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Autophagosome
Autophagosome
Autophagosome
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The autophagic process is divided into five distinct stages: Initiation, phagophore nucleation, autophagosomal formation (elongation), autophagosome-lysosome fusion (autophagolysosome) and cargo degradation.[1]

An autophagosome is a spherical structure with double layer membranes.[2] It is the key structure in macroautophagy, the intracellular degradation system for cytoplasmic contents (e.g., abnormal intracellular proteins, excess or damaged organelles, invading microorganisms). After formation, autophagosomes deliver cytoplasmic components to the lysosomes. The outer membrane of an autophagosome fuses with a lysosome to form an autolysosome. The lysosome's hydrolases degrade the autophagosome-delivered contents and its inner membrane.[3]

The formation of autophagosomes is regulated by genes that are well-conserved from yeast to higher eukaryotes. The nomenclature of these genes has differed from paper to paper, but it has been simplified in recent years. The gene families formerly known as APG, AUT, CVT, GSA, PAZ, and PDD are now unified as the ATG (AuTophaGy related) family.[4]

The size of autophagosomes varies between mammals and yeast. Yeast autophagosomes are about 500-900 nm, while mammalian autophagosomes are larger (500-1500 nm). In some examples of cells, like embryonic stem cells, embryonic fibroblasts, and hepatocytes, autophagosomes are visible with light microscopy and can be seen as ring-shaped structures.[3]

Autophagosome formation

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Autophagosome formation is initiated by assembly and recruitment of the core autophagy machinery to distinct cellular sites, known as phagophore assembly sites (PAS) in yeast or autophagosome formation sites in other organisms. The process is tightly regulated by multiple autophagy-related (ATG) proteins.[5]

The ULK1/ATG1 complex is the initial activator of autophagy in response to nutrient starvation, and it recruits other ATG proteins to the PAS. The class III phosphatidylinositol 3-kinase (PI3K) complex, including VPS34 and Beclin-1, produces phosphatidylinositol 3-phosphate (PI3P), which is essential for phagophore membrane dynamics. Membrane sources for phagophore expansion may include the endoplasmic reticulum, mitochondria, Golgi apparatus, and recycling endosomes.

After the formation of the spherical structure, ATG12-ATG5:ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane.[6]

LC3 is cleaved by ATG4 protease to generate cytosolic LC3. The cleavage is required for the terminal fusion of an autophagosome with its target membrane. LC3 is cleaved and lipidated to form LC3-II, which associates with the autophagosomal membraneand is used as a marker of autophagosomes in immunocytochemistry, because it is the essential part of the vesicle and stays associated until the last moment before its fusion. At first, autophagosomes fuse with endosomes or endosome-derived vesicles and stays associated until the last moment before its fusion.[7]

After the phagophore fully encloses its cargo, it seals and becomes a mature autophagosome. This structure then fuses with lysosomes to form an autolysosome, where the contents are degraded and recycled.

This process is similar in yeast, however the gene names differ. For example, LC3 in mammals is Atg8. In yeast autophagosomes are generated from Pre-Autophagosomal Structure (PAS) which is distinct from the precursor structures in mammalian cells. The pre-autophagosomal structure in yeast is described as a complex localized near the vacuole. However the significance of this localization is not known. Mature yeast autophagosomes fuse directly with vacuoles or lysosomes, and do not form amphisomes as in mammals. In yeast autophagosome maturation, there are also other known players such as Atg1, Atg13 and Atg17. Atg1 is a kinase upregulated upon induction of autophagy. Atg13 regulates Atg1 and together they form a complex called Atg13:Atg1, which receives signals from the master of nutrient sensing – Tor. Atg1 is also important in late stages of autophagosome formation.[8]

Function in neurons

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In neurons, autophagosomes are generated at the neurite tip and mature (acidify) as they travel towards the cell body along the axon.[9] This axonal transport is disrupted if huntingtin or its interacting partner HAP1, which colocalize with autophagosomes in neurons, are depleted.[10]

Clinical relevance

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Autophagosomes serve as essential carriers in the autophagy pathway, enabling cells to adapt to metabolic stress and maintain homeostasis. They are responsible for the selective degradation of damaged organelles (for example, mitophagy (mitochondria), pexophagy (peroxisomes), aggrephagy (protein aggregates), glycophagy (glycogens), lipophagy (lipids), ribophagy (ribosome), xenophagy (pathogens), and ER-phagy).[11]

This selective removal is crucial for cellular quality control, helping prevent the accumulation of toxic proteins and damaged components. Autophagy also plays roles in development, immunity, and cell differentiation. For instance, autophagosomes assist in antigen presentation, regulate inflammatory signaling, and contribute to the elimination of intracellular bacteria and viruses.

