Autophagy, ER-phagy and ER Dynamics During Cell Differentiation

Graphical abstract

Highlights

• •Autophagy is fundamental during development and cell differentiation.
• •ER-phagy has a role in neuron and myotubes differentiation final step.
• •ER dynamics are a crucial part in cell differentiation.

Abstract

The endoplasmic reticulum (ER) is a multifunctional organelle essential for protein and lipid synthesis, ion transport and inter-organelle communication. It comprises a highly dynamic network of membranes that continuously reshape to support a wide range of cellular processes. During cellular differentiation, extensive remodelling of both ER architecture and its proteome is required to accommodate alterations in cell morphology and function. Autophagy, and ER-phagy in particular, plays a pivotal role in reshaping the ER, enabling cells to meet their evolving needs and adapt to developmental cues. Despite the ER’s critical role in cellular differentiation, the mechanisms responsible for regulating its dynamics are not fully understood. Emerging evidence suggests that transcriptional and post-translational regulation play a role in fine-tuning ER-phagy and the unfolded protein response (UPR).

This review explores the molecular basis of autophagy and ER-phagy, highlighting their role in ER remodelling during cellular differentiation. A deeper understanding of these processes could open new avenues for targeted therapeutic approaches in conditions where ER remodelling is impaired.

Introduction

Throughout their lifecycle, cells must continuously eliminate damaged or senescent organelles and protein aggregates both, under steady-state conditions, and even more so when adapting to changing energy demands. Autophagy is the most primordial and efficient mechanism employed by cells to meet these evolving needs, enabling cells to recycle cellular components and rapidly generate energy and building blocks under stressful conditions [1]. The term autophagy refers to a lysosomal degradative process in which portions of cytosolic material (including organelles, protein aggregates, pathogens) are engulfed by a double membrane structure named autophagosome. The fusion of the autophagosomes with the lysosomes promotes the degradation of the cargos and the recycling of the amino acids, which can then be used to fulfil energy needs [2]. Due to its importance to cell biology, autophagy is a well-conserved and tightly regulated pathway across the kingdom of life [3]. Although traditionally associated with maintaining homeostasis, over the past two decades, autophagy has also been demonstrated to play pivotal roles in embryogenesis, tissue development and differentiation [4]. Of note, selective autophagy has been gaining support as a key player with ER-phagy, the selective degradation of the ER, demonstrating a fundamental role in reshaping ER architecture under both physiological and pathological conditions [[5], [6], [7]].

The ER is the largest organelle in the cell and is the premier site for protein synthesis, folding and transport, lipid and steroid synthesis, and calcium storage [8]. A diverse array of proteins and structural domains is required for the ER to perform its specialized functions and adapt to dynamic intracellular environments. Moreover, ER functions are closely linked to the proteins that govern its shape. However, how various cellular signals, influenced by the cellular biological state, regulate ER shaping proteins to alter the ER morphology remains unclear [9]. Studies have demonstrated that during mitosis, the ER undergoes a significant transformation, from a network of sheets and tubules to predominantly sheet-like structures [10,11].

Since the ER is the most interconnected organelle in the cell, ER-phagy likely plays a pivotal role in cell differentiation and tissue development [6]. This review aims to provide an overview of the roles of autophagy and ER-phagy in tissue development, homeostasis, and regeneration, with a specific focus on skeletal muscle.

Molecular biology of autophagy

Autophagy is a highly conserved process across eukaryotes; therefore, the molecular pathways that regulate autophagosome formation, expansion, closure and fusion with lysosomes are well-preserved from yeast to mammals [3]. Autophagy can be classified into three distinct processes based on how cargo is engulfed and delivered to lysosomes: macro-autophagy, micro-autophagy and chaperone-mediated autophagy (CMA) [12]. Macro-autophagy, or simply autophagy, refers to the classical degradation of cytosolic components through the formation of autophagosomes and their fusion with lysosomes [13]. Micro-autophagy is the degradation of cytosolic material via a direct invagination of the lysosomal membrane [14]. In contrast, CMA selectively degrades proteins harbouring a KFERQ-like motif, via the lysosome, upon the recognition by the protein HSC70 [15]. These three pathways are not mutually exclusive and can act synergistically to enhance autophagic flux and target various cargoes simultaneously [16].

