Autophagosome closure requires membrane scission

During the intracellular process of macroautophagy (hereafter autophagy), a membrane-bound organelle, the autophagosome, is generated de novo. The remodeling of the autophagic membrane during the life cycle of the organelle is a complex multistep process and involves several changes in the topology of the autophagic membrane. Here, we focus on the final step of autophagosome formation, the closure of the phagophore, during which the inner and outer autophagic membranes become separate entities. We argue that this topological membrane transformation is a membrane scission event. Surprisingly, not a single recent review describes this substep as membrane scission (or membrane fission). In contrast, a number of publications imply that membrane fusion is involved. We discuss the potential sources for misinterpretation and recommend to consistent use of the unambiguous term “membrane scission.”     

Topological analysis of the fusion process between cellular and subcellular compartments

The study presents an application of the theory of homeomorphic transformations of topological manifolds and the operation of the connected sum of manifolds for topological analysis of membrane transformations during the fusion process between cellular and subcellular compartments. The biological cell and the subcellular structures in the form of vesicles are modelled by an arrangement of two concentric spheres corresponding to the inner and outer layer of the membrane bounding the vesicles. The analysis shows eight succeeding topological stages of membrane transformations during the fusion process and these stages are characterized. It is concluded that there is a vectorial translocation of lipid molecules from the outer layers of the membranes before the fusion process to the internal layer of the membrane bounding the vesicle after the fusion process and there is no lipid translocation in the reverse direction.     

The HOPS/Class C Vps Complex Tethers High‐Curvature Membranes via a Direct Protein–Membrane Interaction

Membrane tethering is a physical association of two membranes before their fusion. Many membrane tethering factors have been identified, but the interactions that mediate inter‐membrane associations remain largely a matter of conjecture. Previously, we reported that the homotypic fusion and protein sorting/Class C vacuolar protein sorting (HOPS/Class C Vps) complex, which has two binding sites for the yeast vacuolar Rab GTPase Ypt7p, can tether two low‐curvature liposomes when both membranes bear Ypt7p. Here, we show that HOPS tethers highly curved liposomes to Ypt7p‐bearing low‐curvature liposomes even when the high‐curvature liposomes are protein‐free. Phosphorylation of the curvature‐sensing amphipathic lipid‐packing sensor (ALPS) motif from the Vps41p HOPS subunit abrogates tethering of high‐curvature liposomes. A HOPS complex without its Vps39p subunit, which contains one of the Ypt7p binding sites in HOPS, lacks tethering activity, though it binds high‐curvature liposomes and Ypt7p‐bearing low‐curvature liposomes. Thus, HOPS tethers highly curved membranes via a direct protein–membrane interaction. Such high‐curvature membranes are found at the sites of vacuole tethering and fusion. There, vacuole membranes bend sharply, generating large areas of vacuole‐vacuole contact. We propose that HOPS localizes via the Vps41p ALPS motif to these high‐curvature regions. There, HOPS binds via Vps39p to Ypt7p in an apposed vacuole membrane.      

Lysosomal Membrane Proteins and Their Central Role in Physiology

The lysosomal membrane was thought for a long time to primarily act as a physical barrier separating the luminal acidic milieu from the cytoplasmic environment. Meanwhile, it has been realized that unique lysosomal membranes play essential roles in a number of cellular events ranging from phagocytosis, autophagy, cell death, virus infection to membrane repair. This review provides an overview about the most interesting emerging functions of lysosomal membrane proteins and how they contribute to health and disease. Their importance is exemplified by their role in acidification, transport of metabolites and ions across the membrane, intracellular transport of hydrolases and the regulation of membrane fusion events. Studies in patient cells, non‐mammalian model organisms and knockout mice contributed to our understanding of how the different lysosomal membrane proteins affect cellular homeostasis, developmental processes as well as tissue functions. Because these proteins are central for the biogenesis of this compartment they are also considered as attractive targets to modulate the lysosomal machinery in cases where impaired lysosomal degradation leads to cellular pathologies. We are only beginning to understand the complex composition and function of these proteins which are tightly linked to processes occurring throughout the endocytic and biosynthetic pathways.      

Lipids and membrane-associated proteins in autophagy

Autophagy is essential for the maintenance of cellular homeostasis and its dysfunction has been linked to various diseases.Autophagy is a membrane driven process and tightly regulated by membrane-associated proteins.Here,we summarized membrane lipid compo-sition,and membrane-associated proteins relevant to autophagy from a spatiotemporal perspective.In par-ticular,we focused on three important membrane remodeling processes in autophagy,lipid transfer for phagophore elongation,membrane scission for phago-phore closure,and autophagosome-lysosome mem-brane fusion.We discussed the significance of the discoveries in this field and possible avenues to follow for future studies.Finally,we summarized the mem-brane-associated biochemical techniques and assays used to study membrane properties,with a discussion of their applications in autophagy.     

