As the site of photosynthesis and carbohydrate metabolism, chloroplasts are essential for phototrophic plant growth. The selective elimination of damaged chloroplasts by autophagy, a process known as chlorophagy, is crucial for chloroplast quality control. However, the mechanisms that activate chlorophagy and that underline its specificity remain poorly understood. Our recent study identified changes in maltose metabolism as a previously unrecognized trigger of a micro-chlorophagy program that depends on components of the core autophagy machinery. These findings provide a new starting point for uncovering the molecular principles that govern micro-chlorophagy. Here, we discuss emerging evidence showing that metabolic changes induce autophagic chloroplast turnover and outline the key challenges in defining the signals that initiate micro-chlorophagy.
Pancreatic ductal adenocarcinoma (PDAC) cells rely on autophagy to adapt to microenvironmental stress. In our recent work, we showed that baseline autophagy flux levels shape the proliferative capacity of human PDAC cells. Here, we extend these findings by demonstrating that low baseline autophagy is consistently associated with enhanced proliferation in vitro and in vivo across multiple human and murine PDAC models. While this inverse relationship between autophagy flux and proliferation is conserved across species, the underlying regulatory mechanisms diverge. Namely, changes in baseline autophagy levels relied predominantly on canonical nutrient-sensing pathways in murine but not human PDAC cells. Additionally, we found that autophagy flux was regulated independently of the cell cycle or the genetic status of p53 (i.e., allelic deletion, point mutation). Collectively, our results reveal conserved phenotypic outcomes but divergent mechanisms, underscoring the need for parallel comparative approaches before extrapolating findings from mouse PDAC models to human disease.
Autophagy promotes tumor progression and immune evasion by numerous mechanisms, representing a promising target for novel (immuno)therapeutic interventions in multiple oncological indications. An expanding literature, however, suggests that safely and effectively targeting autophagy in the clinic remains challenging, especially in populations as complex to manage as patients with pancreatic cancer.
Birt-Hogg-Dubé syndrome (BHD) is an autosomal, dominant condition caused by Folliculin (FLCN) mutation and characterized by enhanced risk for kidney tumors. Previous studies have shown constitutive nuclear localization of the transcription factor TFEB and simultaneous hyperactivation of canonical MTORC1 signaling in the absence of FLCN. Here we assess the impact on autophagy under the situation of combined anabolic and catabolic activation. Using an established BHD patient-derived kidney cancer cell line, we confirmed that TFEB was permanently localized in the nucleus combined with an increase in canonical MTORC1 signaling, whereas bulk autophagy flux and LC3 lipidation were unaffected by FLCN status. However, we found that the autophagy receptor SQSTM1/p62 accumulated in enlarged puncta in the absence of FLCN. Finally, we recapitulate aberrant p62 accumulation in a Norwegian cohort of BHD kidney tumor samples. Our results demonstrate that FLCN loss is characterized by SQSTM1/p62 accumulation, although SQSTM1/p62 appears dispensable for anchorage-independent growth in cell models.
Retinal ganglion cells (RGCs) are the sole projection neurons of the retina and the only direct link between retinal circuitry and the brain. Maintaining this lifelong connection requires constitutive autophagy to preserve organelle quality control and neuronal homeostasis. Although autophagy has been widely studied following ocular hypertension and optic nerve injury, its physiological role in healthy RGCs has remained unclear. We have recently revealed that basal autophagy is highly active in RGCs and that conditional deletion of Atg5 or Atg7 is sufficient to induce progressive RGC dysfunction, optic nerve degeneration, and neurodegeneration. Autophagy deficiency caused the accumulation of swollen mitochondria, distended endoplasmic reticulum, fragmented Golgi, synaptic vesicles, and incomplete autophagosomes accompanied by increased p62 and LC3B levels. These findings establish basal autophagy as an essential housekeeping mechanism that preserves organelle quality control and long-term RGC integrity.
