ER-driven Contact Sites in Autophagosome Biogenesis Sequence

Abstract

Autophagosome biogenesis is a highly coordinated membrane remodeling process that relies on the de novo formation and expansion of the phagophore, yet the cellular principles governing its spatial and temporal organization remain incompletely understood. Accumulating evidence now places the endoplasmic reticulum (ER) at the center of this process, not merely as a membrane source, but as a dynamic scaffold that organizes phagophore assembly through extensive membrane contact sites with multiple organelles. ER-mediated contacts with endosomes, mitochondria, the plasma membrane, and ER–Golgi intermediates create specialized microenvironments that integrate signaling, lipid transfer, vesicle formation and trafficking, and biophysical constraints to drive phagophore nucleation and growth. These contact sites enable the coordinated mobilization of diverse membrane carriers and autophagy regulators in a stress- and context-dependent manner. In this review, we discuss how ER-driven membrane contact sites orchestrate autophagosome biogenesis, highlight emerging mechanistic and biophysical concepts, and consider their broader implications for cellular stress adaptation and disease.

Introduction

The Endoplasmic Reticulum as a Central Organizational Hub of the Eukaryotic Cell

The endoplasmic reticulum (ER) is a highly dynamic and continuous membrane network composed of sheets and tubules that extends throughout the cytoplasm and is physically connected to the nuclear envelope (Figure 1). Historically, the ER has been viewed primarily as the site of secretory and transmembrane protein synthesis, folding, and quality control, as well as a major hub for lipid biosynthesis and intracellular Ca²⁺ storage. Indeed, ribosome-associated rough ER supports protein translocation and post-translational modifications such as N-glycosylation, while smooth ER domains contribute to phospholipid, sterol, and steroid synthesis, detoxification reactions, and Ca²⁺ homeostasis. These canonical functions place the ER at the core of membrane biogenesis and proteostasis in eukaryotic cells.

Figure 1.

Diversity of ER-mediated contact sites and endomembranes in mammalian cells. Schematic representation of a mammalian cell illustrating the extensive network formed by the ER and its physical contacts with multiple organelles, including the nucleus, plasma membrane, mitochondria, Golgi apparatus, endolysosomes, lipid droplets, peroxisomes, and components of the autophagic pathway. The ER establishes single membrane contact sites with individual organelles, as well as higher-order or multiple contact sites where different organelles are closely associated within the same subcellular region. In addition to its central roles in protein and lipid biosynthesis, calcium signaling, and organelle dynamics, the ER contributes to autophagy through its specialization into omegasome structures. These PI3P-enriched ER subdomains serve as platforms for phagophore nucleation and expansion, leading to the formation of autophagosomes and their subsequent maturation into autolysosomes. This figure highlights the spatial organization and functional diversity of ER-derived contact sites in coordinating cellular homeostasis and stress responses.

Beyond these classical roles, it is now clear that the ER acts as a central organizer of cellular architecture, signaling and membrane dynamics. From an evolutionary perspective, the ER is considered one of the earliest organelles to emerge in eukaryotes (Dacks and Field, 2007; Almeida and Amaral, 2020; Kontou et al., 2022). This ancient origin, together with its extensive cytoplasmic distribution, enables the ER to serve as a spatial and functional scaffold upon which many other membrane-bound compartments are generated, maintained, and coordinated. Consistent with this view, the ER participates directly in the biogenesis and dynamics of multiple organelles, including peroxisomes, lipid droplets, lipoprotein particles, and components of the early secretory pathway (Morel, 2020), as well as in the organization of membraneless compartments such as stress granules and P-bodies (Joshi et al., 2017; Lee et al., 2020).

A defining feature of the ER network is its ability to form direct physical contacts with virtually all other organelles within the cell (Figure 1). These regions of close – but transient and dynamic - membrane apposition, typically separated by distances of 10–30 nm and lacking membrane fusion, are termed membrane contact sites. They are now recognized as critical platforms for non-vesicular communication between organelles, enabling the exchange of lipids, ions, and metabolites, as well as the coordination of signaling pathways and membrane remodeling events (Cohen et al., 2018; Scorrano et al., 2019; Prinz et al., 2020). Among all cellular membranes, the ER is the principal and most versatile MCS-forming organelle, owing to its pervasive distribution and its capacity to dynamically remodel its morphology.