Plasma membrane helps autophagosomes grow

The membrane origin of autophagosomes has long been a mystery and it may involve multiple sources. In this punctum, we discuss our recent finding that the plasma membrane contributes to the formation of pre-autophagic structures via clathrin-mediated endocytosis. Our study suggests that Atg16L1 interacts with clathrin heavy-chain/AP2 and is also localized on vesicles (positive for clathrin or cholera toxin B) close to the plasma membrane. Live-cell imaging studies revealed that the plasma membrane contributes to Atg16L1-positive structures and that this process and autophagosome formation are impaired by knockdowns of genes regulating clathrin-mediated endocytosis.Key words: autophagy, plasma membrane, endocytosis, phagophore, originWhere do autophagosomes get their membrane from? Although the field of autophagy has grown tremendously since its discovery a few decades ago, the origin(s) of the membranes that contribute to autophagosome biogenesis has been a mystery among autophagy researchers until recently. Mammalian autophagosomes are formed randomly throughout the cytoplasm via a process that involves elongation and fusion of phagophores to form double-membraned autophagosomes. This process involves two ubiquitin-like conjugation systems: conjugation of Atg12 to Atg5 that later forms a macromolecular complex with Atg16L1, and conjugation of phosphatidylethanolamine (PE) with Atg8/LC3-I. The Atg12-Atg5-Atg16L1 complex is targeted to the preautophagic structures, which then acquire Atg8. Atg12-Atg5-Atg16L1 dissociates from completed autophagosomes, while LC3-PE (LC3-II) is associated both with pre-autophagic structures and completed autophagosomes.Some recent studies have explored the contribution of membranes from different organelles supporting the general idea that autophagosomes derive membranes from pre-existing organelles. It is quite possible that there may be multiple membrane sources involved. A few groups have revisited the hypothesis that the endoplasmic reticulum (ER) may be one of the membrane donors. High-resolution 2D electron microscopy (EM) and 3D EM-tomography studies have revealed connections between the ER and the growing autophagosomes. Whether the ER contributes to general autophagy or a specific form of autophagy, reticulophagy, remains to be determined. In addition, it has not been shown if ER membrane is required for autophagosome formation. Recently another study has reported that autophagosomes receive lipids from the outer mitochondrial membrane, but only under starvation conditions, again fueling the multiple-membrane source hypothesis.We have now found evidence for plasma membrane contribution to pre-autophagic structures via endocytosis. Unlike the previous studies, which have focused on LC3- positive structures, we looked specifically at the Atg5-, Atg12- and Atg16-positive pre-autophagic structures, an idea that stemmed from our finding that clathrin heavy-chain immunoprecipitates with Atg16L1. We think that this interaction is partly mediated by the adaptor protein AP2, since knockdown of AP2 decreases the clathrin heavy-chain-Atg16L1 interaction. Immunogold EM also shows clathrin localization on Atg16L1-labeled vesicles close to the plasma membrane.These findings led us to test whether knockdown of proteins involved in clathrin-mediated endocytosis affected Atg16L1-positive pre-autophagic structures. Indeed, knockdown of key proteins in the clathrin-mediated endocytic pathway results in a decrease in the formation of Atg16L1-positive structures both under basal or autophagy-induced conditions (starvation or trehalose treatment). This correlates with a decrease in the number of LC3-labeled autophagosomes. When we directly analyzed vesicle fusion by livecell microscopy, we observed that vesicles endocytosed from the plasma membrane fuse to the Atg16L1-positive vesicles close to the plasma membrane. This was confirmed by immuno-EM when we found cholera toxin B-labeling (used to label plasma membrane that is subsequently internalized by endocytosis) on Atg16L1-vesicles. We noticed that overexpression of an Atg16L1 mutant that does not bind clathrin heavy-chain does not form Atg16L1-vesicular structures in the way we see with wild-type Atg16L1, suggesting that the binding of Atg16L1 to AP2/clathrin is required for the subsequent formation of the Atg16L1 vesicles.When we blocked endocytic vesicle scission (using both genetic and chemical inhibitors) we found that Atg16L1 strongly immunoprecipitates with clathrin-heavy chain probably due to the accumulation of clathrin-Atg16L1 structures at the plasma membrane that failed to pinch off. This was strongly supported by our fluorescence microscopy and immuno-EM studies that showed what we predicted—accumulation of Atg16L1 at the plasma membrane. This suggests that Atg16L1 in a complex with AP2/clathrin is targeted to the plasma membrane and subsequently internalized as Atg16L1-positive structures. Thus, our data strongly suggest that plasma membrane contributes to early autophagic precursors that subsequently mature to form phagophores (Fig. 1).Open in a separate windowFigure 1Plasma membrane contributes to the formation of early autophagic precursors. Previous studies show that delivery of fully formed autophagosomes to lysosomes requires fusion of such autophagosomes with early or late endosomes to form amphisomes, which are Atg16L1-negative, LC3-positive and are also positive for endosomal markers. We show that blocking clathrin-mediated endocytosis inhibits formation of Atg16L1-positive structures that mature to form phagophores and later autophagosomes. These Atg16L1-vesicles are positive for other early autophagosomal markers like Atg5 and Atg12, but are negative for early endosomal markers like EEA1, suggesting that they are high up in the autophagosome biogenesis cascade. Inhibition of dynamin with Dynsasore or the use of a dominant negative K44A mutant blocks scission and results in Atg16L1 accumulation on the plasma membrane, suggesting that endosomal scission is critical for this process.Although previous studies suggest that completely formed autophagosomes need to fuse with early or late endosomes in order for subsequent autophagosomelysosome fusion to occur, they did not look at the formation of pre-autophagic structures. Our study shows that active endocytosis is required both for the formation of autophagosomes, when very early endocytic intermediates immediately pinching off the plasma membrane (not early endosomes) fuse with Atg16L1-positive structures to form phagophores, and also for maturation of autophagosomes when early or late endosomes fuse with Atg16L1-negative but LC3-positive autophagosomes to form amphisomes. Since blocking clathrin-mediated endocytosis does not completely abrogate autophagosome formation, we believe that other endocytic pathways may have a similar role. Depending on the cell type or the physiological conditions, the contributions from the different endocytic pathways may vary accordingly. It will be interesting to know if the endocytic pathway continuously delivers membrane for early steps in autophagy as the preautophagic structures grow and mature to form autophagosomes, deriving membrane from other sources.     