Autophagy was first discovered as a response to nutrient deprivation in yeast [17]. It has since become evident that autophagy can be activated by a variety of stimuli [18]. Regardless of the trigger, autophagy is initiated by “core” autophagy-related genes (ATGs), whose impairment completely abolishes autophagic flux [3]. The initiation complex, first discovered in yeast, is termed the Atg1 complex and comprises Atg1, Atg13, Atg17, Atg29 and Atg31 [19]. The human equivalent is the ULK complex, which is composed of the ULK1 or ULK2 kinases, ATG13, FIP200 and ATG101 [20]. This complex is regulated by nutrient sensors such as mTOR and AMPK [21]. Upon activation, the ULK complex engages the PI3K complex, composed of BECLIN-1, ATG14L, VSP34 and VSP15, to initiate autophagy [22]. The ULK1 complex recruits ATG9 vesicles, which serve as the foundation for autophagosomes formation [23]. These vesicles establish contact sites with ER membranes, which are mediated by the lipid transfer protein ATG2. ATG2 facilitates lipids shuttling from the ER to the growing phagophore [3,24]. These nascent phagophores ultimately mature to form a complete autophagosome to be delivered to the lysosome for degradation [25].

The ATG5/ATG12/ATG16L complex functions similarly to an E3 ubiquitin-ligase, conjugating ATG8 to the autophagosomal membrane. In humans, ATG8 is encoded by six distinct genes generally classified into the LC3 (MAP1LC3A, MAP1LC3B and MAP1LC3C) and GABARAP (GABARAP, GABARAPL1, and GABARAPL2) families [26]. These ubiquitin-like modifiers are crucial during autophagy and their lipidation with phosphatidylethanolamine (PE), following its proteolytic activation by ATG4 [27,28], allows for their insertion into autophagosomal membranes, facilitating their role in the degradation of cargo.

Selective Autophagy

Selective autophagy is a targeted and specialized process mediated by specific receptors which typically resides with the cargo, allowing for extreme precision during selection. Cargoes destined for degradation are often marked with poly-ubiquitin chains, serving as a “code” to be recognized by autophagy receptors, which possess an Ub-binding domain [29]. In addition to Ub-binding domains, these receptors also share a conserved LC3-interacting region (LIR) motif, characterized by the sequence W/F/YxxL/I/V (where X can be any amino acid), frequently flanked by negatively charged amino acids that enhance ATG8 protein binding [30]. Through their LIR motifs, these receptors facilitate the recruitment of the ubiquitin like modifiers and mediate the formation of autophagosomes around the tagged cargo via their UBA domain [24,31]. Therefore, the receptors themselves are degraded along with the cargo within the lysosomes. To date, dozens of selective autophagy receptors have been described, enabling the precise degradation of various cellular compartments [32,33]. Several criteria are used to classify selective autophagy receptors. For instance, some of them are free cytosolic proteins while others are closely embedded in the membranes of the targeted organelles [33]. The most well-studied and characterized cytosolic autophagy receptors include p62/SQSTM1, Optineurin (OPTN), NBR1, NDP52 and TAX1BP1 [34]. Additionally, there is a growing number of specialized autophagy receptors, that are resident solely in specific cellular organelles. Their high selectivity confers to the selective autophagy they mediate a distinctive nomenclature matching the targeted elements. These selective types of autophagy, include endoplasmic reticulum (ER-phagy) [35], mitochondria (mitophagy) [36], lysosome (lysophagy) [37], ribosome (ribophagy) [38], nuclear portions (nucleophagy) [39], aggregated proteins (aggrephagy) [40], invading bacteria (xenophagy) [41,42] and many others.

ER-phagy: When the endoplasmic reticulum is the cargo

ER-phagy a is fundamental form of selective autophagy which involves the engulfment of discrete portions of the ER into autophagosomes via specific receptors. The vast majority of the ER-phagy receptors are ER resident proteins such as ATL3 [43], CCPG1 [44], FAM134 family [35,45], RTN3 [46], SEC62 [47], TEX264 [48,49], UBAC2 [50] and C53 [51]. Moreover, SQSTM1/p62 and CALCOCO1 have been demonstrated to act as cytosolic ER-phagy receptors working in concert with ER-bound proteins such as VAPA/VAPB [[52], [53], [54]]. The key feature of these receptors is the presence of at least one LC3-interacting region (LIR) domain, which is essential for the binding of LC3/GABARAP protein families. Some receptors, such as the FAM134 family and RTN3, also possess a Reticulon-Homology Domain (RHD). This distinct 200aa intramembrane domain is composed of two hairpin motifs (α helices) connected by a hydrophilic bridge [55]. The clustering of RHDs is the driving force behind the bending of ER membranes, facilitating their vesiculation and fragmentation [56,57].