Defects in autophagosome function have been linked to several human diseases. In addition to neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's, impaired autophagy is associated with cancer, infectious diseases, and metabolic disorders like type 2 diabetes.[12]

References

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Autophagosome

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An autophagosome is a double--bound vesicle, typically 0.3–1 μm in diameter, that forms during macroautophagy to sequester cytoplasmic components such as damaged organelles, aggregated proteins, and pathogens for subsequent degradation and recycling within the cell. The biogenesis of autophagosomes initiates at specific cellular sites, such as endoplasmic reticulum-mitochondria contact points, where a cup-shaped isolation membrane known as the phagophore nucleates and elongates around targeted , driven by conserved autophagy-related (ATG) proteins including the ULK1 complex, the class III 3-kinase (PI3K) complex with Beclin 1, and lipidation of LC3 (an Atg8 homolog) to facilitate membrane expansion and closure. This process draws lipids from diverse sources like the , Golgi apparatus, mitochondria, and plasma , culminating in the closure of the phagophore to form a sealed, spherical autophagosome with an outer that remains largely devoid of ribosomes and intramembrane particles. Once formed, the autophagosome matures by fusing with a —mediated by SNARE proteins (e.g., STX17, SNAP29, VAMP8), Rab7 , and tethering complexes like and EPG5—to generate an autolysosome, where lysosomal hydrolases degrade the inner membrane and engulfed contents into reusable building blocks such as and . This catabolic pathway is essential for maintaining cellular , responding to nutrient or stress, clearing toxic aggregates, and defending against infections, with dysregulation implicated in diseases including neurodegeneration, cancer, and metabolic disorders.

Overview and Morphology

Definition and Discovery

The autophagosome is a double-membrane-bound vesicular that plays a central role in macroautophagy, a conserved catabolic process in eukaryotic cells. It forms to sequester portions of the , including damaged s, protein aggregates, or invading pathogens, enclosing them within its lumen for subsequent delivery to the (or in ) where the contents are degraded and recycled. This sequestration enables the cell to maintain under stress conditions such as deprivation or organelle damage. The discovery of the autophagosome emerged from early electron microscopy studies of cellular degradation processes. In 1962, Thomas P. Ashford and Keith R. Porter first described these structures in rat liver cells subjected to glucagon-induced , observing double-membrane vesicles enclosing cytoplasmic components. The following year, formalized the broader concept of during a symposium on lysosomes, integrating these observations into the understanding of lysosomal degradation pathways. These seminal works laid the foundation for recognizing macroautophagy as a distinct mechanism separate from other vesicular trafficking routes. Autophagosomes exhibit characteristic morphological features that distinguish them from other intracellular vesicles, such as endosomes or multivesicular bodies. They typically appear as spherical, double-membraned structures measuring 0.5–1.5 μm in diameter in mammalian cells, though sizes can vary slightly by and condition. Unlike vesicles derived from pre-existing organelles, autophagosomes form de novo, primarily involving contributions from the (ER) and other membrane sources, which provide lipids for their biogenesis without relying on endocytic intermediates. This de novo assembly ensures their isolation from endosomal acidification machinery until fusion with lysosomes.01316-9) The presence and core machinery of autophagosomes are evolutionarily conserved across eukaryotes, from unicellular organisms like to complex multicellular animals. In the yeast , autophagy-related processes were first genetically dissected through mutants in APG (autophagy) genes, many of which have orthologs in mammals that perform analogous functions in autophagosome formation and maturation. This conservation underscores the fundamental importance of autophagosomes in and survival, with the process retained despite divergences in regulatory inputs across .

Structural Features

The autophagosome is defined by its unique double-membrane architecture, comprising an outer and an inner that isolates cytoplasmic cargo from the surrounding . This structure forms a closed vesicle, with the inner membrane directly enclosing the sequestered while the outer remains in contact with the . The lipid bilayers of the autophagosome derive from multiple cellular compartments, including the (ER), Golgi apparatus, plasma , and mitochondria, which contribute to membrane expansion through lipid transfer mechanisms. Key lipids in the membrane composition include phosphatidylinositol 3-phosphate (PI3P), which is enriched during early nucleation, primarily from mitochondrial sources, and phosphatidylethanolamine (PE) conjugated to microtubule-associated protein 1A/1B-light chain 3 (LC3-PE), which anchors LC3 to the and stabilizes the structure. The inner effectively isolates such as damaged organelles, protein aggregates, and other cytoplasmic components by fully enclosing them within the vesicle, preventing premature degradation or interference with cellular processes. During maturation, the outer selectively fuses with lysosomes via SNARE-mediated mechanisms, delivering the inner and its enclosed into the lysosomal lumen for degradation, while ensuring no direct mixing of autophagosomal contents with the or lysosomal enzymes prior to fusion. Visualization of autophagosomes relies on electron microscopy, which distinctly reveals the double-membrane profile and cup-shaped phagophores in early stages, providing high-resolution insights into their morphology. Complementary fluorescence microscopy employs GFP-tagged LC3 (GFP-LC3) to monitor puncta formation, enabling real-time tracking of autophagosome assembly and distribution in living cells. Autophagosome size varies from approximately 0.5 to 1.5 μm in , scaling with the volume and type of enclosed to optimize sequestration efficiency. These vesicles maintain a neutral cytosolic upon formation but develop an acidic gradient (around 4.5–5.0) in the resulting autolysosome after lysosomal fusion, which activates hydrolytic enzymes for breakdown.