ER membrane contact sites (ERCS) have been described with a wide range of organelles, including mitochondria, the plasma membrane, endosomes, lysosomes, peroxisomes, lipid droplets, and the Golgi apparatus (Friedman et al., 2013; De Matteis and Rega, 2015; Raiborg et al., 2015; Costello et al., 2017; Atakpa et al., 2018). These contacts are not static but highly regulated in space and time, adapting to metabolic state, stress conditions, and developmental cues. Functionally, ERCS serve as hubs for lipid synthesis and transfer, Ca²⁺ signaling, organelle positioning, and membrane fission or growth. A well-characterized example is the ER–mitochondria contact site, which regulates mitochondrial fission, bioenergetics, and apoptosis, in part through localized Ca²⁺ transfer and lipid exchange (Friedman and Voeltz, 2011).

At the molecular level, ERCS are defined by a specialized protein and lipid composition. They are stabilized by tethering proteins that physically bridge opposing membranes, either through direct interactions or via multiprotein complexes. These include conserved tether families such as VAPs, extended synaptotagmins, and various organelle-specific adaptors. In addition to structural tethers, ERCS are enriched in lipid transfer proteins that mediate the non-vesicular exchange of phospholipids, sterols, and other hydrophobic molecules between membranes, thereby supporting rapid membrane expansion and compositional remodeling without relying on vesicular trafficking (Neuman et al., 2022; Hanna et al., 2023). Furthermore, many ERCS harbor Ca²⁺ channels and transporters, allowing the ER to act as a master regulator of localized Ca²⁺ fluxes that influence downstream signaling pathways and organelle function (Stefan, 2020).

Importantly, the ER can engage simultaneously multiple contact sites with different organelles, creating local microdomains where complex signaling and metabolic integration occur (Figure 1). This multiplexing capacity enables the ER to coordinate processes such as lipid metabolism, organelle dynamics, and stress responses across the endomembrane system. As a result, ERCS have emerged as central nodes in cellular homeostasis, with growing evidence implicating their dysfunction in metabolic diseases, neurodegeneration, cancer, and aging (Cohen et al., 2018; Scorrano et al., 2019; Prinz et al., 2020).

Together, these observations position the ER not merely as a biosynthetic factory but as an ancient, highly adaptable platform that orchestrates inter-organelle communication through membrane contact sites. Understanding the molecular architecture and regulation of ERCS is therefore essential for deciphering how cells integrate membrane dynamics, signaling, and metabolic control, processes that are increasingly recognized as fundamental to autophagy.

The Autophagy Pathway and the Hallmarks of Autophagosome Biogenesis

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic pathway that mediates the degradation and recycling of cytosolic components. This process relies on the formation of a double-membrane organelle, the autophagosome, which sequesters cargoes and delivers them to the lysosome for degradation (Boya et al., 2013) (Figure 1, right-lower panel). In most mammalian cells, autophagy occurs at low basal levels but can be strongly induced in response to diverse stress conditions, including nutrient deprivation, infection, and mechanical, physical, or chemical stresses. Because autophagy is a central regulator of cellular homeostasis and survival, its dysregulation has been implicated in numerous human diseases, such as cancer, inflammation, neurodegenerative disorders, and metabolic syndromes (Yang and Klionsky, 2010).

Stimulated autophagy, notably in response to nutrient deprivation, involves a tightly coordinated sequence of signaling and membrane remodeling events that culminate in the sequestration of cytoplasmic material within newly formed autophagosomes and their subsequent fusion with lysosomes. Autophagosome biogenesis initiates with the emergence of a cup-shaped membrane structure termed the phagophore or isolation membrane, which expands and ultimately closes to form a mature autophagosome. These membrane dynamics are largely controlled by autophagy-related (ATG) proteins, while non-ATG factors mainly contribute to upstream signaling and membrane trafficking processes (Walker and Ktistakis, 2019). Although the precise origin of the phagophore remains debated, accumulating evidence supports its derivation from multiple membrane sources, including the endoplasmic reticulum (ER), endosomes, Golgi-derived vesicles, and mitochondria.