Mechanical analyses of morphological and topological transformation of liposomes

Liposomes are micro-compartments made of lipid bilayer membranes possessing the characteristics quite similar to those of biological membranes. To form artificial cell-like structures, we made liposomes that contained subunit proteins of cytoskeletons: tubulin or actin. Spherical liposomes were transformed into bipolar or cell-like shapes by mechanical forces generated by the polymerization of encapsulated subunits of microtubules. On the other hand, disk- or dumbbell-shaped liposomes were developed by the polymerization of encapsulated actin. Dynamic processes of morphological transformations of liposomes were visualized by high intensity dark-field light microscopy. Topological changes, such as fusion and division of membrane vesicles, play an essential role in cellular activities. To investigate the mechanism of these processes, we visualized the liposomes undergoing topological transformation in real time. A variety of novel topological transformations were found, including the opening-up of liposomes and the direct expulsion of inner vesicles.     

Effects of sodium chloride on the plasma membrane of halotolerant Dunaliella primolecta: an electron spin resonance study

Electron spin resonance spectroscopy was used to monitor the in vivo microviscosity of the plasma membrane and lipid extracts of the salt tolerant alga, Dunaliella primolecta. The fluidity of the plasma membrane decreased as the algae were adapted to and suspended in higher sodium chloride concentrations [2–24% (w/v)]. Both biochemical modification and a physical interaction between Na⁺ and lipids were implicated.When the microviscosity of the plasma membrane and that of lipid extracts were determined as a function of temperature, two or three lipid phase transformations were observed. There were always transformations at 9–14° C and 39–43° C. These were interpreted as the onset and completion of the lipid phase transition of at least a major lipid component of the membrane, possibly the entire membrane. These transformation temperatures were independent of the salt concentration to which the algae were adapted or suspended. This suggests that D. primolecta exists with some of its membrane in the solid-fluid mixed lipid state. With a NaCl concentration of 8% (w/v) or greater in the growth medium, a third transformation occurred around 20–22° C. It was the result of a lipid-lipid interaction and was not related to adaptation.Abbreviations ESR electron spin resonance spectroscopy - 2 T hyperfine splitting - S order parameter - 5-DS or 5-doxyl-stearate 2-(3-carboxylpropyl)-4,4-dimethyl-2-tridecyl-3-oxazolidinyloxyl     

