role for the autophagy machinery in maintaining early endosome function and growth factor sensing.

Alpha-synuclein fibrils recruit TBK1 and OPTN to lysosomal damage sites and induce autophagy in microglial cells.

Autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, but the exact mechanisms and causal connections are not clear and most previous work was done in neurons and not in microglial cells. Here, we report that exogenous fibrillary, but not monomeric, alpha-synuclein (AS, also known as SNCA) induces autophagy in microglial cells. We extensively studied the dynamics of this response using both live-cell imaging and correlative light-electron microscopy (CLEM), and found that it correlates with lysosomal damage and is characterised by the recruitment of the selective autophagy-associated proteins TANK-binding kinase 1 (TBK1) and optineurin (OPTN) to ubiquitylated lysosomes. In addition, we observed that LC3 (MAP1LC3B) recruitment to damaged lysosomes was dependent on TBK1 activity. In these fibrillar AS-treated cells, autophagy inhibition impairs mitochondrial function and leads to microglial cell death. Our results suggest that microglial autophagy is induced in response to lysosomal damage caused by persistent accumulation of AS fibrils. Importantly, triggering of the autophagic response appears to be an attempt at lysosomal quality control and not for engulfment of fibrillar AS.This article has an associated First Person interview with the first author of the paper.

ERES: sites for autophagosome biogenesis and maturation?

Autophagosomes are the hallmark of autophagy, but despite their central role in this degradative pathway that involves vesicle transport to lysosomes or vacuoles, the mechanism underlying their biogenesis still remains largely unknown. Our current concepts about autophagosome biogenesis are based on models suggesting that a small autonomous cisterna grows into an autophagosome through expansion at its extremities. Recent findings have revealed that endoplasmic reticulum (ER) exit sites (ERES), specialized ER regions where proteins are sorted into the secretory system, are key players in the formation of autophagosomes. Owing to the morphological connection of nascent autophagosomes with the ER, this has raised several questions that challenge our current perception of autophagosome biogenesis, such as are ERES the compartments where autophagosome formation takes place? What is the functional relevance of this connection? Are these compartments providing essential molecules for the generation of autophagosomes and/or are they structural platforms where these vesicles emerge? In this Hypothesis, we discuss recent data that have implicated the ERES in autophagosome biogenesis and we propose two models to describe the possible role of this compartment at different steps in the process of autophagosome biogenesis. This article is part of a Focus on Autophagosome biogenesis. For further reading, please see related articles: 'Membrane dynamics in autophagosome biogenesis' by Sven R. Carlsson and Anne Simonsen (J. Cell Sci. 128, 193-205) and 'WIPI proteins: essential PtdIns3P effectors at the nascent autophagosome' by Tassula Proikas-Cezanne et al. (J. Cell Sci. 128, 207-217).

Live-cell imaging for the assessment of the dynamics of autophagosome formation: focus on early steps.

Autophagy is a cytosolic degradative pathway, which through a series of complicated membrane rearrangements leads to the formation of a unique double membrane vesicle, the autophagosome. The use of fluorescent proteins has allowed visualizing the autophagosome formation in live cells and in real time, almost 40 years after electron microscopy studies observed these structures for the first time. In the last decade, live-cell imaging has been extensively used to study the dynamics of autophagosome formation in cultured mammalian cells. Hereby we will discuss how the live-cell imaging studies have tried to settle the debate about the origin of the autophagosome membrane and how they have described the way different autophagy proteins coordinate in space and time in order to drive autophagosome formation.

Dynamic association of the ULK1 complex with omegasomes during autophagy induction.

Induction of autophagy requires the ULK1 protein kinase complex and the Vps34 lipid kinase complex. PtdIns3P synthesised by Vps34 accumulates in omegasomes, membrane extensions of the ER within which some autophagosomes form. The ULK1 complex is thought to target autophagosomes independently of PtdIns3P, and its functional relationship to omegasomes is unclear. Here we show that the ULK1 complex colocalises with omegasomes in a PtdIns3P-dependent way. Live-cell imaging of Atg13 (a ULK1 complex component), omegasomes and LC3 establishes and annotates for the first time a complete sequence of steps leading to autophagosome formation, as follows. Upon starvation, the ULK1 complex forms puncta associated with the ER and sporadically with mitochondria. If PtdIns3P is available, these puncta become omegasomes. Subsequently, the ULK1 complex exits omegasomes and autophagosomes bud off. If PtdIns3P is unavailable, ULK1 puncta are greatly reduced in number and duration. Atg13 contains a region with affinity for acidic phospholipids, required for translocation to punctate structures and autophagy progression.

Dynamics of autophagosome formation: a pulse and a sequence of waves.

Autophagosomes form in eukaryotic cells in response to starvation or to other stress conditions brought about by the unwanted presence in the cytosol of pathogens, damaged organelles or aggregated protein assemblies. The uniqueness of autophagosomes is that they form de novo and that they are the only double-membraned vesicles known in cells, having arisen from flat membrane sheets which have expanded and self-closed. The various steps describing their formation as well as most of the protein and lipid components involved have been identified. Furthermore, the hierarchical relationships among the components are well documented, and the mechanistic rationale for some of these hierarchies has been revealed. In the present review, we try to provide a current view of the process of autophagosome formation in mammalian cells, emphasizing along the way gaps in our knowledge that need additional work.

Proteostasis in health and disease: a conversation with Professor Rick Morimoto.