Among the signaling pathways regulating autophagy, the mTORC1 and AMPK complexes play central roles in coordinating autophagic initiation under stress conditions (Molino et al., 2017). Inhibition of mTORC1 leads to activation of the ULK1 complex—comprising ULK1/2, FIP200, ATG13, and ATG101—which in turn stimulates the class III phosphatidylinositol 3-kinase (PI3 K) complex (VPS34, Beclin1, VPS15, and ATG14L1). This complex generates localized pools of phosphatidylinositol-3-phosphate (PI3P) on specialized ER subdomains known as omegasomes or pre-autophagosomal membranes (Axe et al., 2008) (Figure 2).

Figure 2.

Molecular Machineries of ER Membrane Contact Sites Involved in Autophagosome Biogenesis. Schematic representation of the main protein complexes and pathways operating at ER-associated membrane contact sites during autophagosome formation in mammalian cells. The figure illustrates ER contacts with the phagophore (a), mitochondria (b), plasma membrane (c), Golgi/ERGIC (d), and endosomes (e). Autophagy initiation is coordinated by the ULK complex and the class III phosphatidylinositol 3-kinase complex, leading to local production of PI3P and recruitment of downstream effectors such as DFCP1 and WIPI proteins. ER-resident proteins, vesicular trafficking pathways, and lipid transfer mechanisms contribute to membrane supply and phagophore expansion. Together, these coordinated machineries ensure efficient and spatially organized autophagosome biogenesis at ER-associated contact sites.

PI3P enrichment at pre-autophagosomal membranes is essential for phagophore nucleation and expansion, as it promotes the recruitment of PI3P-binding ATG proteins such as members of the WIPI family. WIPI2 binds PI3P via its PROPPIN domain (Baskaran et al., 2012) and recruits the ATG16L1-ATG5/ATG12 conjugation system (Dooley et al., 2014), thereby enabling the lipidation and membrane association of LC3 (the mammalian homolog of yeast ATG8). LC3 lipidation (conversion of LC3-I to LC3-II) requires several ATG proteins, including ATG4, ATG3, ATG7, and ATG10, and remains a defining hallmark of autophagic membranes (Nishimura et al., 2013; Dooley et al., 2014; Wilson et al., 2014). The combined presence of PI3P and LC3-positive membranes defines sites of autophagic activity and drives phagophore growth and closure (Boya et al., 2013).

The membrane remodeling events that support omegasome transition to expanding phagophores are still incompletely understood but likely require the coordinated contribution of multiple membrane sources. Consistent with this view, endosomes, Golgi-derived membranes, ER exit sites, ER-Golgi intermediate compartment (ERGIC) vesicles, and the plasma membrane have all been implicated in autophagosome biogenesis (Molino et al., 2017). ATG9-positive vesicles, which may originate from endosomal and Golgi-associated compartments, further support a multi-membrane origin model for autophagosomes (Hurley and Young, 2017). In this context, membrane trafficking regulators such as the recycling endosome-associated small GTPase Rab11 have emerged as important contributors to phagophore assembly and expansion (Puri et al., 2018), highlighting the repurposing of canonical trafficking pathways during autophagy. Ultimately, phagophore closure generates a mature double-membrane autophagosome, which fuses with lysosomes in a process dependent on endo-lysosomal regulators, including SNARE proteins and Rab GTPases (Molino et al., 2017).

ERCS in Autophagosomal Membranes Assembly

ER-Phagophore Contact Sites

Three-dimensional electron tomography has revealed that the phagophore and the ER are physically connected through narrow membrane continuities. These connections were observed between the phagophore/autophagosomal membrane and ER cisternae located both outside and inside the forming autophagosome (Ylä-Anttila et al., 2009; Biazik et al., 2015), suggesting a role of the ER in the expansion of the phagophore membrane (Figure 2a).

