Multifarious roles of lipid droplets in autophagy - Target, product, and what else?

Lipid droplets (LDs) are not an inert storage of excessive lipids, but play various roles in cellular lipid metabolism. Autophagy involves several mechanisms for the degradation of cellular components, and is related to many aspects of lipid metabolism. LD and autophagic membranes often distribute in proximity, but their relationship is complex. LDs can be degraded by autophagy, but LDs are also generated as a result of autophagy or support the execution of autophagy. Moreover, several proteins crucial for autophagy were shown to affect different aspects of LD formation. This article aims to categorize this multifaceted and seemingly entangled LD-autophagy relationship and to discuss unresolved issues.

TRAPPC11 functions in autophagy by recruiting ATG2B-WIPI4/WDR45 to preautophagosomal membranes.

TRAPPC11 has been implicated in membrane traffic and lipid-linked oligosaccharide synthesis, and mutations in TRAPPC11 result in neuromuscular and developmental phenotypes. Here, we show that TRAPPC11 has a role upstream of autophagosome formation during macroautophagy. Upon TRAPPC11 depletion, LC3-positive membranes accumulate prior to, and fail to be cleared during, starvation. A proximity biotinylation assay identified ATG2B and its binding partner WIPI4/WDR45 as TRAPPC11 interactors. TRAPPC11 depletion phenocopies that of ATG2 and WIPI4 and recruitment of both proteins to membranes is defective upon reduction of TRAPPC11. We find that a portion of TRAPPC11 and other TRAPP III proteins localize to isolation membranes. Fibroblasts from a patient with TRAPPC11 mutations failed to recruit ATG2B-WIPI4, suggesting that this interaction is physiologically relevant. Since ATG2B-WIPI4 is required for isolation membrane expansion, our study suggests that TRAPPC11 plays a role in this process. We propose a model whereby the TRAPP III complex participates in the formation and expansion of the isolation membrane at several steps.

A "Lamellar structure" contributes to autophagosome biogenesis and mitophagy in zebrafish hepatocytes.

Despite many studies being conducted over the past few decades, the origin of autophagosomal membranes remains unclear. The present study aimed to uncover the formation process of autophagosomal membranes in hepatocytes of zebrafish (Danio rerio), a model organism in medical science. Immunohistochemistry of zebrafish hepatocytes indicated that light chain 3-like protein 2 (LC3-II) is highly active in some hepatocytes, but poorly expressed in others. Under transmission electron microscopy, the amount of autophagosomes (APs) varied in different hepatocytes. When the endoplasmic reticulum (ER) is dispersed in the cytoplasm, few isolation membranes (IMs) and APs were observed. Subsequently, when the ER assembles into a particular "lamellar structure" (LS), IMs arise from it and extend to enwrap the mitochondria. With further aggregation of the ER, the LS developed into an over twenty-layered structure, and mitophagy was more obvious in the hepatocytes and cavities appeared in mitochondria. Finally, most ERs were assembled into several LSs. At this point, mitophagy was most active in the hepatocytes. Thereafter, glycogen and lipid droplet increased gradually, while the LS degenerated and ER scatter increased. Then, the glycogen and lipid droplets dominated the hepatocellular cytoplasm. After suppressing the formation of autophagosomes using 3-Methyladenine (3-MA), the LS could no longer be visualized in the hepatocellular cytoplasm, and mitophagy decreased drastically. Taken together, the results suggested that this LS in the hepatocytes of zebrafish, might be another manifestation of a pre-autophagosomal structure in zebrafish liver, analogous to the omegasome in yeast or the ER-IM complex in mammalian cell lines. Furthermore, selective mitophagy and consequent cyclic utilization of its products were probably relevant to dynamic cycle of the hepatocellular cytoplasm.

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).

Monitoring and Measuring Autophagy.

Autophagy is a cytoplasmic degradation system, which is important for starvation adaptation and cellular quality control. Recent advances in understanding autophagy highlight its importance under physiological and pathological conditions. However, methods for monitoring autophagic activity are complicated and the results are sometimes misinterpreted. Here, we review the methods used to identify autophagic structures, and to measure autophagic flux in cultured cells and animals. We will also describe the existing autophagy reporter mice that are useful for autophagy studies and drug testing. Lastly, we will consider the attempts to monitor autophagy in samples derived from humans.

Visualization of Atg3 during autophagosome formation in Saccharomyces cerevisiae.

