Evidence for the involvement of GD3 ganglioside in autophagosome formation and maturation

Sphingolipids are structural lipid components of cell membranes, including membrane of organelles, such as mitochondria or endoplasmic reticulum, playing a role in signal transduction as well as in the transport and intermixing of cell membranes. Sphingolipid microdomains, also called lipid rafts, participate in several metabolic and catabolic cell processes, including apoptosis. However, the defined role of lipid rafts in the autophagic flux is still unknown. In the present study we analyzed the role of gangliosides, a class of sphingolipids, in autolysosome morphogenesis in human and murine primary fibroblasts by means of biochemical and analytical cytology methods. Upon induction of autophagy, by using amino acid deprivation as well as tunicamycin, we found that GD3 ganglioside, considered as a paradigmatic raft constituent, actively contributed to the biogenesis and maturation of autophagic vacuoles. In particular, fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses revealed that this ganglioside interacts with phosphatidylinositol 3-phosphate and can be detected in immature autophagosomes in association with LC3-II as well as in autolysosomes associated with LAMP1. Hence, it appears as a structural component of autophagic flux. Accordingly, we found that autophagy was significantly impaired by knocking down ST8SIA1/GD3 synthase (ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 1) or by altering sphingolipid metabolism with fumonisin B1. Interestingly, exogenous administration of GD3 ganglioside was capable of reactivating the autophagic process inhibited by fumonisin B1. Altogether, these results suggest that gangliosides, via their molecular interaction with autophagy-associated molecules, could be recruited to autophagosome and contribute to morphogenic remodeling, e.g., to changes of membrane curvature and fluidity, finally leading to mature autolysosome formation.     

Evidence for the involvement of GD3 ganglioside in autophagosome formation and maturation

Sphingolipids are structural lipid components of cell membranes, including membrane of organelles, such as mitochondria or endoplasmic reticulum, playing a role in signal transduction as well as in the transport and intermixing of cell membranes. Sphingolipid microdomains, also called lipid rafts, participate in several metabolic and catabolic cell processes, including apoptosis. However, the defined role of lipid rafts in the autophagic flux is still unknown. In the present study we analyzed the role of gangliosides, a class of sphingolipids, in autolysosome morphogenesis in human and murine primary fibroblasts by means of biochemical and analytical cytology methods. Upon induction of autophagy, by using amino acid deprivation as well as tunicamycin, we found that GD3 ganglioside, considered as a paradigmatic raft constituent, actively contributed to the biogenesis and maturation of autophagic vacuoles. In particular, fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses revealed that this ganglioside interacts with phosphatidylinositol 3-phosphate and can be detected in immature autophagosomes in association with LC3-II as well as in autolysosomes associated with LAMP1. Hence, it appears as a structural component of autophagic flux. Accordingly, we found that autophagy was significantly impaired by knocking down ST8SIA1/GD3 synthase (ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase 1) or by altering sphingolipid metabolism with fumonisin B1. Interestingly, exogenous administration of GD3 ganglioside was capable of reactivating the autophagic process inhibited by fumonisin B1. Altogether, these results suggest that gangliosides, via their molecular interaction with autophagy-associated molecules, could be recruited to autophagosome and contribute to morphogenic remodeling, e.g., to changes of membrane curvature and fluidity, finally leading to mature autolysosome formation.     

Lipid droplets regulate autophagosome biogenesis

The source of the autophagic membrane and the regulation of autophagosome biogenesis are still elusive open issues in the field of autophagy. In our recent study of the role of lipid droplets (LDs) and their constituents in autophagy, we provided evidence that both the biogenesis of LDs and its lipolysis by specific lipases are important for autophagosome biogenesis. Our study sheds new light on the source of the autophagic membrane and suggests that a flow of membranes from the endoplasmic reticulum (ER) to LDs, and from LDs to the ER, is essential for autophagosome biogenesis.     

Autophagosome formation is initiated at phosphatidylinositol synthase‐enriched ER subdomains

The autophagosome, a double‐membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy‐initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy‐initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy‐initiation complex localizes to phosphatidylinositol synthase (PIS)‐enriched ER subdomains. Then, the initiation complex translocates to the ATG9A‐positive autophagosome precursors in a PI3P‐dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI‐specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy‐initiation complex, the PIS‐enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation.     