Biogenesis and Formation

Initiation and Nucleation

The initiation of autophagosome formation is primarily triggered by cellular stress signals, particularly nutrient deprivation such as , which inhibits the mechanistic target of rapamycin complex 1 (). Under nutrient-rich conditions, phosphorylates and suppresses the unc-51 like autophagy activating 1 (ULK1) complex, preventing onset; upon , dissociation allows ULK1 . Concurrently, the (AMPK), an energy sensor, becomes activated during low cellular energy states and directly phosphorylates ULK1 at Ser317 and Ser777, promoting its activity and further driving initiation. Nucleation follows initiation and involves the assembly of the ULK1 complex, consisting of ULK1, autophagy-related protein 13 (ATG13), focal adhesion kinase family-interacting protein of 200 kDa (FIP200), and ATG101 in mammals (equivalent to the Atg1 complex in ). This complex phosphorylates downstream effectors to recruit the phosphatidylinositol 3-kinase (PI3K) class III complex, which includes vacuolar protein sorting 34 (Vps34), Vps15, Beclin-1 (BECN1), and ATG14L. The PI3K complex generates phosphatidylinositol 3-phosphate (PI3P) at specific platforms, marking the site and facilitating the recruitment of effector proteins like WD-repeat protein interacting with phosphoinositides (WIPI) family members, which expand the nascent . In mammals, this occurs at the omegasome, an (ER)-associated subdomain enriched in PI3P and double FYVE-containing protein 1 (DFCP1), serving as the cradle for phagophore emergence. The pre-autophagosomal structure (PAS) in yeast, analogous to the mammalian isolation membrane (IM) or phagophore, represents the dedicated site for ATG protein recruitment and early membrane assembly. In yeast, the PAS forms near the vacuole upon Atg1 complex activation, concentrating core ATG machinery for nucleation; in mammals, the IM/phagophore initiates at ER subdomains without a singular fixed site, dynamically incorporating lipids from multiple sources. This structure ensures spatially organized progression from a flat cisterna to a cup-shaped phagophore, primed for subsequent expansion. Beyond nutritional cues, non-canonical triggers such as hypoxia, ER stress, and infection can independently initiate nucleation. Hypoxia activates autophagy via hypoxia-inducible factor 1α (HIF-1α)-mediated signaling, which intersects with the ULK1 pathway to promote PI3P production under oxygen limitation. ER stress engages the unfolded protein response (UPR), leading to IRE1-dependent JNK activation that inhibits and stimulates ULK1 recruitment to ER membranes. infection, as in xenophagy, recruits the ULK1 complex to bacterial entry sites, enhancing PI3P generation for targeted phagophore nucleation around invaders like . These signals underscore the adaptability of initiation to diverse stressors, maintaining cellular .