Macroautophagy (autophagy) is a highly conserved cellular recycling process involved in degradation of eukaryotic cellular components. During autophagy, macromolecules and organelles are sequestered into the double-membrane autophagosome and degraded in the vacuole/lysosome. Autophagy-related 8 (Atg8), a core Atg protein essential for autophagosome formation, is a marker of several autophagic structures: the pre-autophagosomal structure (PAS), isolation membrane (IM), and autophagosome. Atg8 is conjugated to phosphatidylethanolamine (PE) through a ubiquitin-like conjugation system to yield Atg8-PE; this reaction is called Atg8 lipidation. Although the mechanisms of Atg8 lipidation have been well studied in vitro, the cellular locale of Atg8 lipidation remains enigmatic. Atg3 is an E2-like enzyme that catalyzes the conjugation reaction between Atg8 and PE. Therefore, we hypothesized that the localization of Atg3 would provide insights about the site of the lipidation reaction. To explore this idea, we constructed functional GFP-tagged Atg3 (Atg3-GFP) by inserting the GFP portion immediately after the handle region of Atg3. During autophagy, Atg3-GFP transiently formed a single dot per cell on the vacuolar membrane. This Atg3-GFP dot colocalized with 2× mCherry-tagged Atg8, demonstrating that Atg3 is localized to autophagic structures. Furthermore, we found that Atg3-GFP is localized to the IM by fine-localization analysis. The localization of Atg3 suggests that Atg3 plays an important role in autophagosome formation at the IM.

Fine mapping of autophagy-related proteins during autophagosome formation in Saccharomyces cerevisiae.

Autophagy is a bulk degradation system mediated by biogenesis of autophagosomes under starvation conditions. In Saccharomyces cerevisiae, a membrane sac called the isolation membrane (IM) is generated from the pre-autophagosomal structure (PAS); ultimately, the IM expands to become a mature autophagosome. Eighteen autophagy-related (Atg) proteins are engaged in autophagosome formation at the PAS. However, the cup-shaped IM was visualized just as a dot by fluorescence microscopy, posing a challenge to further understanding the detailed functions of Atg proteins during IM expansion. In this study, we visualized expanding IMs as cup-shaped structures using fluorescence microscopy by enlarging a selective cargo of autophagosomes, and finely mapped the localizations of Atg proteins. The PAS scaffold proteins (Atg13 and Atg17) and phosphatidylinositol 3-kinase complex I were localized to a position at the junction between the IM and the vacuolar membrane, termed the vacuole-IM contact site (VICS). By contrast, Atg1, Atg8 and the Atg16-Atg12-Atg5 complex were present at both the VICS and the cup-shaped IM. We designate this localization the 'IM' pattern. The Atg2-Atg18 complex and Atg9 localized to the edge of the IM, appearing as two or three dots, in close proximity to the endoplasmic reticulum exit sites. Thus, we designate these dots as the 'IM edge' pattern. These data suggest that Atg proteins play individual roles at spatially distinct locations during IM expansion. These findings will facilitate detailed investigations of the function of each Atg protein during autophagosome formation.

Phospholipid Supply for Autophagosome Biogenesis.

Autophagy is a cellular degradation pathway where double-membrane autophagosomes form de novo to engulf cytoplasmic material destined for lysosomal degradation. This process requires regulated membrane remodeling, beginning with the initial autophagosomal precursor and progressing to its elongation and maturation into a fully enclosed, fusion-capable vesicle. While the core protein machinery involved in autophagosome formation has been extensively studied over the past two decades, the role of phospholipids in this process has only recently been studied. This review focuses on the phospholipid composition of the phagophore membrane and the mechanisms that supply lipids to expand this unique organelle.

The COPII coat protein SEC24D is required for autophagosome closure in mammals.

Macroautophagy involves the encapsulation of cellular components within double-membrane autophagosomes for subsequent degradation in vacuoles or lysosomes. Coat protein complex II (COPII) vesicles serve as a membrane source for autophagosome formation. However, the specific role of SEC24D, an isoform of the COPII coat protein SEC24, in the macroautophagy pathway remains unclear. In this study, we demonstrate that SEC24D is indispensable for macroautophagy and important for autophagosome closure. Depletion of SEC24D leads to the accumulation of unsealed isolation membranes. Furthermore, under conditions of starvation, SEC24D interacts with casein kinase1 delta (CK1δ), a member of the casein kinase 1 family, and autophagy-related 9A (ATG9A). Collectively, our findings unveil the indispensable role of SEC24D in starvation-induced autophagy in mammalian cells.

Detection of autophagy-related structures in fruiting bodies of edible mushroom, Pleurotus ostreatus.