Osh6 links yeast vacuolar functions to lifespan extension and TOR

Comment on: Gebre S, et al. Cell Cycle 2012; 11:2176-88.Almost all organisms age–the aging process is both genetically determined and can be modified by the environment. Lifespan extension by dietary restriction (DR) is observed in evolutionarily distant species from yeast to mammals. Not only are the phenomena of aging and DR conserved, but at least some mechanisms and genes are evolutionarily conserved, which may pave the way to manipulate human aging.¹ For example, TOR (target of rapamycin) mediates aging and, when suppressed, triggers anti-aging processes in many species. Moreover, identifying genes that modulate the potential for cell division is of great interest, given that changes in the number of times that cells divide have been associated with longevity manipulations in mammals (including DR).²Sterols are hydrophobic molecules present in all cellular organisms. For instance, cholesterol is an essential structural component of cellular membranes of mammals and several of its derivates have additional hormonal and signaling functions. Oxysterols are oxygenated derivates of cholesterol. Oxysterol-binding protein (OSBP)-related protein (ORP) family members are present in numerous copies from yeast to man, suggesting that this protein family has fundamental functions in eukaryotes. OSBP and ORPs regulate lipid metabolism, vesicle transport and various signaling pathways³ and may specifically mediate lipid exchange at membrane contact sites.The lifespan-extending effect of DR has often been shown to be mediated by specific genes and to be accompanied by discrete changes in gene expression as well as metabolic reprogramming. Both lipid metabolism and cellular recycling activities have been demonstrated to be essential for lifespan extension in numerous species. For example, DR suppresses sterol synthesis from yeast to mammals,⁴ while it induces some form of autophagy, a mighty housekeeping mechanism utilizing lysosomes within its power to recycle various kinds of molecules and cellular structures. Vacuoles, the yeast equivalent of mammalian lysosomes, are highly dynamic organelles that fuse and divide in response to environmental or intrinsic cues. Mutants with defects in vacuolar fusion (such as ypt7Δ, nyv1Δ, vac8Δ, or erg6Δ) are either short-lived or do not appear to respond to DR.⁵While mammals have 12 OSBPs, the yeast genome encodes seven oxysterol-binding protein sequence homologs (OSH). Deletion of any OSH gene alone does not impact on vacuolar morphology, yet deletion of all results in highly fragmented vacuoles, a sign of defective vacuole fusion. Gebre et al. now show that overexpression of OSH family member OSH6 in yeast can complement the vacuole fusion defect of nyv1Δ but not erg6Δ or vac8Δ. Thus, Osh6 mediates vacuolar fusion, which depends on ergosterol (Erg6), and the protein anchor Vac8. In contrast, overexpression of another OSH-family member, OSH5, exacerbated fragmentation and decreased lifespan in wild-type cells. It is interesting to note that OSH5 expression progressively increases with age, and Osh6 overexpression blocked this age-dependent change in OSH5 levels. Also, elevated Osh6 maintains the enrichment of Vac8 in microdomains of vacuolar membranes with advancing age, which is required for vacuole fusion. Intriguingly, exactly at the age when the longevity protein Sir2 declines, Osh6 protein levels also decline.⁶Furthermore, Gebre et al. showed that P_ERG6-OSH6 (ERG6 promoter driving OSH6 overexpression) dramatically extends the lifespan of wild-type and nyv1Δ mutants. tor1Δ mutants are also long-lived, though not so long as P_ERG6-OSH6. Surprisingly, P_ERG6-OSH6 tor1Δ double mutant had a very short lifespan. P_ERG6-OSH6 mutants were more sensitive to TOR inhibitors, indicating that TOR is less active in this strain.⁶ OSH6 overexpression downregulates total cellular sterol levels, just like DR. Osh6 binds PI3P and PI(3,5)P2 which are vacuole-specific lipids.⁷ As such, Osh6 might promote vacuole fusion by regulating the transports and/or distribution of sterols to the vacuolar membranes. But where are the sterols coming from? Numerous overexpression mutants with effects in vacuolar morphology are involved in endocytosis.⁸ Similarly, Osh6’s coiled-coil domain interacts with Vps4, which is located in endosomes. TOR complex 1 (TORC1) also sits on endosomes as well as on vacuoles and actively catalyzes vacuolar scission.⁹ Osh6 may therefore (1) transport sterols from late endosomes to the vacuolar membrane (Fig. 1), which increases the homototypic fusion ability of vacuoles, and (2) averaging the lipids between late endosome and vacuoles promotes also late-endosome-to-vacuole fusion.Open in a separate windowFigure 1. Putative mechanism of the lifespan extension conferred by Osh6 overexpression. TORC1 promotes vacuolar scission and therefore fragments vacuoles. In contrast, Osh6 enhances vacuolar fusion and might be doing this by transporting sterols from the endosomes to the vacuolar membrane. Improved vacuolar morphology then promotes autophagy. Thus, Osh6 appears to counteract TORC1 activity.Overall, Gebre and colleagues link the vacuole to lifespan extension, perhaps via TOR, and reveal that vacuole fusion is both necessary and sufficient for lifespan extension.     