Professor Richard (Rick) Morimoto is the Bill and Gayle Cook Professor of Biology and Director of the Rice Institute for Biomedical Research at Northwestern University. He has made foundational contributions to our understanding of how cells respond to various stresses, and the role played in those responses by chaperones. Working across a variety of experimental models, from C. elegans to human neuronal cells, he has identified a number of important molecular components that sense and respond to stress, and he has dissected how stress alters cellular and organismal physiology. Together with colleagues, Professor Morimoto has coined the term "proteostasis" to signify the homeostatic control of protein expression and function, and in recent years he has been one of the leaders of a consortium trying to understand proteostasis in healthy and disease states. I took the opportunity to talk with Professor Morimoto about proteostasis in general, the aims of the consortium, and how autophagy is playing an important role in their research effort.

International consensus guidelines for the definition, detection, and interpretation of autophagy-dependent ferroptosis.

Macroautophagy/autophagy is a complex degradation process with a dual role in cell death that is influenced by the cell types that are involved and the stressors they are exposed to. Ferroptosis is an iron-dependent oxidative form of cell death characterized by unrestricted lipid peroxidation in the context of heterogeneous and plastic mechanisms. Recent studies have shed light on the involvement of specific types of autophagy (e.g. ferritinophagy, lipophagy, and clockophagy) in initiating or executing ferroptotic cell death through the selective degradation of anti-injury proteins or organelles. Conversely, other forms of selective autophagy (e.g. reticulophagy and lysophagy) enhance the cellular defense against ferroptotic damage. Dysregulated autophagy-dependent ferroptosis has implications for a diverse range of pathological conditions. This review aims to present an updated definition of autophagy-dependent ferroptosis, discuss influential substrates and receptors, outline experimental methods, and propose guidelines for interpreting the results.Abbreviation: 3-MA:3-methyladenine; 4HNE: 4-hydroxynonenal; ACD: accidentalcell death; ADF: autophagy-dependentferroptosis; ARE: antioxidant response element; BH2:dihydrobiopterin; BH4: tetrahydrobiopterin; BMDMs: bonemarrow-derived macrophages; CMA: chaperone-mediated autophagy; CQ:chloroquine; DAMPs: danger/damage-associated molecular patterns; EMT,epithelial-mesenchymal transition; EPR: electronparamagnetic resonance; ER, endoplasmic reticulum; FRET: Försterresonance energy transfer; GFP: green fluorescent protein;GSH: glutathione;IF: immunofluorescence; IHC: immunohistochemistry; IOP, intraocularpressure; IRI: ischemia-reperfusion injury; LAA: linoleamide alkyne;MDA: malondialdehyde; PGSK: Phen Green™ SK;RCD: regulatedcell death; PUFAs: polyunsaturated fatty acids; RFP: red fluorescentprotein;ROS: reactive oxygen species; TBA: thiobarbituricacid; TBARS: thiobarbituric acid reactive substances; TEM:transmission electron microscopy.

Autophagy in the commercial arena: a conversation with Dr. Leon Murphy, Chief Scientific Officer at Casma therapeutics.

As a maturing field that continues to provide fundamental insights into cell physiology, autophagy is also beginning to attract considerable interest from the biotechnology/pharmaceutical sector. For this Editor's corner, I thought it would be both useful and interesting to talk with somebody who has spent a lot of time in the commercial sphere, working on autophagy and related processes. I was fortunate that Dr. Leon Murphy, Chief Scientific Officer at Casma therapeutics, was willing and able to answer my questions. In addition to his insights on the commercial interest for autophagy, Dr. Murphy also shared his personal experience on the scientific life working in large and small pharmaceutical companies.

Evaluating Autophagy Levels in Two Different Pancreatic Cell Models Using LC3 Immunofluorescence.

Autophagy is a specialized catabolic process that selectively degrades cytoplasmic components, including proteins and damaged organelles. Autophagy allows cells to physiologically respond to stress stimuli and, thus, maintain cellular homeostasis. Cancer cells might modulate their autophagy levels to adapt to adverse conditions such as hypoxia, nutrient deficiency, or damage caused by chemotherapy. Ductal pancreatic adenocarcinoma is one of the deadliest types of cancer. Pancreatic cancer cells have high autophagy activity due to the transcriptional upregulation and post-translational activation of autophagy proteins. Here, the PANC-1 cell line was used as a model of pancreatic human cancer cells, and the AR42J pancreatic acinar cell line was used as a physiological model of highly differentiated mammalian cells. This study used the immunofluorescence of microtubule-associated protein light chain 3 (LC3) as an indicator of the status of autophagy activation. LC3 is an autophagy protein that, in basal conditions, shows a diffuse pattern of distribution in the cytoplasm (known as LC3-I in this condition). Autophagy induction triggers the conjugation of LC3 to phosphatidylethanolamine on the surface of newly formed autophagosomes to form LC3-II, a membrane-bound protein that aids in the formation and expansion of autophagosomes. To quantify the number of labeled autophagic structures, the open-source software FIJI was utilized with the aid of the "3D Objects Counter" tool. The measure of the autophagic levels both in physiological conditions and in cancer cells allows us to study the modulation of autophagy under diverse conditions such as hypoxia, chemotherapy treatment, or the knockdown of certain proteins.

Modulation of local PtdIns3P levels by the PI phosphatase MTMR3 regulates constitutive autophagy.

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant-negative inactive mutant of Myotubularin-related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy-related PtdIns3P-binding proteins, GFP-DFCP1 and GFP-WIPI-1alpha (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock-down of MTMR3 increased autophagosome formation, and overexpression of wild-type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3-kinase and PI 3-phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.