Autophagy is involved in various fungal morphogenetic processes. However, there are limited reports regarding the role of autophagy in mushroom fruiting body formation. The purpose of this study was to reveal the autophagy-related structures in mushroom-forming fungi. The edible mushroom Pleurotus ostreatus was used in this study. Transmission electron microscopy revealed double-membrane bounded structures containing cytoplasmic components in the fruiting bodies of this fungus. Some of these double-membrane structures were observed to interact with the vacuoles. Additionally, curved flat cisternae of various lengths were detected in the cytoplasm. The shape, size and thickness of the limiting membrane of the double-membrane structures and the flat cisternae corresponded well with those of the autophagosomes and the isolation membranes, respectively. Regarding autophagosome formation, a membrane-bound specific zone was detected near the isolation membrane, which appeared to expand along the novel membrane. This is the first detailed report showing autophagy-related structures in P. ostreatus and provides a possible model for autophagosome formation in these filamentous fungi.

Morphology of Phagophore Precursors by Correlative Light-Electron Microscopy.

Autophagosome biogenesis occurs in the transient subdomains of the endoplasmic reticulum that are called omegasomes, which, in fluorescence microscopy, appear as small puncta, which then grow in diameter and finally shrink and disappear once the autophagosome is complete. Autophagosomes are formed by phagophores, which are membrane cisterns that elongate and close to form the double membrane that limits autophagosomes. Earlier electron-microscopy studies showed that, during elongation, phagophores are lined by the endoplasmic reticulum on both sides. However, the morphology of the very early phagophore precursors has not been studied at the electron-microscopy level. We used live-cell imaging of cells expressing markers of phagophore biogenesis combined with correlative light-electron microscopy, as well as electron tomography of ATG2A/B-double-deficient cells, to reveal the high-resolution morphology of phagophore precursors in three dimensions. We showed that phagophores are closed or nearly closed into autophagosomes already at the stage when the omegasome diameter is still large. We further observed that phagophore precursors emerge next to the endoplasmic reticulum as bud-like highly curved membrane cisterns with a small opening to the cytosol. The phagophore precursors then open to form more flat cisterns that elongate and curve to form the classically described crescent-shaped phagophores.

Key Regulators of Autophagosome Closure.

Autophagy is an evolutionarily conserved pathway, in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and then transported into lysosomes or vacuoles for degradation. Over 40 conserved autophagy-related (ATG) genes define the core machinery for the five processes of autophagy: initiation, nucleation, elongation, closure, and fusion. In this review, we focus on one of the least well-characterized events in autophagy, namely the closure of the isolation membrane/phagophore to form the sealed autophagosome. This process is tightly regulated by ESCRT machinery, ATG proteins, Rab GTPase and Rab-related proteins, SNAREs, sphingomyelin, and calcium. We summarize recent progress in the regulation of autophagosome closure and discuss the key questions remaining to be addressed.

Should I bend or should I grow: the mechanisms of droplet-mediated autophagosome formation.

Phase-separated droplets with liquid-like properties can be degraded by macroautophagy/autophagy, but the mechanism underlying this degradation is poorly understood. We have recently derived a physical model to investigate the interaction between autophagic membranes and such droplets, uncovering that intrinsic wetting interactions underlie droplet-membrane contacts. We found that the competition between droplet surface tension and the increasing tendency of growing membrane sheets to bend determines whether a droplet is completely engulfed or isolated in a piecemeal fashion, a process we term fluidophagy. Intriguingly, we found that another critical parameter of droplet-membrane interactions, the spontaneous curvature of the membrane, determines whether the droplet is degraded by autophagy or - counterintuitively - serves as a platform from which autophagic membranes expand into the cytosol. We also discovered that the interaction of membrane-associated LC3 with the LC3-interacting region (LIR) found in the autophagic cargo receptor protein SQSTM1/p62 and many other autophagy-related proteins influences the preferred bending directionality of forming autophagosomes in living cells. Our study provides a physical account of how droplet-membrane wetting underpins the structure and fate of forming autophagosomes.

Membrane supply and remodeling during autophagosome biogenesis.

The de novo generation of double-membrane autophagosomes is the hallmark of autophagy. The initial membranous precursor cisterna, the phagophore, is very likely generated by the fusion of vesicles and acts as a membrane seed for the subsequent expansion into an autophagosome. This latter step requires a massive convoy of lipids into the phagophore. In this review, we present recent advances in our understanding of the intracellular membrane sources and lipid delivery mechanisms, which principally rely on vesicular transport and membrane contact sites that contribute to autophagosome biogenesis. In this context, we discuss lipid biosynthesis and lipid remodeling events that play a crucial role in both phagophore nucleation and expansion.

Casein Kinase 1 Family Member CK1δ/Hrr25 Is Required for Autophagosome Completion.