Beyond serving as a structural cradle platform via DFCP1 protein (Axe et al., 2008), the ER actively participates in phagophore expansion through specialized contact sites and resident proteins. The vacuole membrane protein 1 (VMP1) is a conserved ER transmembrane protein (Molejon et al., 2013), associated with the ER transmembrane protein 41B (TMEM41B), which regulate cholesterol and phosphatidylserine in ER membrane thanks to their scramblase activity (T. Li et al., 2023). VMP1 is localized on ER tubules and organizes ER microdomains that mark the initiation sites for autophagosome biogenesis (Tábara et al., 2016). VMP1-enriched regions on the ER can connect with other organelles such as mitochondria, endosomes or lipid droplets, but also with autophagosomes in starvation (Tábara et al., 2016). VMP1 deficiency induces accumulation of large omegasomes in C. elegans (Tian et al., 2010) and immature autophagosomes in HeLa cells (Kishi-Itakura et al., 2014).

These VMP1-enriched ER microdomains not only define the structural platform for autophagosome formation, but also create the interface through which lipids can be supplied to the expanding phagophore. In this context, ER-phagophore contact sites are functionally mediated by the lipid transfer protein ATG2A/B, which bridges the two membranes and ensures continuous membrane growth during autophagosome biogenesis. In mammalian cells, depletion of ATG2A strongly impairs autophagic flux, whereas depletion of ATG2B has little effect; simultaneous knockdown of both ATG2A and ATG2B, however, further exacerbates the defect, indicating that ATG2B can partially compensate for the loss of ATG2A (Velikkakath et al., 2012). The ATG2A N-terminal part containing the chorein domain is interacting with VMP1 and TMEM41B (Ghanbarpour et al., 2021) on the ER membrane while the C-terminal part of ATG2A interacts with the scramblase ATG9A on the phagophore membrane. Interestingly, ATG9A can also interact with the N-terminal region of ATG2A, suggesting multiple points of contact between these proteins (Van Vliet et al., 2024). VMP1 and TMEM41B redistribute lipids within the ER membrane, while ATG9A performs a similar function in the phagophore membrane, together ensuring efficient lipid flow across the ER–phagophore interface. ATG2A also directly interacts with LC3/GABARAP via its LC3 interacting region (LIR) (Bozic et al., 2020).

ATG2A can bind a variety of glycerophospholipids including phosphatidylethanolamine, phosphatidylcholine, and phosphatidylserine with low specificity. Moreover, lipid transfer assays using liposomes have shown that, thanks to its elongated hydrophobic cavity, ATG2A can efficiently transfer glycerophospholipids from a donor to an acceptor membrane in vitro (Valverde et al., 2019). ATG2A also associates with the PI3P-binding proteins WIPIs (Ren et al., 2020; Wang et al., 2025) on the phagophore membrane, which facilitates its recruitment and enhances the efficiency of lipid transfer between the ER and the phagophore (Maeda et al., 2019). Both the N-terminal and C-terminal regions of ATG2A are required for its recruitment to the phagophore, mediating interactions with ER components and ATG9A vesicles (Nishimura et al., 2017). ATG2A depletion leads to the accumulation of unclosed autophagosomes ((Velikkakath et al., 2012; Kishi-Itakura et al., 2014; Tang, Takahashi and Wang, 2019), disrupts PI3P enrichment on ATG9 vesicles but also reduce lysosomal LC3-II turnover and impair p62 degradation (Holzer et al., 2025).

While the role of additional tethers, organelle contacts, and the energetic regulation of lipid transfer remains to be fully elucidated, these ER-phagophore contacts are clearly central to autophagosome biogenesis.

ER-Mitochondria Contact Sites

In addition to ER-phagophore contacts, several other ER-mediated contact sites contribute to phagophore biogenesis or priming (Figure 2b). ER forms contacts with mitochondria, called MERCS, which mediate the transfer of reactive oxygen species, calcium (Ca²⁺) and lipids (Vance, 1990; Rizzuto et al., 1998; Marchi et al., 2014) between the two organelles, contributing to lipid supply and signaling events.