Secretory membranes of the lactating mammary gland

Summary The lactating mammary gland is one of the most highly differentiated and metabolically active organs in the body. Membranes of the lactating mammary cell have important roles in transmitting from one membrane to another of hormonal information and in milk secretion, which is the final event. During milk secretion, the projection of the surface membrane into the alveolar lumen by enveloping intracellular lipid droplets with the apical plasma membrane is one of the most remarkable aspects of biological membrane action throughout nature.This review focuses on current knowledge about membranes in the lactating mammary gland. (1) Advances in the isolation and properties of membranes, especially the plasma membrane and Golgi-derived secretory vesicles, concerned with milk secretion from the lactating mammary gland are described. (2) Milk serum components are secreted by fusing the membranes of secretory vesicles that condense milk secretions with the plasma membrane in the apical regions. This occurs through the formation of a tubular-shaped projection and vesicular depression in a ball-and-socket configuration, as well as by simple fusion. (3) Intracellular lipid droplets are directly extruded from the mammary epithelial cells by progressive envelopment of the plasma membranes in the apical regions. (4) The balance between the surface volume lost in enveloping lipid droplets and that provided by fusion of the secretory vesicle and other vesicles with the apical plasma membrane is discussed. (5) The membrane surrounding a milk fat globule, which is referred to as the milk fat globule membrane (MFGM), is composed of at least the coating membrane of an intracellular lipid droplet, of the apical plasma membrane and secretory vesicle membrane, and of a coat material. Consequently, MFGM is molecularly different from the plasma membrane in composition. (6) MFGM of bovine milk is structurally composed of an inner coating membrane and outer plasma membrane just after segregation. These two membranes are fused and reorganized through a process of vesiculation and fragmentation to stabilize the fat globules. Hypothetical structural models for MFGM from bovine milk fat globules just after secretion and after rearrangement are proposed.Abbrevations MFGM milk fat globule membrane - HEPES N-2-hydroxylpiperazine-N-2-ethanesulfonic acid - INT 2-(p-indophenyl)-3-(p-nitrophenyl)-5-phenyltetrazolium - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - Sph sphingomyelin - PC phosphatidyl choline - PE phosphatidyl ethanolamine - PS phosphatidyl serine - PI phosphatidyl inositol - PAS periodic acid-Schiff reagent - CB Coomassie brilliant blue R-250 Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Phospholipid Association Is Essential for Dynamin-related Protein Mgm1 to Function in Mitochondrial Membrane Fusion