Autophagy requires endoplasmic reticulum targeting of the PI3-kinase complex via Atg14L

Autophagy is a catabolic process that allows cells to digest their cytoplasmic constituents via autophagosome formation and lysosomal degradation. Recently, an autophagy-specific phosphatidylinositol 3-kinase (PI3-kinase) complex, consisting of hVps34, hVps15, Beclin-1, and Atg14L, has been identified in mammalian cells. Atg14L is specific to this autophagy complex and localizes to the endoplasmic reticulum (ER). Knockdown of Atg14L leads to the disappearance of the DFCP1-positive omegasome, which is a membranous structure closely associated with both the autophagosome and the ER. A point mutation in Atg14L resulting in defective ER localization was also defective in the induction of autophagy. The addition of the ER-targeting motif of DFCP1 to this mutant fully complemented the autophagic defect in Atg14L knockout embryonic stem cells. Thus, Atg14L recruits a subset of class III PI3-kinase to the ER, where otherwise phosphatidylinositol 3-phosphate (PI3P) is essentially absent. The Atg14L-dependent appearance of PI3P in the ER makes this organelle the platform for autophagosome formation.     

New functions of mitochondria associated membranes in cellular signaling

In all eukaryotic cells, the endoplasmic reticulum (ER) and the mitochondria establish a tight interplay, which is structurally and functionally modulated through a proteinaceous tether formed at specific subdomains of the ER membrane, designated mitochondria-associated membranes or MAMs. The tethering function of the MAMs allows the regulation of lipid synthesis and rapid transmission of calcium (Ca^2 +) signals between the ER and mitochondria, which is crucial to shape intracellular Ca^2 + signaling and regulate mitochondrial bioenergetics. Research on the molecular characterization and function of MAMs has boomed in the last few years and the list of signaling and structural proteins dynamically associated with the ER–mitochondria contact sites in physiological and pathological conditions, is rapidly increasing along with the realization of an unprecedented complexity underlying the functional role of MAMs. Besides their established role as a signaling hub for Ca^2 + and lipid transfer between ER and mitochondria, MAMs have been recently shown to regulate mitochondrial shape and motility, energy metabolism and redox status and to be central to the modulation of various key processes like ER stress, autophagy and inflammasome signaling. In this review we will discuss some emerging cell-autonomous and cell non-autonomous roles of the MAMs in mammalian cells and their relevance for important human diseases. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Trafficking of cholera toxin-ganglioside GM1 complex into Golgi and induction of toxicity depend on actin cytoskeleton

Intestinal epithelial lipid rafts contain ganglioside G_M1 that is the receptor for cholera toxin (CT). The ganglioside binds CT at the plasma membrane (PM) and carries the toxin through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER). In the ER, a portion of the toxin unfolds and translocates to the cytosol to activate adenylyl cyclase. Activation of the cyclase leads to an increase in intracellular cAMP, which results in apical chloride secretion. Here, we find that an intact actin cytoskeleton is necessary for the efficient transport of CT to the Golgi and for subsequent activation of adenylyl cyclase. CT bound to G_M1 on the cell membrane fractionates with a heterogeneous population of lipid rafts, a portion of which is enriched in actin and other cytoskeletal proteins. In this actin-rich fraction of lipid rafts, CT and actin colocalize on the same membrane microdomains, suggesting a possible functional association. Depolymerization or stabilization of actin filaments interferes with transport of CT from the PM to the Golgi and reduces the levels of cAMP generated in the cytosol. Depletion of membrane cholesterol, which also inhibits CT trafficking to the TGN, causes displacement of actin from the lipid rafts while CT remains stably raft associated. On the basis of these observations, we propose that the CT-G_M1 complex is associated with the actin cytoskeleton via the lipid rafts and that the actin cytoskeleton plays a role in trafficking of CT from the PM to the Golgi/ER and the subsequent activation of adenylyl cyclase. membrane microdomains; membrane lipids; bacterial toxins; endocytosis; intestinal mucosa     

Ferroptotic agent-induced endoplasmic reticulum stress response plays a pivotal role in the autophagic process outcome

Ferroptosis has been reported as a unique form of cell death. However, in recent years, researchers have increasingly challenged the uniqueness of ferroptosis compared to other types of cell death. In this study, we examined whether ferroptosis shares cell death pathways with other types of cell death, especially autophagy, via the autophagic process. Here, we observed that ferroptosis inducers (artesunate [ART] and erastin [ERA]) and autophagy inducers (bortezomib [BOR] and XIE62-1004) led to autophagosome formation via the endoplasmic reticulum (ER) stress response. Unlike XIE62-1004, ART, ERA, and BOR, which affect glutathione production or utilization, induced oxidative stress responses—an increase in the levels of heme oxygenase-1 and lipid peroxidation. Oxidative stress responses were attenuated by deletion of autophagy-related gene-5 or treatment with autophagy inhibitors (bafilomycin and chloroquine). Our studies provide an overview of common death pathways—the ER stress response-associated autophagic process in ferroptosis and autophagy. We also highlight the role played by glutathione redox system in the outcome of the autophagic process.     