The importance of the RHD has been extensively studied at both the biochemical and biophysical levels [56,58]. The downregulation of RHD-containing ER-phagy receptors, such as FAM134B or RTN3, results in ER enlargement and impaired ER membrane fragmentation [35]. Interestingly, RHD-containing proteins, which are not classified as ER-phagy receptors, are often found within ER-phagy receptor clusters, and their presence strongly enhances clustering. For instance, knockout of the FAM134B interacting protein ARL6IP2, has been reported to significantly impair ER-phagy flux [59]. Of note, ER-phagy receptors also possess intrinsically disordered regions (IDRs) at their C- and/or N-termini, where LIR domains are located. IDRs were initially thought to primarily enhance interactions with ATG8s, however, they are now recognized to play an active role in ER-phagy receptor clustering and in facilitating ER membrane bending, acting synergistically with RHDs to remodel ER membranes [60].

ER-phagy plays a pivotal role in maintaining ER homeostasis and vitality as well as in reshaping its network architecture. While ER-phagy is basally active in all cells, its flux can be significantly upregulated when needed, for example during starvation and ER stress [7,[61], [62], [63]].

Transcriptional regulation of ER-phagy receptors

ER-phagy flux can be regulated by fine-tuning specific receptors at both the transcriptional and post-translational levels. Most insights regarding the regulation of ER-phagy come from studies on the FAM134 receptor family. During starvation or upon pharmacological inhibition of mTOR, FAM134B expression is induced by transcription factors TFEB and TFE3, coupling ER-phagy to lysosomal activity [64]. Notably, ER-phagy receptor transcription is differentially regulated depending on the tissue. The most striking example of this phenomenon is represented by FAM134B-2, an N-terminal truncated isoform of FAM134B [65,66]. While FAM134B-2 expression is induced by C/EBPβ during starvation in the liver [65], in HeLa cells, amino-acid deprivation induces its expression through the cooperation between MEF2D and NR4A1 [67]. In terminally differentiated muscle, the expression of this isoform is driven by MYF6 [68]. In addition to FAM134B, the ER-phagy receptor CCPG1, is transcriptionally regulated in both HeLa and A549 cells [44]. During ER stress, both the mRNA and protein levels of CCPG1 are upregulated, as part of the UPR, in a PERK-dependent manner [69]. Although the transcription factors responsible for this induction remain elusive, evidence suggests that MIST1, a transcription factor active during ER stress, can bind the CCPG1 promoter [70]. This tissue- and context-specific regulation is intriguing because it underscores the potential for targeted modulation of ER-phagy.

Post-translational regulation of ER-phagy receptors

While the transcriptional regulation of FAM134B and CCPG1 contributes to the genetic control of ER-phagy, its flux is primarily governed by post-translational modifications (PTMs) to its receptors, including phosphorylation, (poly)ubiquitination, and acetylation [7].

Phosphorylation plays a critical role in ER-phagy with several receptors undergoing this PTM. Casein kinase 2 (CK2) has been demonstrated to phosphorylate FAM134B at Ser149, Ser151 and Ser153 [71]. These residues can also be phosphorylated by CAMK2B, and this phosphorylation is crucial for receptor clustering and the induction of ER-phagy [72]. CK2 also phosphorylates FAM134C at several residues, including Ser258, which impacts Torin1-induced ER-phagy flux [71]. On the other hand, phosphorylation of Ser435, Ser436 and Thr440, located close to FAM134C’s LIR domain, weakens its interaction with LC3B [73]. Interestingly, CK2 also phosphorylates four Ser residues on TEX264 (Ser266, Ser269, Ser271, and Ser272) [74], which appears to enhance its affinity for ATG8s [74].