Early evidence suggested that mitochondrial membranes supply lipids, notably PE, for autophagosome biogenesis (Hailey et al., 2010), highlighting the cooperative role of mitochondria membrane in this process. Building on this observation, subsequent work demonstrated that starvation induces the recruitment and the enrichment of autophagy related protein such as ATG14L1 and ATG5, the omegasome marker DFCP1, as well as components of PI3 K complex, at mitochondrial-associated membranes (Hamasaki et al., 2013), demonstrating that MERCS functions as a platform for autophagosome biogenesis. Consistently, ATG2A has been shown to be recruited at ER-mitochondria contact sites in starvation, via an interaction between the MLD domain of ATG2A and TOMM40 (Tang, Takahashi, He et al., 2019). The MLD domain (MAM localization domain) corresponds to a 45 amino acid sequence located in the C-terminal region of ATG2A, which is part of a larger region previously shown to be required for autophagosome and lipid droplet localization (Velikkakath et al., 2012). Importantly, this small MLD domain alone is sufficient to recruit ATG2A to ER-mitochondria contact sites (Tang, Takahashi, He et al., 2019). At MERCS, ATG2A is also interacting with WIPI4, acting as a local scaffold, to form a membrane-tethering complex that bridges PI3P-enriched autophagosomal membranes with the ER to facilitate lipid transfer for phagophore expansion (Chowdhury et al., 2018; Zhu et al., 2024).

In addition, the VAPB–PTPIP51 complex contributes to the structural and functional organization of ER–mitochondria contact sites. The ER-resident protein VAPB interacts with the mitochondrial outer membrane protein PTPIP51 to mediate tethering and regulate lipid and Ca²⁺ exchange between the two organelles. Disruption of this interaction alters mitochondrial homeostasis and signaling, which can secondarily impact autophagy (Gomez-Suaga et al., 2017). However, PTPIP51 is now primarily recognized as a lipid transfer/tethering factor at MERCS, and its effects on autophagy are likely indirect and context-dependent.

ER-Plasma Membrane Contact Sites

The ER forms dynamic contact sites with the plasma membrane (PM), which serve as regulatory platforms for lipid signaling and membrane remodeling in response to metabolic cues. Under starvation conditions, a potent inducer of autophagy, ER-PM contact sites are actively remodeled through the localized recruitment of extended synaptotagmin (E-Syt) proteins (Nascimbeni et al., 2017), linking nutrient sensing to membrane organization (Figure 2c). Mechanistically, starvation promotes the accumulation of E-Syts at ER-PM interface, where they stabilize contact sites and create a permissive environment for autophagosome initiation. Functional disruption of E-Syts significantly impairs starvation-induced autophagy, whereas their enforced expression enhances autophagic flux, indicating that E-Syt-dependent ER-PM contacts are not merely structural but actively instructive for autophagosome biogenesis. These sites were identified as hotspots for phagophore nucleation. At the molecular level, a transient interaction between VMP1, E-Syt2, and Beclin-1 assembles specifically at ER-PM contact sites during starvation. This complex facilitates the localized activation of the class III phosphatidylinositol 3-kinase (PI3KC3) machinery, resulting in the production of PI3P on ER membranes engaged in PM contacts. The spatially restricted enrichment of PI3P acts as a key trigger for phagophore formation by recruiting downstream PI3P-binding autophagy effectors, thereby initiating the membrane nucleation and expansion steps required for autophagosome biogenesis. Collectively, these findings establish ER-PM contact sites as metabolically regulated signaling hubs that spatially coordinate PI3P production and autophagy initiation in response to nutrient deprivation (Nascimbeni et al., 2017).

