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.     

Biogenesis of autophagosomes from the ERGIC membrane system

<正>Introduction Macroautophagy (hereafter referred as autophagy) is a process of cellular self-degradation. In response to nutrient deprivation or other stimuli, a nascent double-membrane autophagosome, encapsulating intracellular materials or damaged organelles, is generated. The autophagosome is transported toward and eventually fuses with the lysosome (or the vacuole in yeast and plant cells),     

Yeast autophagosomes: de novo formation of a membrane structure

Autophagy - the degradation of organelles and cytoplasmic material - occurs through dynamic rearrangements of cellular membrane structures. Following the induction of autophagy, newly formed autophagosomes transfer cytosolic materials to the lysosome or vacuole for degradation. The autophagosome is an organelle destined for degradation, suggesting that the membrane structure is formed de novo many times. The autophagosome is formed through the nucleation, assembly and elongation of membrane structures. The concerted action of several Apg/Aut/Cvt proteins around a characteristic subcellular structure (the preautophagosomal structure) is the key to understanding this novel type of membrane-formation process.     

Plasma membrane microdomains

Several lines of evidence indicate that the lipids in the plasma membrane of animal cells are inhomogeneously distributed, and that various types of specialized lipid domains play an important role in many biological processes. The characteristics of these domains, such as size, composition and dynamics, are currently under active investigation. It appears that there are many different types of membrane domains in the plasma membrane, and perhaps the entire membrane should be viewed as a mosaic of microdomains.     

Plasma membrane tubulin

The association of tubulin with the plasma membrane comprises multiple levels of penetration into the bilayer: from integral membrane protein, to attachment via palmitoylation, to surface binding, and to microtubules attached by linker proteins to proteins in the membrane. Here we discuss the soundness and weaknesses of the chemical and biochemical evidence marshaled to support these associations, as well as the mechanisms by which tubulin or microtubules may regulate functions at the plasma membrane.     

Inclusion bodies and autophagosomes: Are ER-derived protective organelles different than classical autophagosomes?

A hallmark of some endoplasmic reticulum (ER)-storage diseases is the formation of inclusion bodies (IBs) that are membrane-limited. The nature and function of the IBs has started to be investigated. We have recently found that sequestration of mutated α1-antitrypsin (ATZ) into IBs is a cell protective mechanism that maintains ER function. We also found that IBs are ER-derived and yet separate from the main ER and do not have markers of autophagosomes and lysosomes. We propose that formation of the IBs is a quality control mechanism that leads to storage of unwanted proteins outside the secretory pathway by a mechanism different than direct autophagosome formation from the ER.

Addendum to: Granell S, Baldini G, Mohammad S, Nicolin V, Narducci P, Storrie B, Baldini G. Sequestration of mutated Éø-1-antitrypsin into inclusion bodies is a cell protective mechanism to maintain endoplasmic reticulum function. Mol Biol Cell 2008; 19:571-85.     

Scaffolding the expansion of autophagosomes

The conjugation of the small ubiquitin (Ub)-like protein Atg8 to autophagic membranes is a key step during the expansion of phagophores. This reaction is driven by 2 interconnected Ub-like conjugation systems. The second system conjugates the Ub-like protein Atg12 to Atg5. The resulting conjugate catalyzes the covalent attachment of Atg8 to membranes. Atg12–Atg5, however, constitutively associates with the functionally less well-characterized coiled-coil protein Atg16. By reconstituting the conjugation of Atg8 to membranes in vitro, we showed that after Atg8 has been attached to phosphatidylethanolamine (PE), it recruits Atg12–Atg5 to membranes by recognizing a noncanonical Atg8-interacting motif (AIM) within Atg12. Atg16 crosslinks Atg8–PE-Atg12–Atg5 complexes to form a continuous 2-dimensional membrane scaffold with meshwork-like architecture. Apparently, scaffold formation is required to generate productive autophagosomes and to deliver autophagic cargo to the vacuole in vivo.     

Mitotic membrane helps to focus and stabilize the mitotic spindle

During mitosis, microtubules (MTs), aided by motors and associated proteins, assemble into a mitotic spindle. Recent evidence supports the notion that a membranous spindle matrix aids spindle formation; however, the mechanisms by which the matrix may contribute to spindle assembly are unknown. To search for a mechanism by which the presence of a mitotic membrane might help spindle morphology, we built a computational model that explores the interactions between these components. We show that an elastic membrane around the mitotic apparatus helps to focus MT minus ends and provides a resistive force that acts antagonistically to plus-end-directed MT motors such as Eg5.     

Freeze-fracture replica immunolabelling reveals human WIPI-1 and WIPI-2 as membrane proteins of autophagosomes

Autophagy defines the lifespan of eukaryotic organisms by ensuring cellular survival through regulated bulk clearance of proteins, organelles and membranes. Pathophysiological consequences of improper autophagy give rise to a variety of age-related human diseases such as cancer and neurodegeneration. Rational therapeutic implementation of autophagy modulation remains problematic, as fundamental molecular details such as the generation of autophagosomes, unique double-membrane vesicles formed to permit the process of autophagy, are insufficiently understood. Here, freeze-fracture replica immunolabelling reveals WD-repeat protein interacting with phosphoinositides 1 and 2 (WIPI-1 and WIPI-2) as membrane components of autophagosomes and the plasma membrane (PM). In addition, WIPI-1 is also present in membranes of the endoplasmic reticulum (ER) and WIPI-2 was further detected in membranes close to the Golgi cisternae. Our results identify WIPI-1 and WIPI-2 as novel protein components of autophagosomes, and of membrane sites from which autophagosomes might originate (ER, PM, Golgi area). Hence therapeutic modulation of autophagy could involve approaches that functionally target human WIPI proteins.     

Plasma membrane ATPase of sugarbeet

A membrane fraction enriched with magnesium-dependent ATPase activity was isolated from sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI and sucrose density gradient centrifugation. This activity was inhibited by vanadate, N,N′-dicyclohexylcarbodiimide and diethylstilbestrol, but was insensitive to molybdate, azide, oligomycin, ouabain, and nitrate, suggesting enrichment in plasma membrane ATPase. The enzyme was substrate specific for ATP, had a pH optimum of 7.0, but showed little stimulation by 50 mM KCl. The sugarbeet ATPase preparation contained endogenous protein kinase activity which could be reduced by extraction of the membranes with 0.1% (w/v) sodium deoxycholate. Reduction of protein kinase activity allowed the demonstration of a rapidly turning over phosphorylated intermediate on a M_r 105000 polypeptide, most likely representing the catalytic subunit of the ATPase. Phosphorylation was magnesium dependent, sensitive to diethylstilbestrol and vanadate but insensitive to oligomycin and azide. Neither the ATPase activity nor phosphoenzyme level were affected by combinations of sodium and potassium in the assay. These results argue against the presence of a synergistically stimulated NaK-ATPase at the plasma membrane of sugarbeet.