Essential Roles of Gangliosides in the Formation and Maintenance of Membrane Microdomains in Brain Tissues

Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported. Here we summarized roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues by comparing complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among several mutant mice of ganglioside synthases. Depending on the defects in ganglioside compositions, corresponding up-regulation of complement-related genes, proliferation of astrocytes and infiltration of microglia were found with gradual severity. Immunoblotting of fractions separated by sucrose density gradient ultracentrifugation revealed that DAF and NCAM having GPI-anchors tended to disappear from the raft fraction with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > WT. The lipid raft markers tended to disperse from the raft fractions with similar intensities. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. All these results indicate that GEM/rafts architecture is destroyed by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions. Implication of inflammation caused by deficiency of gangliosides in various neurodegenerative diseases was discussed.     

Autophagy Induced by Calcium Phosphate Precipitates Involves Endoplasmic Reticulum Membranes in Autophagosome Biogenesis

Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.     

MTOR,PIK3C3, and autophagy: Signaling the beginning from the end

A key point in starvation-induced autophagy occurs at the end of the process, where lysosomes are regenerated from autolysosomes through a pathway termed autophagic lysosome reformation (ALR). ALR occurs when autolysosomal MTOR becomes reactivated by amino acids derived from the autophagic delivery of protein cargo. This activation not only turns off autophagosome formation but also leads to reformation of lysosomes, ready for the next round of autophagy, through a series of events involving autolysosomal tubulation. We have now found that MTOR regulates multiple steps of ALR including direct activation of the PIK3C3-UVRAG lipid kinase complex to enable autolysosomal tubules to break away and regenerate lysosomes.     

Membrane Delivery to the Yeast Autophagosome from the Golgi�CEndosomal System

While many of the proteins required for autophagy have been identified, the source of the membrane of the autophagosome is still unresolved with the endoplasmic reticulum (ER), endosomes, and mitochondria all having been evoked. The integral membrane protein Atg9 is delivered to the autophagosome during starvation and in the related cytoplasm-to-vacuole (Cvt) pathway that occurs constitutively in yeast. We have examined the requirements for delivery of Atg9-containing membrane to the yeast autophagosome. Atg9 does not appear to originate from mitochondria, and Atg9 cannot reach the forming autophagosome directly from the ER or early Golgi. Components of traffic between Golgi and endosomes are known to be required for the Cvt pathway but do not appear required for autophagy in starved cells. However, we find that pairwise combinations of mutations in Golgi-endosomal traffic components apparently only required for the Cvt pathway can cause profound defects in Atg9 delivery and autophagy in starved cells. Thus it appears that membrane that contains Atg9 is delivered to the autophagosome from the Golgi-endosomal system rather than from the ER or mitochondria. This is underestimated by examination of single mutants, providing a possible explanation for discrepancies between yeast and mammalian studies on Atg9 localization and autophagosome formation.     

TMEM41B is a novel regulator of autophagy and lipid mobilization

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well‐characterized autophagy‐related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2‐ and DFCP1‐positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and β‐oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER‐localized regulator of autophagosome biogenesis and lipid mobilization.     

Ca2+ transfer via the ER-mitochondria tethering complex in neuronal cells contribute to cadmium-induced autophagy

Mitochondrial-associated endoplasmic reticulum (ER) membranes (MAMs) play a key role in several physiological functions, including calcium ion (Ca²⁺) transfer and autophagy; however, the molecular mechanism controlling this interaction in cadmium (Cd)-induced neurotoxicity is unknown. This study shows that Cd induces alterations in MAMs and mitochondrial Ca²⁺ levels in PC12 cells and primary neurons. Ablation or silencing of mitofusin 2 (Mfn2) in PC12 cells or primary neurons blocks the colocalization of ER and mitochondria while reducing the efficiency of mitochondrial Ca²⁺ uptake. Moreover, Mfn2 defects reduce interactions or colocalization between GRP75 and VDAC1. Interestingly, the enhancement of autophagic protein levels, colocalization of LC3 and Lamp2, and GFP-LC3 puncta induced by Cd decreased in Mfn2^?/? or Grp75^?/? PC12 cells and Mfn2- or Grp75-silenced primary neurons. Notably, the specific Ca²⁺ uniporter inhibitor RuR blocked both mitochondrial Ca²⁺ uptake and autophagy induced by Cd. Finally, this study proves that the mechanism by which IP3R-Grp75-VDAC1 tethers in MAMs is associated with the regulation of autophagy by Mfn2 and involves their role in mediating mitochondrial Ca²⁺ uptake from ER stores. These results give new evidence into the organelle metabolic process by demonstrating that Ca²⁺ transport between ER-mitochondria is important in autophagosome formation in Cd-induced neurodegeneration.