ER-Golgi Contact Sites

Autophagosome biogenesis can occur at contact sites between the endoplasmic reticulum (ER) and the Golgi apparatus, particularly at subdomains involving the ER-Golgi intermediate compartment (ERGIC) and ER exit sites (ERES) (Figure 2e). Notably, vesicles derived from ERES and ERGIC have been shown to contribute directly to autophagosome formation. Indeed, COPII-coated vesicles, which bud from ERES, are enriched in autophagy-related machinery. Specifically, ATG9A physically interacts with Sec24, a core COPII component, to promote autophagosome biogenesis (Davis et al., 2016). In vitro reconstitution experiments further demonstrate that starvation enhances COPII vesicle formation and enables LC3 lipidation on these vesicles, indicating their functional role as membrane precursors (Ge, Zhang et al., 2014). Starvation also triggers remodelling of ERES components: Sec12 is relocalized to ERGIC compartments in a CTAGE5-dependent manner, facilitating autophagosome initiation. This process is mediated by a direct interaction between Sec12 and FIP200, a core component of the ULK1 complex (Ge et al., 2017). Most ATG9A molecules reside in Golgi-derived vesicles (∼30–60 nm in diameter) that continuously traffic between the Golgi, plasma membrane, and endosomal compartments (Mari et al., 2010; Puri et al., 2014). Upon autophagy induction, ATG9A vesicle formation increases and these vesicles are recruited to nascent autophagosome sites, partly through PI3KC3-C1 activity (Reggiori et al., 2004; Reggiori and Tooze, 2012; Yamamoto et al., 2012). Beyond serving as membrane seeds, ATG9A vesicles also deliver key regulators of autophagosome formation. Starvation-induced ATG9A vesicles associate with PI4KIIIβ, which generates phosphatidylinositol 4-phosphate (PI4P) at autophagosome biogenesis sites (Judith et al., 2019). Local PI4P accumulation stabilizes ATG13 binding, thereby promoting recruitment of the ULK complex to the omegasome (Karanasios et al., 2013; Judith et al., 2019).

Although the contribution of the Golgi and ERGIC to autophagosome biogenesis is established, the molecular mechanisms required for the formation of ER-Golgi membrane contacts are less characterized. Interestingly, starvation-induced ERGIC-ERES contacts are mediated by a direct interaction between TMED9 and SEC12 (Li et al., 2022), however these proteins can shuttle between the two compartments suggesting that other tethering regulators strictly associated to each organelle may be involved.

ER-Endosomes Contact Sites

ER-endosomes contact sites are mutually important for each partner: the ER supports endosomal maturation and regulation of receptor signaling (Eden et al., 2010; Wu and Voeltz, 2021), while endosomes influence ER dynamics and mobility (Audhya et al., 2007; Jang et al., 2022). On the other side, the endolysosomal and autophagic pathways are highly interconnected (Ao et al., 2014; Tooze et al., 2014; Da Graça et al., 2024). Indeed, canonical intracellular trafficking is essential for recruiting core ATG proteins at sites of autophagosome formation to enable its regulation, and multiple Rab GTPases regulate autophagic processes (Ao et al., 2014; Zhao et al., 2025). Specifically, Rab1 promotes activation of the ULK initiation complex, while Rab5 enhances PI3 K activity and favors autophagy (Dou et al., 2013; Webster et al., 2016). Autophagy induction by starvation is associated with mobilization of Rab11-positive compartments, resulting in reduced recycling capacity (e.g., transferrin receptor recycling) (Longatti et al., 2012; Knævelsrud et al., 2013; Puri et al., 2013, 2025). Rab11/SNX18 positive compartments have been shown to traffic core ATG proteins, such as ATG9A and ATG16L1, to sites of autophagosome biogenesis (Knævelsrud et al., 2013; Søreng et al., 2018). Other studies suggest that Rab11 compartments serve as direct membrane platforms for autophagosome formation, mediated by the Rab11-WIPI2 interaction (Puri et al., 2013, 2018). Additional findings indicate that early endosomes lose PI3P upon starvation via MTM1 phosphatase activation, leading to reduced ER-early endosome contacts mediated by the ER-shaping and tethering proteins RRBP1 and KTN1 (Jang et al., 2022).