Elongation, Closure, and Maturation

Following , the phagophore elongates through the action of two parallel ubiquitin-like conjugation systems that promote membrane expansion and modification. The first system involves the covalent conjugation of ATG12 to ATG5, catalyzed by the E1-like ATG7 and the E2-like ATG10, resulting in an ATG12–ATG5 conjugate. This conjugate non-covalently associates with ATG16L1 via its coiled-coil domain, forming the ATG12–ATG5–ATG16L1 complex, which localizes to the convex (cytosolic) side of the phagophore membrane and acts as an E3-like to facilitate subsequent membrane elongation. The second system processes the ubiquitin-like protein LC3 (a mammalian homolog of ATG8) through proteolytic cleavage by ATG4 proteases to expose a residue at its . ATG7 then activates LC3 as an E2-like , transferring it to ATG3, where the ATG12–ATG5–ATG16L1 complex promotes the conjugation of LC3 to (PE) in the phagophore membrane, driving cisternal expansion and curvature essential for enclosing cytosolic . These lipidated LC3 molecules coat both the inner and outer membranes of the elongating phagophore, stabilizing its structure and enabling further growth. Elongation culminates in phagophore closure to form a sealed double-membrane autophagosome, a regulated to avoid premature lysosomal interaction. ATG9A facilitates this closure by interacting with IQGAP1 to recruit ESCRT-III components like CHMP2A, promoting membrane scission. Syntaxin 17 (STX17), a tail-anchored , is recruited specifically to the outer membrane of completed autophagosomes but not to open phagophores, ensuring temporal control over membrane sealing. STX17's hairpin structure and glycine zipper motifs facilitate its insertion into the post-closure, where it assembles with SNAP29 and lysosomal VAMP7 or VAMP8 to form trans-SNARE complexes that drive membrane fusion only after sealing, thereby preventing leakage or untimely degradation of unfinished structures. This selective recruitment maintains autophagosome integrity during the transition from phagophore to closed vesicle. during closure is enforced by the endosomal sorting complex required for transport () machinery, which seals any remaining openings in the phagophore membrane to prevent aberrant structures. VPS37A, an ESCRT-I subunit, recruits ESCRT-III components like CHMP2A to the phagophore via its PUEV domain, enabling VPS4 ATPase-driven membrane scission for complete enclosure. In ESCRT-deficient conditions, unclosed phagophores accumulate as cup- or oval-shaped intermediates, impairing autophagic flux and triggering their degradation to avert cellular stress, thus ensuring only sealed autophagosomes proceed to maturation. Mature autophagosomes then undergo trafficking and fusion events to deliver for degradation. Centripetal movement along toward the microtubule-organizing center is powered by the dynein-dynactin motor complex, often in coordination with Rab7 effectors like FYCO1 for directed transport, positioning autophagosomes near perinuclear lysosomes to enhance fusion efficiency. Fusion occurs either directly with lysosomes to generate autolysosomes or sequentially with late endosomes/multivesicular bodies to form amphisomes, followed by lysosomal merger. For direct fusion, the default SNARE complex is STX17-SNAP47-VAMP7/VAMP8, while SNAP29 is involved in amphisome-lysosome fusion. These events are orchestrated by Rab GTPases, including Rab7 for and Rab2 for autophagosome localization, alongside SNARE-mediated docking, stabilized by multi-subunit complexes such as (which binds Rab7 and STX17) and EPG5.

Molecular Mechanisms and Regulation

Key Proteins and Pathways

The formation of autophagosomes relies on a suite of core autophagy-related (Atg) proteins that orchestrate distinct stages of the process, from initiation to encapsulation. The ULK1/2 complex, comprising ULK1 or ULK2, Atg13, FIP200, and Atg101, initiates by phosphorylating downstream targets to promote phagophore under stress conditions. Recent research indicates that the ULK1/2 complex facilitates phagophore via liquid-liquid phase separation (LLPS), with components like FIP200 forming biomolecular condensates on the to concentrate initiation factors. involves the Beclin-1-Vps34 complex, where Vps34 (a class III phosphatidylinositol 3-kinase) generates phosphatidylinositol 3-phosphate (PI3P) to recruit effector proteins, while Beclin-1 acts as a scaffold to assemble the complex with Atg14L for phagophore formation. Atg9, a , delivers bilayers from peripheral sources to the nascent isolation , facilitating membrane expansion. The LC3 family of proteins, including LC3A/B and GABARAPs, drives elongation and closure by integrating into the phagophore and tagging for sequestration. Two ubiquitin-like conjugation systems underpin autophagosome membrane dynamics. In the Atg12 system, Atg12 is activated by the E1-like enzyme Atg7 and transferred to the E2-like Atg10, then conjugated to Atg5, forming an Atg12-Atg5 conjugate that associates with Atg16L1 to act as an E3-like ligase for the second system. The LC3-PE lipidation system parallels ubiquitination: LC3 is processed by Atg4, activated by Atg7 (E1), and conjugated to phosphatidylethanolamine (PE) on the phagophore via Atg3 (E2), with the Atg12-Atg5-Atg16L1 complex enhancing specificity and efficiency. Atg7 (E1)+LC3→Atg7-LC3(activation)\text{Atg7 (E1)} + \text{LC3} \rightarrow \text{Atg7-LC3} \quad \text{(activation)} Atg7-LC3+Atg3 (E2)→Atg3-LC3+Atg7(transfer)\text{Atg7-LC3} + \text{Atg3 (E2)} \rightarrow \text{Atg3-LC3} + \text{Atg7} \quad \text{(transfer)} Atg3-LC3+PE→Atg12-Atg5-Atg16L1LC3-PE+Atg3(conjugation)\text{Atg3-LC3} + \text{PE} \xrightarrow{\text{Atg12-Atg5-Atg16L1}} \text{LC3-PE} + \text{Atg3} \quad \text{(conjugation)}
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