Most proteins synthesized in the endoplasmic reticulum (ER) are covalently modified upon addition of pre-assembled oligosaccharides to side chains of asparagine (N) residues. Processing of N-linked oligosaccharides by ER-resident glucosidases, mannosidases and glucosyltransferases determines the fate of the associated polypeptides. Terminally glucose residues are removed from N-glycans to interrupt the engagement of ER-resident glucose-binding chaperones and promote secretion of native polypeptides. Mannose residues are removed to target terminally misfolded proteins for dislocation across the ER membrane and clearance by the cytoplasmic ubiquitin proteasome system (ER-associated degradation, ERAD). Recent evidence highlights the role of persistent N-glycan glucosylation as a signal that promotes ER lectins-driven segregation of misfolded proteins in ER subdomains that are eventually delivered to endolysosomal compartments for ER-to-Lysosome-Associated Degradation (ERLAD). Here we show that the polymerization-prone Portland variant of Neuroserpin (NS_PL) associated with familial encephalopathy with NS inclusion bodies (FENIB) is a client of the ERLAD machinery. Its lysosomal clearance relies on the LC3-dependent delivery branch of ERLAD involving the lectin chaperone Calnexin (CNX), the ERphagy receptor FAM134B and the SNARE protein Syntaxin17 (STX17), which is engaged upon persistent glucosylation of the NS_PL oligosaccharide linked at the asparagine residue at position 321.
Macroautophagy is an intracellular degradation process that relies on autophagosomes and lysosomes to maintain cellular and organismal homeostasis. Actin cytoskeletal rearrangements driven by the Arp2/3 (actin-related protein 2/3) complex, an essential actin nucleator, impact multiple steps of this pathway, but where and when Arp2/3-mediated actin assembly is most influential has remained unclear. Recent work now shows that the Arp2/3 complex is crucial in the later stages of autophagy due to its function in maintaining lysosomal integrity. WHAMM (WASP homolog associated with actin, membranes, and microtubules) is the key nucleation-promoting factor that activates Arp2/3 at permeabilized lysosomes, uncovering new roles for actin, the Arp2/3 complex, and WHAMM in lysosomal damage responses.
Parkinson's disease-associated proteins PINK1 and Parkin collaboratively regulate stress-induced mitophagy. While in vitro human neuronal cultures are valuable for studying the roles of PINK1 and Parkin in a disease-relevant context, the impact of culture conditions on these processes remains largely underexplored. Here, it is shown that human induced neurons (iNeurons) cultured in N2B27 and BrainPhys medium exhibit distinct PINK1-Parkin-dependent mitophagy phenotypes. Specifically, BrainPhys-cultured iNeurons show greater resistance to PINK1-dependent mitophagy initiation, linked to a reduction in glucose availability and reduced PINK1 protein availabilities, leading to decreases in stress-induced and basal mitophagy fluxes. These findings highlight the critical impact of culture conditions on mitophagy dynamics and emphasize the need to account for media-specific differences when using in vitro models to investigate mitophagy mechanisms in human neurons.
Lipophagy, the selective autophagic degradation of lipid droplets (LDs), is central to cellular lipid homeostasis, yet its dynamic regulation in living organisms has remained largely unobservable in real time. To overcome this barrier, we recently developed the mCherry‑eGFP‑LiveDrop (tfLiveDrop) reporter mice, in which a pH‑sensitive tandem fluorescent probe targeted to LDs via the GPAT4‑derived LiveDrop domain enables real‑time, single‑cell visualization of lipophagic flux in vivo. Using tfLiveDrop mice, we uncovered pronounced organ heterogeneity in basal lipophagy, identified an organ‑specific lipophagic inhibition in type 2 diabetes, and revealed a previously unrecognized developmentally programmed lipophagy induction that drives renal metabolic maturation. Together, these findings demonstrate that tfLiveDrop is a sensitive and versatile reporter for in vivo lipophagy research.