Phosphorylation and ubiquitination are closely connected through a phenomenon known as phosphorylation-dependent ubiquitination [71]. While RHD clustering drives membrane vesiculation, it alone is insufficient to complete the process. A functional autophagy flux is also required [46,57]. Of note, ubiquitination of ER-phagy receptors downstream of phosphorylation significantly enhances receptor clustering and ER-phagy flux [71]. Specifically, the formation of high-density FAM134B and FAM134C clusters is significantly amplified when their RHDs are heavily ubiquitinated [58]. E3 ubiquitin ligases and deubiquitinating enzymes are often found in FAM134B clusters, potentially fine-tuning ER-phagy flux through its ubiquitination status, further highlighting the importance of ubiquitination as a regulatory PTM [75,76].

Another PTM gaining momentum in ER-phagy is UFMylation [77]. DDRGK1, a protein identified in a genetic screen, acts to recruit the E3 ligase UFL1 to the ER surface, targeting RPN1 [78], a known interactor of the ER-phagy receptor SEC62. This interaction suggests a link between UFMylation and SEC62-driven ER-phagy. Additionally, the ribosomal protein RPL26 is also UFMylated by the DDRGK1/UFL1 axis and the C53 receptor is involved in the clearance of stalled ribosomes by promoting their UFMylation [51,79].

Acetylation can also modulate ER-phagy flux. Deacetylated ATG9A interacts with SEC61 and FAM134B, facilitating autophagosome formation and LC3B recruitment [80]. This is further supported by findings from pancreatic acinar cells expressing reduced levels of the Acetyl-CoA transporter AT-1, where reduced global protein acetylation within the ER lumen leads to the accumulation of secretory proteins as a result of diminished ER turnover [81].

Spatial distribution of ER-phagy receptors

Another regulatory layer lies in the tissue-specific expression and intracellular localization of the ER-phagy receptors. While, ATL3, SEC62, FAM134B and TEX264 are expressed in most tissue [49,66], the FAM134B-2 isoform is predominantly found in testis, heart, mature muscle and fasted liver [65,66]. Moreover, the subcellular localization of these receptors also seems specific, where RTN3L is predominantly associated with ER tubules [43,46], while ATL3 and TEX264 mainly reside at 3-way ER junctions [48]. In contrast, FAM134A and FAM134C are more widely distributed across the ER, while, FAM134B is enriched in ER sheets [35]. The differential expression and compartmentalization of ER-phagy receptors likely represents a critical regulatory node; however, further studies are needed to better elucidate this mechanism.

Autophagy and selective autophagy in development and differentiation

Since the early 2000s, studies conducted in mammalian models have demonstrated that the first significant surge in autophagy flux occurs a few hours following oocyte fertilization. This is a critical step in embryo development prior to implantation [82]. A noteworthy example of this is the mitophagy-mediated degradation of paternal mitochondria to prevent heteroplasmy and ensure that only maternal mtDNA is inherited [83]. The selective elimination of paternal mitochondria is indispensable for embryonic development [83]. Autophagy also plays a role in the zygote-to-embryo transition, enabling progression from 4 to 8 cells [82,84]. The fundamental role of autophagy in embryonic development is further highlighted by the mid-embryonic lethality observed when key autophagy regulators, such as Beclin1 and Ambra1, are deleted [85,86]. Additionally, ATG knockout animals that survive embryonic development typically die shortly after birth. Autophagy remains essential for early post-natal survival, where autophagy flux is once again increased to cope with the starvation period that occurs when transplacental nutrient supply ceases [87]. Newborn mice lacking Atg5 [87], Atg7 [88], Atg12 [89] or Atg16l1 [90] die within a few hours after birth. This postnatal autophagy flux is primarily regulated mainly by mTORC1 and is crucial for neonatal survival [91].

Moreover, autophagy is also implicated in cellular differentiation processes such as haematopoiesis. In Drosophila, Atg2 regulates hematopoietic homeostasis by facilitating the proliferation and differentiation of mature plasmatocytes [92]. Similarly, in murine models, the deletion of Atg5 in endothelial cells disrupts the endothelial to hematopoietic transition, thus impairing embryonic hematopoietic stem cell development [93].

Autophagy is also essential for the selective elimination of mitochondria during RBC differentiation, where disruption of autophagy, or mitophagy in particular, results in mature RBCs retaining mitochondria [94]. The biological role of NIX/BNIP3L, as mitophagy receptors, was initially established in the context of erythropoiesis [95,96].