Graphical abstract

     

Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells

Plasma membrane lipid microdomains have been considered as a sort of 'closed chamber', where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-beta-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.     

ER–plasma membrane contact sites contribute to autophagosome biogenesis by regulation of local PI3P synthesis

The double‐membrane‐bound autophagosome is formed by the closure of a structure called the phagophore, origin of which is still unclear. The endoplasmic reticulum (ER) is clearly implicated in autophagosome biogenesis due to the presence of the omegasome subdomain positive for DFCP1, a phosphatidyl‐inositol‐3‐phosphate (PI3P) binding protein. Contribution of other membrane sources, like the plasma membrane (PM), is still difficult to integrate in a global picture. Here we show that ER–plasma membrane contact sites are mobilized for autophagosome biogenesis, by direct implication of the tethering extended synaptotagmins (E‐Syts) proteins. Imaging data revealed that early autophagic markers are recruited to E‐Syt‐containing domains during autophagy and that inhibition of E‐Syts expression leads to a reduction in autophagosome biogenesis. Furthermore, we demonstrate that E‐Syts are essential for autophagy‐associated PI3P synthesis at the cortical ER membrane via the recruitment of VMP1, the stabilizing ER partner of the PI3KC3 complex. These results highlight the contribution of ER–plasma membrane tethers to autophagosome biogenesis regulation and support the importance of membrane contact sites in autophagy.     

The size of lipid rafts: an atomic force microscopy study of ganglioside GM1 domains in sphingomyelin/DOPC/cholesterol membranes

Atomic force microscopy has been used to study the distribution of ganglioside GM1 in model membranes composed of ternary lipid mixtures that mimic the composition of lipid rafts. The results demonstrate that addition of 1% GM1 to 1:1:1 sphingomyelin/dioleoylphosphatidylcholine/cholesterol monolayers leads to the formation of small ganglioside-rich microdomains (40-100 nm in size) that are localized preferentially in the more ordered sphingomyelin/cholesterol-rich phase. With 5% GM1 some GM1 microdomains are also detected in the dioleoylphosphatidylcholine-rich phase. A similar preferential localization of GM1 in the ordered phase is observed for bilayers with the same ternary lipid mixture in the upper leaflet. The small GM1-rich domains observed in these experiments are similar to the sizes for lipid rafts in natural membranes but considerably smaller than the ordered bilayer domains that have been shown to be enriched in GM1 in recent fluorescence microscopy studies of lipid bilayers. The combined data from a number of studies of model membranes indicate that lateral organization occurs on a variety of length scales and mimics many of the properties of natural membranes.     

Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.     

FKBP8 is a novel molecule that participates in the regulation of the autophagic pathway

Autophagy is a homeostatic process by which misfolded proteins, organelles and cytoplasmic material are engulfed in autophagosomal vesicles and degraded through a lisosomal pathway. FKBP8 is a member of the FK506-binding proteins family (FKBP) usually found in mitochondria and the endoplasmic reticulum. This protein plays a critical role in cell functions such as protein trafficking and folding. In the present report we demonstrate that the depletion of FKBP8 abrogated autophagy activation induced by starvation, whereas the overexpression of this protein triggered the autophagy cascade. We found that FKBP8 co-localizes with ATG14L and BECN1, both members of the VPS34 lipid kinase complex, which regulates the initial steps in the autophagosome formation process. We have also demonstrated that FKBP8 is necessary for VPS34 activity. Our findings indicate that the regulatory function of FKBP8 in the autophagy process depends of its transmembrane domain. Surprisingly, this protein was not found in autophagosomal vesicles, which reinforces the notion that the FKBP8 only participates in the initial steps of the autophagosome formation process. Taken together, our data provide evidence that FKBP8 modulates the early steps of the autophagosome formation event by interacting with the VPS34 lipid kinase complex.SummaryIn this article, the protein FKBP38 is reported to be a novel modulator of the initial steps of the autophagic pathway, specifically in starvation-induced autophagy. FKBP38 interacts with the VPS34 lipid kinase complex, with the transmembrane domain of FKBP38 being critical for its biological function.