Mgm1, the yeast ortholog of mammalian OPA1, is a key component in mitochondrial membrane fusion and is required for maintaining mitochondrial dynamics and morphology. We showed recently that the purified short isoform of Mgm1 (s-Mgm1) possesses GTPase activity, self-assembles into low order oligomers, and interacts specifically with negatively charged phospholipids (Meglei, G., and McQuibban, G. A. (2009) Biochemistry 48, 1774–1784). Here, we demonstrate that s-Mgm1 binds to a mixture of phospholipids characteristic of the mitochondrial inner membrane. Binding to physiologically representative lipids results in ∼50-fold stimulation of s-Mgm1 GTPase activity. s-Mgm1 point mutants that are defective in oligomerization and lipid binding do not exhibit such stimulation and do not function in vivo. Electron microscopy and lipid turbidity assays demonstrate that s-Mgm1 promotes liposome interaction. Furthermore, s-Mgm1 assembles onto liposomes as oligomeric rings with 3-fold symmetry. The projection map of negatively stained s-Mgm1 shows six monomers, consistent with two stacked trimers. Taken together, our data identify a lipid-binding domain in Mgm1, and the structural analysis suggests a model of how Mgm1 promotes the fusion of opposing mitochondrial inner membranes.Mitochondrial dynamics have been implicated in neurodegenerative diseases such as dominant optic atrophy and Parkinson disease (1, 2). Mitochondrial morphology is regulated by balanced membrane fusion and fission reactions that are orchestrated by members of the highly conserved dynamin-related protein family (3). Dynamin-related proteins are large GTPases that can self-assemble and promote membrane remodeling (4, 5). We have shown previously that the dynamin-related protein Mgm1 has GTPase activity, self-assembles into low order oligomers, and binds to negatively charged phospholipids (6). Mgm1 exists as two isoforms in the mitochondria; l-Mgm1² is anchored to the IM via a transmembrane domain, and s-Mgm1 is peripherally associated with the IM and also found in the intermembrane space. s-Mgm1 results from the regulated cleavage by the mitochondrial rhomboid protease (7, 8). It was shown recently that both isoforms are essential but have distinct roles in mitochondrial membrane fusion whereby only s-Mgm1 requires its GTPase activity (9). It is proposed that l-Mgm1 serves as a receptor for s-Mgm1 to mediate fusion of opposing membranes upon GTP hydrolysis. Here, we provide molecular data indicating that lipid binding of s-Mgm1 is required for proper membrane fusion. Furthermore, structural analysis of s-Mgm1 assembled onto liposomes suggests a model whereby stacked trimers of s-Mgm1 on opposing membranes would facilitate fusion.     

Common Properties of Fusion Peptides from Diverse Systems

Although membrane fusion occurs ubiquitously and continuously in alleukaroytic cells, little is known about the mechanism that governs lipidbilayer fusion associated with any intracellular fusion reactions. Recentstudies of the fusion of enveloped viruses with host cell membranes havehelped to define the fusion process. The identification and characterizationof key proteins involved in fusion reactions have mainly driven recent advancesin our understanding of membrane fusion. The most important denominator amongthe fusion proteins is the fusion peptide. In this review, work done in thelast few years on the molecular mechanism of viral membrane fusion will behighlighted, focusing in particular on the role of the fusion peptide and themodification of the lipid bilayer structure. Much of what is known regardingthe molecular mechanism of viral membrane fusion has been gained using liposomesas model systems in which the molecular components of the membrane and the environmentare strictly controlled. Many amphilphilic peptides have a high affinity forlipid bilayers, but only a few sequences are able to induce membrane fusion. Thepresence of -helical structure in at least part of the fusion peptideis strongly correlated with activity whereas, -structure tends to beless prevalent, associated with non-native experimental conditions, and morerelated to vesicle aggregation than fusion. The specific angle of insertionof the peptides into the membrane plane is also found to be an importantcharacteristic for the fusion process. A shallow penetration, extending onlyto the central aliphatic core region, is likely responsible for the destabilization ofthe lipids required for coalescence of the apposing membranes and fusion.     