Similarly, lymphocyte development also relies on autophagy. B cells lacking Atg5 have an impaired ability to differentiate into plasma cells, produce less immunoglobulins, and are more susceptible to apoptosis [97,98]. In T cells, mTORC1 suppression results in their differentiation into regulatory T cells (T_reg) [99], while memory CD8⁺ T Atg5^null or Atg7^null cells have a compromised lifespan [100]. Autophagy also facilitates macrophage polarization, towards a pro-inflammatory (M1) or anti-inflammatory (M2) phenotype [101,102].

Adipocyte differentiation is also dependent on autophagy, and this process is impaired in cells and mouse models lacking the core autophagy genes, Atg5 or Atg7. Specifically, Atg5^null MEFs fail to differentiate into adipocytes following the stimulation of adipogenesis [103], while Atg7^null 3T3L-1 pre-adipocytes show markedly reduced triglyceride synthesis and storage [104]. Moreover, Atg7^-/- mice display reduced fat mass, smaller adipocytes, and lower body weight [105].

Furthermore, several studies elucidated the role of autophagy in both embryonic and adult neurogenesis. Deficiencies in either Ambra1 or Atg5 impair central nervous system development [106,107]. Knockdown of Atg5 alone is sufficient to stall cortical neurogenesis during early brain development [108], while Atg16l1 hypomorphic mice and primary neurons further highlight the importance of autophagy machinery in modulating neurogenesis [109]. Moreover, conditional knockout of Atg5 in mice leads to the accumulation of tubular ER in axons, culminating in increased excitatory neurotransmission and premature death [110]. Neurogenesis is particularly interesting because it depends on two types of selective autophagy. First, differentiation of human stem cells into neurons (iNeurons) is associated with an increase in PINK1-independent mitophagy, which correlates with changes in the cellular metabolic status. BNIP3L also plays a key role in this context [111]. Second, ER-phagy is critical for reshaping the ER morphology and proteomic landscape during neurogenesis. During axonal differentiation, ER-phagy receptors FAM134A/C and CCPG1 are responsible for remodelling the ER proteome and morphology [112].

In skeletal muscle, autophagy plays an essential role in differentiation, homeostasis, and regeneration [113]. During myogenesis, the transition from myoblasts to myotubes [114], muscle cells undergo extensive cytoplasmic and organelle remodelling. This includes the transformation of the ER into the sarcoplasmic reticulum (SR). While compromised autophagy flux, contributes to the loss of muscle mass seen in elderly individuals, while enhanced autophagy prevents the senescent phenotype in muscle stem cells (MSCs) [115,116]. Studies on low and high passage C2C12 cells demonstrated that autophagy and the redox sensor p66Shc are crucial for myoblast differentiation and cell lineage commitment [117]. Autophagy flux is particularly high in the early stages of myoblast differentiation [118], possibly due to the extensive cellular remodelling and the high energy demands of this process. Disrupting autophagy flux during this critical period, through the deletion of core Atg genes such as Atg5, Atg7 or Beclin-1, results in impaired myoblasts fusion.

Studies in animal models further corroborate the importance of autophagy in myogenesis. Muscle regeneration was impaired in Atg16l1 hippomorphic mice injured with cardiotoxin (CTX). Muscle repair was slower and regenerated fibres were significantly smaller in diameter in Atg16l1^low mice compared to their WT counterparts [119]. Similarly, Ambra1 zebrafish morphants and Ambra1^gt/gt mouse embryos exhibited significant abnormalities in myogenesis [113].

In sum, autophagy is indispensable during development and cellular differentiation, playing critical roles in embryogenesis, post-natal survival, as well as the development and differentiation of tissues (Figure 1).

Figure 1.

The role of autophagy in development and cell differentiation. Autophagy contributes to the organism’s development in several ways and at multiple time points. Autophagy is also essential for terminal differentiation step acting to facilitate cellular remodelling.

ER dynamics in cell differentiation

The ER is a highly complex organelle, composed of a dynamic network of sheets and tubules, encompassing the nuclear envelope and the cortical ER, which extends throughout the cytosol to the plasma membrane. Proper ER architecture is fundamental for cellular function, as mutations in ER shaping proteins are implicated in several human diseases [120]. The peripheral structure of the ER is shaped by membrane-associated proteins that regulate the ratio between sheets and tubules while also coordinating the homotypic fusion between ER membranes. Peripheral ER membranes are characterized by a high degree of curvature, with their tubular morphology maintained by wedge-shaped proteins such as reticulons and REEPs, whereas sheets are stabilized by different proteins like CLIMP63 [121,122].