Direct evidence demonstrates that SNX1-positive early endosomal structures are recruited to omegasomes via the formation of endosome-ER contact sites under starvation conditions. This starvation-induced interaction between SNX1 + endosomes and the ER requires direct binding between SNX2, an SNX1 endosomal partner, via its FFAT motif and the ER-resident protein VAP-B via its MSP domain (Da Graça et al., 2023). Further analysis of the dynamic interplay between endosomes and the ER during autophagosome formation reveals that endosomes are recruited in sequential waves: early endosomes (Rab5+) arrive within 15 min of starvation, while recycling endosomes (Rab11+) are recruited after 1 h of starvation (Da Graça et al., 2025; Da Graça and Morel, 2025), consistent with studies highlighting Rab11's role in phagophore formation (Puri et al., 2013, 2018, 2025). PI3P-positive endosomes are recruited to ER-exit sites via the ER protein KTN1. At these endosome-ER contact sites, contact sites not only facilitate ATG protein delivery via endosomal trafficking but, more specifically, create a local microenvironment promoting de novo phagophore membrane seeding (Figure 2d). This process occurs through localized Ca²⁺ elevation via TPC1-ITPR1/3 activity which favors local liquid-liquid phase separation of proteins. Thus, such combination leads to the formation of Rab3 + nano-sized vesicles whose subsequent fusion is regulated by RAB3GAP1/2 (Da Graça et al., 2025). Moreover, the ATG2 lipid transfer protein can shuttle lipids from PI3P + endosomes, via ANKFY1 binding, to the growing phagophore (Wei et al., 2024). Altogether, these studies showed that endosomes can also be recruited to the ER to favor phagophore formation.

ER-Mediated Contacts and Biophysical Properties

Beyond their protein composition, ER-mediated contacts are also defined by specific lipid organization, where lipid ordering directly modulates the activity of these contact sites with (King et al., 2020). Given that autophagosome biogenesis relies on both non-vesicular lipid transfer and local liquid–liquid phase separation (LLPS) (Fujioka et al., 2020; Agudo-Canalejo et al., 2021), it is therefore expected that the ER provides distinct biophysical properties that support phagophore formation. In the context of ER-phagophore contact sites and VMP1-ATG2-ATG9 partnership, the directionality of lipid flow at is not solely determined by protein-protein interactions but is sustained by a thermodynamic coupling between lipid transfer and scramblase activity which dissipate the lipid asymmetry generated on each membrane leaflet as lipids are delivered or depleted (King et al., 2020). Thus, such synergetic partnership only becomes apparent when considered at the scale of the full contact site geometry, where donor and acceptor membranes are held in defined proximity.

Local Ca²⁺ burst on the ER membrane surface triggers LLPS of FIP200 (Zheng et al., 2022), leading to the formation and enhanced activity of the initiation ULK1 complex. Recent findings revealed that the spatiotemporally regulation of this Ca²⁺ increase can be mediated by the formation of ER-mediated contacts, especially when associated with endosomes (Da Graça et al., 2025; Da Graça and Morel, 2025), which restricts Ca²⁺ signal to a confined compartment within the cytoplasm. In addition, shortening distances between membranes most likely favour local molecular crowding, a determinant factor to induce LLPS. Such events, positions phase separation not as an independent event but as a downstream biophysical consequence of ERCS-associated ion fluxes, directly linking the structural identity of a given contact site to its capacity to nucleate an autophagy initiation hub.

Together, these principles suggest that ERCS do not merely provide a permissive environment for autophagosome biogenesis but actively encode spatial and thermodynamic information that instructs where, when, and how efficiently phagophore formation can proceed.