Alzheimer disease (AD) pathology is accompanied by increased senescence and reduced levels of autophagy in the brain. We investigated whether pharmacologically inducing autophagy could alter the senescent phenotype and ameliorate AD pathology. We discovered that Bisdemethoxycurcumin (BDMC), a natural compound found in Curcuma longa, stimulates autophagy in primary astrocytes. We found that autophagy and senescence exhibit an inverse relationship in aging astrocytes, with increased expression of senescent proteins and downregulation of autophagic proteins. However, treatment of aged astrocytes with BDMC reversed the senescent phenotype by ameliorating the impaired autophagy. Interestingly, the senescent phenotype persisted when autophagy was downregulated by knockdown of AMPK. Additionally, BDMC-induced autophagy aided in the removal of amyloid beta (Aβ) that was administered externally to the astrocytes. Further, to validate these results in a mouse model of AD, we confirmed that BDMC significantly penetrates the blood-brain barrier (BBB) in mice. Therefore, we administered 50 and 100 mg/kg b.w. of BDMC to transgenic 3xTg-AD mice for two months. In their hippocampus, the Control 3xTg-AD animals showed more senescent cells and lower autophagy levels. In contrast, autophagic proteins were significantly upregulated while senescence indicators, such as senescence-associated secretory phenotype (SASP) proteins, were sharply downregulated in the brain of treated animals. We discovered that the hippocampus of treated mice had a significantly lower Aβ load. These molecular changes in the brain were ultimately reflected in the improved working memory and neuromuscular coordination behavior of mice treated with BDMC. This study warrants further evaluation of BDMC for the management of AD.
p62 is a multi-domain selective autophagy receptor and signaling scaffold conserved across eukaryotes. However, its evolutionary roots and functional significance in early-diverging eukaryotes like Dictyostelium have remained unexplored. In our recent study, we characterized p62 in the social amoeba Dictyostelium discoideum. Loss of p62 significantly delayed the starvation induced development and led to the formation of smaller multicellular structures. Altered p62 levels also disrupted autophagy flux and cell fate commitment in Dictyostelium cells. Treatment of p62 knockout (p62- ) cells with pulses of exogenous cyclic AMP (cAMP) partially rescued developmental defects, suggesting a potential role for p62 in sustaining intracellular cAMP levels necessary for development. Taken together, p62 emerges as an evolutionarily conserved coordinator of nutrient sensing, second messenger signaling, and autophagy that promotes multicellular development in Dictyostelium.
Mitophagy is the selective degradation program for damaged and unnecessary mitochondria to maintain cellular mitostasis and survival. Specific mutations in the mediators for the canonical ubiquitin (ub)-dependent mitophagy pathway have been identified with unique neurological diseases like Parkinson disease and ALS (amyotrophic lateral sclerosis), metabolic diseases, and cancer. Mammalian OPTN (optineurin) has been shown as a SAR (selective autophagy receptor) for ub-dependent mitophagy in vitro with direct connections of its mutations with glaucoma and ALS. Despite the in vitro demonstration of OPTN’s role in mitophagy, the in vivo physiological characterization of OPTN’s mitophagy function is largely unexplored. In our recent study, we provide in vivo evidence that the Drosophila melanogaster (Dm) protein, Kenny, directly mediates the sequestration of target mitochondria for the progression and completion of ub-dependent mitophagy. This result establishes Kenny as the Dm homolog of OPTN. Previously, Kenny had only been characterized for its role in innate immune activation and modulation. The conclusion from this study provides avenues for further understanding the in vivo signaling regulating Kenny’s role in mitophagy and investigating homologous disease-relevant mutations of OPTN in Dm.