Regulation and Function of Autophagy during Cell Survival and Cell Death

Autophagy is an important catabolic process that delivers cytoplasmic material to the lysosome for degradation. Autophagy promotes cell survival by elimination of damaged organelles and proteins aggregates, as well as by facilitating bioenergetic homeostasis. Although autophagy has been considered a cell survival mechanism, recent studies have shown that autophagy can promote cell death. The core mechanisms that control autophagy are conserved between yeast and humans, but animals also possess genes that regulate autophagy that are not present in yeast. These regulatory differences may be explained by the need to control autophagy in a cell context-specific manner in multicellular animals, such as during cell survival and cell death. Autophagy was thought to be a bulk cytoplasmic degradation mechanism, but recent studies have shown that specific cargo is recruited for degradation. This suggests the possibility that either cell survival or death may be regulated by selective autophagic clearance of cytoplasmic material. Here we summarize the mechanisms that regulate autophagy and how they may contribute to cell survival and death.Autophagy (self-eating) is an evolutionarily conserved catabolic process that is used to deliver cytoplasmic materials, including organelles and proteins, to the lysosome for degradation. Three types of autophagy have been described, including macroautophagy, microautophagy, and chaperone-mediated autophagy (Mizushima and Komatsu 2011). Although macroautophagy involves the fusion of the double membrane autophagosome and lysosomes, microautophagy is poorly understood and thought to involve direct uptake of material by the lysosome via a process that appears similar to pinocytosis. By contrast, chaperone-mediated autophagy is a biochemical mechanism to import proteins into the lysosome; it depends on a signature sequence and interaction with protein chaperones. Here we will focus on macroautophagy (hereafter called autophagy) because of our knowledge of this process in cell survival and cell death.Autophagy was likely first observed when electron microscopy was used to observe “dense bodies” containing mitochondria in mouse kidneys (Clark 1957). Five years later, it was reported that rat hepatocytes exposed to glucagon possessed membrane-bound vesicles that were rich in mitochondria and endoplasmic reticulum (Ashford and Porter 1962). Almost simultaneously, it was shown that these membrane-bound vesicles contained lysosomal hydrolases (Novikoff and Essner 1962). In 1965 de Duve coined the term “autophagy” (Klionsky 2008).The delivery of cytoplasmic material to the lysosome by autophagy involves membrane formation and fusion events (Fig. 1). First an isolation membrane, also known as a phagophore, must be initiated from a membrane source known as the phagophore assembly site (PAS). de Duve suggested that the smooth endoplasmic reticulum could be the source of autophagosome membrane (de Duve and Wattiaux 1966), and subsequent studies have supported this possibility (Dunn 1990; Axe et al. 2008). Although controversial, mitochondria and plasma membrane could also supply membranes for the formation of the autophagosomes under different conditions (Hailey et al. 2010; Ravikumar et al. 2010). The elongating isolation membrane surrounds cargo that is ultimately enclosed in the double membrane autophagosome. Once the autophagosome is formed, it fuses with lysosomes (known as the vacuole in yeasts and plants) to form autolysosomes in which the cargo is degraded by lysosomal hydrolases. At this stage lysosomes must reform so that subsequent autophagy may occur (Yu et al. 2010).Open in a separate windowFigure 1.Macroautophagy (autophagy) delivers cytoplasmic cargo to lysosomes for degradation, and involves membrane formation and fusion. The isolation membrane is initiated from a membrane source known as the from the phagophore assembly site (PAS). The isolation membrane surrounds cargo, including organelles and proteins, to form a double membrane autophagosome. Autophagosomes fuse with lysosomes to form autolysosomes in which the cargo is degraded by lysosomal hydrolases.     

Regulation of the high‐affinity choline transporter activity and trafficking by its association with cholesterol‐rich lipid rafts

The sodium‐coupled, hemicholinium‐3‐sensitive, high‐affinity choline transporter (CHT) is responsible for transport of choline into cholinergic nerve terminals from the synaptic cleft following acetylcholine release and hydrolysis. In this study, we address regulation of CHT function by plasma membrane cholesterol. We show for the first time that CHT is concentrated in cholesterol‐rich lipid rafts in both SH‐SY5Y cells and nerve terminals from mouse forebrain. Treatment of SH‐SY5Y cells expressing rat CHT with filipin, methyl‐β‐cyclodextrin (MβC) or cholesterol oxidase significantly decreased choline uptake. In contrast, CHT activity was increased by addition of cholesterol to membranes using cholesterol‐saturated MβC. Kinetic analysis of binding of [³H]hemicholinium‐3 to CHT revealed that reducing membrane cholesterol with MβC decreased both the apparent binding affinity (K_D) and maximum number of binding sites (B_max); this was confirmed by decreased plasma membrane CHT protein in lipid rafts in cell surface protein biotinylation assays. Finally, the loss of cell surface CHT associated with lipid raft disruption was not because of changes in CHT internalization. In summary, we provide evidence that CHT association with cholesterol‐rich rafts is critical for transporter function and localization. Alterations in plasma membrane cholesterol cholinergic nerve terminals could diminish cholinergic transmission by reducing choline availability for acetylcholine synthesis.

     

Topological model of the division process of cellular and subcellular compartments

The application of the theory of homeomorphic transformations of topological manifolds and the operation of the connected sum of manifolds for a formation of a topological model of membrane transformations during the division process of cellular and subcellular compartments, has been shown. The biological cell and the subcellular structures in the form of vesicles are modelled by an arrangement of two concentric spheres corresponding to the inner and outer layer of the membrane bounding the vesicle. The analysis shows eight succeeding topological stages of membrane transformations during the division process and these stages are characterised. It is concluded that there is a vectorial translocation of lipid molecules from the inner layer of the membrane bounding the vesicle before the division process to the outer layer of the membranes after the division process and there is no lipid translocation from the outer layer to the inner layers during the division process.     

Atg27 Tyrosine Sorting Motif is Important for Its Trafficking and Atg9 Localization

During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9‐positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP‐3, which mediates direct transport from the trans‐Golgi to the vacuole. The four C‐terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP‐3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.      