Concluding Remarks

In mammalian cells, the ER is unequivocally essential for phagophore nucleation (Walker and Ktistakis, 2019; Ktistakis, 2020). However, the ER does not act alone: it serves as a central scaffold that recruits diverse organelles, including mitochondria, endosomes, ERGIC material, Golgi-derived vesicles, and recycling compartments, to supply lipids, proteins, or membrane templates required for autophagosome formation. The choice of organelle contribution might appear context-dependent, shaped by the nature of the stress stimulus, the temporal phase of autophagy induction, and the specific cargo targeted for degradation. In addition, multiple organelles can be mobilized simultaneously, probably fulfilling complementary roles. For instance, while endosomes are recruited to ER exit sites under starvation to initiate phagophore formation, components of the ERGIC are also enriched at these contact sites (Da Graça et al., 2025; Da Graça and Morel, 2025), suggesting that autophagosome biogenesis is not a linear process but rather a collaborative effort orchestrated across multiple membrane systems. Further studies will be required to determine whether and how the ER engages in sequential and/or simultaneous contacts with multiple organelles to support autophagosome biogenesis. One attractive possibility is that the spatial origin of autophagosome formation dictates the repertoire of ER contact sites that are mobilized, reflecting pre-existing local interactions such as ER–mitochondria, ER–endosome, or ER–plasma membrane contacts. In this context, distinct contact sites could contribute differentially to autophagosome formation, for instance by supplying lipids, coordinating membrane remodeling, or integrating signaling cues. Moreover, ER contacts may operate in a temporally coordinated manner, with specific interfaces engaged sequentially to sustain phagophore nucleation, expansion, and closure, potentially acting as a relay of membrane and regulatory inputs. Finally, the nature of the inducing stress and the identity of the cargo may further tune this process, raising the possibility that selective forms of autophagy rely on dedicated combinations of ER contact sites. Altogether, this suggests a dynamic and adaptable network of ER–organelle interactions, orchestrated in both space and time, that could generate functionally distinct autophagosomes tailored to specific cellular needs.

De novo membrane formation, as it is the case for the phagophore, demands precise biophysical and biochemical regulation, sustained by spatial and temporal framework. Accumulating evidence indicates that phagophore appearance process is initiated from small, dynamic vesicular carriers, including – exclusively or in coordination - ATG9A-positive vesicles, COPII-coated vesicles, or Rab3-positive compartments (Yamamoto et al., 2012; Ge, Baskaran et al., 2014; Ge, Zhang et al., 2014; Holzer et al., 2024; Da Graça et al., 2025; Hama et al., 2025). Critically, nano-vesicle formation likely arises from confinement and molecular crowding at membrane contact sites. This environment promotes LLPS - essential for autophagosome biogenesis (Fujioka et al., 2020; Agudo-Canalejo et al., 2021) - thereby enhancing local protein activity and non-vesicular lipid transfer. Consequently, ER-associated contact sites could act as dynamic microreactors that coordinate lipid transfer, protein activity, and biophysical constraints. Through these mechanisms, they shape the local environment of participating organelles and thereby contribute to efficient autophagosome formation.

Despite the rapid progress in identifying ER-associated contact sites involved in autophagosome biogenesis, several important limitations still constrain our current understanding. First, the highly dynamic and nanoscale nature of membrane contact sites makes them technically challenging to visualize and quantify in living cells, particularly when/if multiple contacts coexist or rapidly remodel during autophagy induction. This limitation is enhanced by the difficulty of disentangling overlapping or sequential interactions between the ER and distinct organelles, which may occur within the same spatial domain but serve different functional roles over time. As a result, current models likely underestimate the complexity and combinatorial nature of ER contact site engagement during autophagosome formation. In addition, most studies rely on perturbation of individual tethers or pathways, which does not fully capture the integrated behavior of the contact site network as a whole. Looking forward, an important conceptual advance will be to consider ER contact sites not only as contributors to autophagosome biogenesis, but more broadly as central hubs mobilized during acute stress responses to coordinate membrane turnover, membrane biogenesis, and membrane repair. In this view, autophagosome formation would represent one manifestation of a more general ER-driven membrane adaptation program, in which distinct combinations of contact sites are selectively engaged depending on cellular needs. Elucidating how these contact site networks are specified, coordinated, and repurposed across different stress contexts will be essential to define the fundamental principles governing cellular membrane homeostasis.

Altogether, future research will need to shift from a static, organelle-centric view toward a dynamic, systems-level understanding of ER-mediated contact-sites as emergent dynamic organizing hubs. Deciphering how ER membrane plasticity, biophysical constraints, phase behavior, and stress-specific organelle crosstalk collectively shape phagophore initiation and selective autophagy will require innovative experimental paradigms combining cell biology, soft-matter physics, and systems biology. Embracing such interdisciplinary and unconventional approaches will be crucial to uncover general principles of autophagosome biogenesis, cellular stress adaptation, and their dysregulation in pathological contexts such as neurodegeneration and cancer.