Autophagy starts with the initiation and nucleation of isolation membranes, which further expand and seal to form autophagosomes. The regulation of isolation membrane closure remains poorly understood. CK1δ is a member of the casein kinase I family of serine/threonine specific kinases. Although CK1δ is reported to be involved in various cellular processes, its role in autophagy is unknown. Here, we show that CK1δ regulates the progression of autophagy from the formation of isolation membranes to autophagosome closure, and is essential for macroautophagy. CK1δ depletion results in impaired autophagy flux and the accumulation of unsealed isolation membranes. The association of LC3 with ATG9A, ATG14L, and ATG16L1 was found to be increased in CK1δ-depleted cells. The role of CK1δ in autophagosome completion appears to be conserved between yeasts and humans. Our data reveal a key role for CK1δ/Hrr25 in autophagosome completion.

Autophagosomes are formed at a distinct cellular structure.

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes, which deliver bulk cytoplasmic material to the lytic compartment of the cell for degradation. Autophagosome formation is initiated by assembly and recruitment of the core autophagy machinery to distinct cellular sites, referred to as phagophore assembly sites (PAS) in yeast or autophagosome formation sites in other organisms. A large number of autophagy proteins involved in the formation of autophagosomes has been identified; however, how the individual components of the PAS are assembled and how they function to generate autophagosomes remains a fundamental question. Here, we highlight recent studies that provide molecular insights into PAS organization and the role of the endoplasmic reticulum and the vacuole in autophagosome formation.

Atg2: A novel phospholipid transfer protein that mediates de novo autophagosome biogenesis.

The degradation of cytoplasmic components via autophagy is crucial for intracellular homeostasis. In the process of autophagy, a newly synthesized isolation membrane (IM) is developed to sequester degradation targets and eventually the IM seals, forming an autophagosome. One of the most poorly understood autophagy-related proteins is Atg2, which is known to localize to a contact site between the edge of the expanding IM and the exit site of the endoplasmic reticulum (ERES). Recent advances in structural and biochemical analyses have been applied to Atg2 and have revealed it to be a novel multifunctional protein that tethers membranes and transfers phospholipids between them. Considering that Atg2 is essential for the expansion of the IM that requires phospholipids as building blocks, it is suggested that Atg2 transfers phospholipids from the ERES to the IM during the process of autophagosome formation, suggesting that lipid transfer proteins can mediate de novo organelle biogenesis.

The autophagic membrane tether ATG2A transfers lipids between membranes.

An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid transfer protein. ATG2A can extract lipids from membrane vesicles and unload them to other vesicles. Lipid transfer by ATG2A is more efficient between tethered vesicles than between untethered vesicles. The PI3P effectors WIPI4 and WIPI1 associate ATG2A stably to PI3P-containing vesicles, thereby facilitating ATG2A-mediated tethering and lipid transfer between PI3P-containing vesicles and PI3P-free vesicles. Based on these results, we propose that ATG2-mediated transfer of lipids from the ER to the phagophore enables phagophore expansion.

Improved Electron Microscopy Fixation Methods for Tracking Autophagy-Associated Membranes in Cultured Mammalian Cells.

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/OsO4 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.

The ER Contact Proteins VAPA/B Interact with Multiple Autophagy Proteins to Modulate Autophagosome Biogenesis.

The endoplasmic reticulum (ER) is the site of biogenesis of the isolation membrane (IM, autophagosome precursor) and forms extensive contacts with IMs during their expansion into double-membrane autophagosomes. Little is known about the molecular mechanism underlying the formation and/or maintenance of the ER/IM contact. The integral ER proteins VAPA and VAPB (VAPs) participate in establishing ER contacts with multiple membranes by interacting with different tethers. Here, we demonstrate that VAPs also modulate ER/IM contact formation. Depletion of VAPs impairs progression of IMs into autophagosomes. Upon autophagy induction, VAPs are recruited to autophagosome formation sites on the ER, a process mediated by their interactions with FIP200 and PI(3)P. VAPs directly interact with FIP200 and ULK1 through their conserved FFAT motifs and stabilize the ULK1/FIP200 complex at the autophagosome formation sites on the ER. The formation of ULK1 puncta is significantly reduced by VAPA/B depletion. VAPs also interact with WIPI2 and enhance the formation of the WIPI2/FIP200 ER/IM tethering complex. Depletion of VMP1, which increases the ER/IM contact, greatly elevates the interaction of VAPs with these autophagy proteins. The VAPB P56S mutation, which is associated with amyotrophic lateral sclerosis, reduces the ULK1/FIP200 interaction and impairs autophagy at an early step, similar to the effect seen in VAPA/B-depleted cells. Our study reveals that VAPs directly interact with multiple ATG proteins, thereby contributing to ER/IM contact formation for autophagosome biogenesis.