ER morphology and the ratio between sheets and tubules vary across cell types, in accordance with their functional demands. ER sheets contain ribosomes and are therefore enriched in pancreatic and plasma cells, which have high protein synthesis and secretory activity. In contrast, neurons and muscle cells, which have minimal secretory activity but are characterized by an elongated shape, predominantly feature an extended tubular ER network [121,122].

Since ER structure and function are defined by its proteomic landscape, it must be dynamic, system capable of adapting to cellular changes. Cellular differentiation is a key example of this phenomenon. During neurogenesis and myogenesis, ER protein composition undergoes significant adaptations both in quantity and quality, favouring the formation of tubular structures enriched with specialized proteins. In both cases, ER-phagy plays a crucial role in reshaping the ER proteome [68,112]. Conversely, during plasma cell differentiation, the rough ER undergoes a dramatic expansion to accommodate the folding of nascent immunoglobulin chains. This is accompanied by the upregulation of resident ER chaperones and folding enzymes. This process is tightly regulated by a fine-tuning of the three branches of the UPR [123].

ER morphology, dynamics, and function are intertwined and equally important for maintaining ER homeostasis. Although the precise molecular relationship between these three ER properties remains incompletely understood, disruption in ER dynamics and mutations in ER-shaping proteins are linked to several diseases where ER function is impaired [121].

ER-phagy in differentiation and development

Despite extensive research into the role of autophagy in tissue development and differentiation, little is known about the specific contribution of selective autophagy, and particularly ER-phagy to these processes. Recently, studies have begun to elucidate the biological role of ER-phagy in cellular differentiation (Figure 2).

Figure 2.

The role of ER-phagy during cell differentiation. ER-phagy contributes to cellular differentiation by remodelling ER membranes, in adaptation to the changing cellular needs.

During intestinal development in Drosophila, mitochondria and ER are cleared by mitophagy and ER-phagy, which is critical for the larva to prepupa transition. Although these processes are similarly regulated, they remain distinct. Three Drosophila ER-phagy receptors Atl, Rtnl1 and Trp1, which are orthologs of the human ATL3, RTN3 and SEC62, mediate the extensive ER clearance in enterocytes during this transition. Enterocytes with mutations in Atl and Rtnl1 are characterized by abnormal ER structures with excessive rough ER. Notably, the key mammalian ER-phagy receptors, the FAM134 family of proteins, CCPG1 and TEX264 do not have orthologs in flies. Therefore, this developmental ER clearance in Drosophila enterocytes relies on PINK1, which is typically associated with mitophagy. In this context, PINK1 acts as a molecular switch between mitophagy and ER-phagy. On the one hand, PINK1 promotes Parkin-mediated ubiquitination of mitochondrial proteins, thus favouring mitophagy, while on the other hand, PINK1 also influences the KEAP1 and Cullin3 E3 ubiquitin ligase complex, which is required for Rtnl1 ubiquitination and ER-phagy [124]. Even though PINK1 appears to regulate both pathways, the two types of selective autophagy remain independent, where the loss of ER-phagy receptors Atl, Rtnl1 or Trp1 does not affect mitochondrial clearance, while ER-phagy is paradoxically enhanced in the intestines of Parkin mutants [124].

In the context of mammalian neurogenesis, ER-phagy is mainly regulated by the FAM134 receptor family and CCPG1. Neurogenesis requires a substantial rearrangement of ER membranes to form the refined tubular ER network characteristic of dendrites and axons. ER remodelling during neurogenesis is complex and requires a combined and simultaneous action of multiple ER-phagy receptors. Studies in iNeurons demonstrated that deletion of a single ER-phagy receptor does not significantly impair ER degradation, suggesting redundancy among receptors. Indeed, combinatorial deletions of ER-phagy receptors does impairs ER clearance and dynamics [112].