A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis

Autophagy is a well-defined catabolic mechanism whereby cytoplasmic materials are engulfed into a structure termed the autophagosome. In plants, little is known about the underlying mechanism of autophagosome formation. In this study, we report that SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a Bin-Amphiphysin-Rvs domain–containing protein, translocates to the phagophore assembly site/preautophagosome structure (PAS) upon autophagy induction and actively participates in the membrane deformation process. Using the SH3P2–green fluorescent protein fusion as a reporter, we found that the PAS develops from a cup-shaped isolation membranes or endoplasmic reticulum–derived omegasome-like structures. Using an inducible RNA interference (RNAi) approach, we show that RNAi knockdown of SH3P2 is developmentally lethal and significantly suppresses autophagosome formation. An in vitro membrane/lipid binding assay demonstrates that SH3P2 is a membrane-associated protein that binds to phosphatidylinositol 3-phosphate. SH3P2 may facilitate membrane expansion or maturation in coordination with the phosphatidylinositol 3-kinase (PI3K) complex during autophagy, as SH3P2 promotes PI3K foci formation, while PI3K inhibitor treatment inhibits SH3P2 from translocating to autophagosomes. Further interaction analysis shows that SH3P2 associates with the PI3K complex and interacts with ATG8s in Arabidopsis thaliana, whereby SH3P2 may mediate autophagy. Thus, our study has identified SH3P2 as a novel regulator of autophagy and provided a conserved model for autophagosome biogenesis in Arabidopsis.     

SNARE proteins in membrane trafficking

SNAREs are the core machinery mediating membrane fusion. In this review, we provide an update on the recent progress on SNAREs regulating membrane fusion events, especially the more detailed fusion processes dissected by well‐developed biophysical methods and in vitro single molecule analysis approaches. We also briefly summarize the relevant research from Chinese laboratories and highlight the significant contributions on our understanding of SNARE‐mediated membrane trafficking from scientists in China.      

Modeling degranulation with liposomes: Effect of lipid composition on membrane fusion

Degranulation involves the regulated fusion of granule membrane with plasma membrane. To study the role of lipid composition in degranulation, large unilamellar vesicles (LUVs) of increasing complexity in lipid compositions were constructed and tested for Ca²⁺-mediated lipid and contents mixing. Lipid-mixing rates of LUVs composed of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were strongly decreased by the addition of either phosphatidylcholine (PC) or sphingomyelin (SM), while phosphatidylinositol (PI) had little effect. Complex LUVs of PCPESMPIPS (2427201613, designed to emulate neutrophil plasma membranes) also showed very low rates of both lipid mixing and contents mixing. The addition of cholesterol significantly lowered the Ca²⁺ threshold for contents mixing and increased the maximum rates of both lipid and contents mixing in a dose-dependent manner. Membrane remodeling, which occurs in neutrophil plasma membranes upon stimulation, was simulated by incorporating low levels of phosphatidic acid (PA) or a diacylglycerol (DAG) into complex LUVs containing 50% cholesterol. The addition of PA both lowered the Ca²⁺ threshold and increased the rate of contents mixing in a dose-dependent manner, while the DAG had no significant effect. The interaction of dissimilar LUVs was also examined. Contents-mixing rates of LUVs of two different cholesterol contents were intermediate between the rates observed for the LUVs of identical composition. Thus, cholesterol needed to be present in only one fusing partner to enhance fusion. However, for PA to stimulate fusion, it had to be present in both sets of LUVs. These results suggest that the rate of degranulation may be increased by a rise in the cholesterol level of either the inner face of the plasma membrane or the outer face of the granule membrane. Further, the production of PA can promote fusion, and hence degranulation, whereas the subsequent conversion of PA to DAG may reverse this promotional effect.Abbreviations ANTS 8-aminonaphthalene-1,3,6-trisulfonic acid - DiC₈ 1,2-dioctanoyl-sn-glycerol - DPX p-xylene-bis-pyridinium bromide - LUV large unilamellar vesicle - PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - R18 octadecyl rhodamine - SM sphingomyelin     

Distinction between breast cancer cell subtypes using third harmonic generation microscopy

Third Harmonic Generation (THG) microscopy as a non‐invasive, label free imaging methodology, allows linkage of lipid profiles with various breast cancer cells. The collected THG signal arise mostly from the lipid droplets and the membrane lipid bilayer. Quantification of THG signal can accurately distinguish HER2‐positive cells. Further analysis using Fourier transform infrared (FTIR) spectra reveals cancer‐specific profiles, correlating lipid raft‐corresponding spectra to THG signal, associating thus THG to chemical information.

THG imaging of a cancer cell.