FAM134 family members play a dominant and redundant role in shaping ER membranes and controlling the removal of ER morphogens, while CCPG1 regulates the turnover of luminal ER proteins [112]. Whereas FAM134A and FAM134B appear redundant in this scenario, the combined loss of two FAM134 proteins strongly influences ER clearance, leading to the accumulation of ER proteins. FAM134C is particularly critical for ER remodelling and targeting ER morphogens, such us REEPs, for degradation. CCPG1, on the other hand mostly regulates intraluminal proteins, likely due to its intraluminal domain in lieu of a RHD [112]. In vivo, mice with a double knockout of Fam134b and Fam134c exhibit an expanded tubular ER along the axons of motor and sensory neurons [125].

Despite FAM134B’s intrinsic ability to vesiculate ER membranes and its physiological role in the dorsal root ganglia [126], it alone only modestly contributes to ER proteome and network remodelling during neurogenesis [112]. However, in collaboration with CANX, FAM134B controls ER-phagy during cerebral cortex development [126], by ensuring proper trafficking of Tropomyosin receptor kinase B (TrkB). Activation of TrkB by EGF, is critical for its transport to the cell surface, where it mediates cellular signalling downstream of brain-derived neurotrophic factor (BDNF) [127], which is essential for cortical plate formation. FAM134B cannot directly interact with TrkB because it lacks an ER luminal domain, therefore, CANX serves as a co-receptor interacting with both TrkB and FAM134B [127].

Neurogenesis is a complex and finely tuned process that drives the specification of diverse cell types, each with distinct morphological and functional properties. Therefore, the involvement of several ER-phagy receptors, each with its unique characteristics beyond their role as selective autophagy receptors, may play a crucial role in shaping ER morphology, protein composition, and function in different neuronal cell types.

Myogenesis is another striking example of ER remodelling during cellular differentiation. When myoblasts fuse to form myotubes, ER membranes are restructured to form the SR, a specialized organelle unique to skeletal muscle. During this process, autophagy flux is enhanced until myotubes mature, and then progressively decreases, establishing a new steady state [68]. Proteomic analysis revealed that, among ER-phagy receptors, FAM134B protein levels are the only ones that change during myogenesis; where myoblasts express the classic, FAM134B1 isoform, while mature skeletal muscles express the shorter, FAM134B2 isoform [66]. FAM134B1, with its full RHD, facilitates dynamic ER remodelling during differentiation enabling the formation of interconnected SR networks. After differentiation is complete, no further network reshaping is required; consequently, ER-phagy flux returns to steady-state levels. FAM134B2, is a truncated isoform which lacks half of the RHD, rendering it less dynamic. The switch between the two isoforms during myogenesis likely reflects the changing ER dynamics [68]. During myogenesis, the balance between the degradation of FAM134B1 and the transcriptional upregulation of FAM134B2 is finely tuned. This is the first documented case of an ER-phagy receptor being replaced by another to maintain ER homeostasis [68].

The physiological role of FAM134B2 in adult skeletal muscle remains unclear. However, FAM134B (likely FAM134B2) mediated ER-phagy helps to maintain ER homeostasis in skeletal muscles following exercise [128].

ER-phagy and diseases

Dysregulation in ER structure and function is implicated in various sensory and neuromuscular disorders. Several genes involved in axonopathies play critical roles in ER shaping and ER-phagy, suggesting that ER dynamics play a central role in disease pathogenesis [129,130]. For example, in sensory neuropathies, mutations in ATL3 disrupt ER-mitochondria contact sites, leading to aberrant ER membrane morphology [131,132]. Similarly, mutations in FAM134B cause hereditary sensory and autonomic neuropathy type II (HSANII) [126]. This is not surprising given that most of these conditions affect cell types in which the ER undergoes significant developmental remodelling and serves highly specialized functions.

Moreover, hereditary spastic paraplegias (HSPs) are a group of inherited neurological disorders characterized by structural abnormalities in organelles, particularly the ER. HSP pathogenesis primarily affects corticospinal and lower motor neurons [120], two cell types which require a precisely structured ER network to function.

Beyond neurological disorders, FAM134B is also implicated in ER storage diseases [133], esophageal and colorectal carcinoma [134,135], allergic rhinits [136], vascular diseases [137] and viral infection [138]. Moreover, RTN3 plays a role in clearing misfolded proinsulin (Akita mutant) from the ER lumen [139], contributes to the development of neuritic plaques and lipid metabolism in Alzheimer’s disease brains [140], and is involved in ER membrane remodelling during viral infection [141].

While there is no clear-cut evidence regarding the involvement of the other ER-phagy receptors in human diseases, CCPG1 is essential for pancreatic cell homeostasis and may be involved in tumorigenesis [44]. Moreover, FAM134A and FAM134C, which share functional similarities with FAM134B, could also be involved in human pathology. Additionally, TEX264 is broadly expressed across different cell types, suggesting potential disease relevance.

Despite these insights, numerous open questions remain in the field of ER-phagy, and further research is necessary to better elucidate its physiological and pathological significance.

UPR in development and differentiation

The accumulation of misfolded proteins in the ER lumen disrupts organelle function, activating the UPR to mitigate stress and restore balance. However, the UPR is not limited to the regulation of protein folding, it also plays a critical role in cellular differentiation [142]. The interplay between the UPR and ER-phagy serves as a protective mechanism in recovery from ER stress [143], proteotoxicity [44], and infection [144]. Moreover, a physiological level of ER stress is essential for the differentiation of connective, muscle, epithelial and neural tissues [[145], [146], [147], [148]]. While controlled UPR activation is necessary to drive differentiation, excessive ER stress can have detrimental effects [142], highlighting the need for precise regulation of UPR signalling.

Interestingly, it is unknown whether the UPR activation is the cause or consequence of cell differentiation. Three different signalling cascades can activate the UPR: IRE1, PERK and ATF6, all of which play critical roles in differentiation processes, including fibroblasts to myoblasts transition [[149], [150], [151]], adipocyte maturation [[152], [153], [154]], myoblasts to myofibers transformation [[155], [156], [157]], and osteoblasts driven bone formation [[158], [159], [160]]. Suppression of any of these pathways negatively impacts the outcomes of differentiation [142].

The UPR activation is triggered by endothelin-1 and thrombin during fibroblasts to myofibroblast differentiation [151], while adipocytes differentiation is regulated by the phosphorylation of eIF2$\alpha$ phosphorylation, XBP1 splicing (sXBP1), and CHOP synthesis [154]. In skeletal muscle, UPR pathways play distinct roles at different stages of differentiation. During developmental myogenesis, the IRE1 and ATF6 pathways are activated, while regenerative myogenesis in adult muscles requires PERK signalling [156]. In bone development, BMP2 induces ER stress via the IRE1, ATF6, and PERK pathways to stimulate osteogenesis [159]. Conversely, osteoclast differentiation involves the IRE1/XBP1 and PERK/eIF2 signalling cascades [161].

Dysregulation of the UPR can lead to abnormal activation or suppression of differentiation which may contribute to disease pathogenesis. For instance, excessive differentiation of fibroblasts, adipocytes, or osteoclasts may lead to fibrosis, obesity and osteoporosis, respectively [142].

Concluding Remarks and Future Perspectives

The pathogenesis of several axonopathies, muscular dystrophies, and certain types of cancer is intricately linked to altered ER functions, which stem from or contribute to abnormal ER morphologies. ER membrane remodelling plays a key role in cellular differentiation and tissue homeostasis, however, the molecular mechanisms that regulate ER dynamics remain largely obscure. Recent discoveries in the field of ER-phagy have highlighted novel pathways that warrant further exploration. Understanding the distinct roles of individual ER-phagy receptors during cellular differentiation may provide critical insights into ER remodelling in tissue-specific contexts and has the potential to inform the development of targeted therapeutic strategies and diagnostic tools for diseases associated with a malfunction in the ER-shaping machinery.

A particularly promising avenue to explore further is the regulation of ER-phagy. However, this is not trivial as the redundancy, cell-specific expression, and subcellular localization of ER-phagy receptors present significant challenges. Technological advances in highly sensitive techniques such as mass spectrometry offer new tools to tackle these challenges and unravel the complexities of ER-phagy by aiding in the detection of PTMs and other crucial regulatory events. An intriguing, yet poorly explored area, is the role of ER dynamics during cell differentiation and how much ER membrane reshaping depends on ER-phagy receptors and autophagy flux. Gaining further insights into these mechanisms can illuminate novel pathways for modulating ER-phagy and advance our understanding of ER dynamics in health and disease.

CRediT authorship contribution statement

Michele Cillo: Writing – review & editing, Writing – original draft. Viviana Buonomo: Writing – review & editing, Writing – original draft. Anna Vainshtein: Writing – review & editing. Paolo Grumati: Writing – review & editing, Writing – original draft, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.