Selective autophagy of the endoplasmic reticulum (ER-phagy/reticulophagy) is essential for organelle homeostasis and host defense, yet how ER quality control (ERQC) pathways distinguish viral glycoproteins from misfolded host proteins remains poorly understood. Recent work identifies TMEM259/MEMBRALIN (transmembrane protein 259) as a selective ER-phagy receptor containing a non-canonical LC3-interacting region (LIR) motif that assembles a dedicated ER-to-lysosome-associated degradation (ERLAD) complex targeting viral class I fusion glycoproteins. TMEM259 is a multi-pass ER membrane protein with luminal domains that recruit MAN1B1 (mannosyl-oligosaccharide 1,2-α-mannosidase) and cytosolic regions that engage VCP/p97 (valosin-containing protein). This TMEM259-MAN1B1-VCP axis directs diverse viral glycoproteins, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, Ebola virus (EBOV) glycoprotein, influenza A virus (IAV) hemagglutinin (HA), and human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein, to lysosomes in a ubiquitin-independent manner. In contrast, misfolded host glycoproteins are primarily cleared through canonical ER-associated degradation (ERAD) or alternative ERLAD pathways. Preferential recognition of densely glycosylated viral substrates suggests that MAN1B1 may function as a glycan-density sensor, enabling TMEM259 to couple ER proteostasis with intrinsic antiviral immunity. These findings expand the conceptual framework of selective autophagy and uncover a specialized ER-phagy pathway dedicated to eliminating viral glycoproteins.
Mitophagy selectively eliminates damaged or excess mitochondria to maintain mitochondrial homeostasis. During this process, mitochondria need to be fragmented to allow their sequestration within autophagosomes. However, the well-known dynamin-related fission factors, Dnm1 in yeasts and DNM1L/DRP1 in mammals, are dispensable for mitophagy, leaving the underlying mechanism unresolved. In the yeast Saccharomyces cerevisiae, the identification of the mitochondrial intermembrane space protein Atg44 (autophagy-related 44) uncovered the existence of a new class of proteins, mitofissin, involved in mitochondrial fission during mitophagy. Whether Atg44 alone is sufficient for mitophagy-associated fission remained unclear. Our recent study identified Mfi2 (mitofissin 2) as a mitochondrial outer membrane-resident mitofissin that is required for efficient mitophagy and acts independently of Dnm1. Our findings indicate that mitophagy-associated mitochondrial fission is driven by mitofissins acting from both the inner and outer mitochondrial membranes. Here, we discuss remaining issues, including how mitofissin activities are regulated and how their function is modulated by mitochondrial lipids such as cardiolipin.
Microautophagy (MI-autophagy) is an umbrella term for intracellular degradative pathways that entail the invagination or protrusion of the limiting membranes of endolysosomal compartments, that is, late endosomes and mammalian lysosomes or yeast and plant vacuoles, followed by pinching-off of the membrane into the lumen of the organelle. During these processes, the material specifically and nonspecifically targeted for degradation is sequestered within the invaginating or protuberating membrane. In contrast to macroautophagy, the molecular mechanisms underlying MI-autophagy are largely unknown due to their diversity and complexity in location, regulation and molecular machinery requirements. Here, we review recent progress in the field of MI-autophagy, describing the molecular basis and functions of the MI-autophagic pathways reported to date in eukaryotic cells, from yeast to mammalian and plant cells.
Mitochondria are central hubs for cellular fitness, empowered by plastic remodeling of their shape, proteome composition, and/or metabolic state. MFN2 (mitofusin 2) mediates mitochondrial fusion and ensures adaptations in response to metabolic changes and stresses. Besides this canonical role, MFN2 serves as a communication hub with other organelles. It tethers mitochondria to the endoplasmic reticulum (ER), lipid droplets, and peroxisomes, regulating calcium buffering, apoptosis, lipid biosynthesis, and lipolysis. Dysfunctional MFN2 causes the hereditary neuropathy Charcot-Marie-Tooth type 2A (CMT2A) and is linked to several metabolic diseases. In a recent publication, we described another fusion-independent role of MFN2 in proteostasis and mitophagy. MFN2 binds the chaperone HSPA8/HSC70 (heat shock protein family A [Hsp70] member 8) and the proteasome, a key function in maintaining mitochondrial and cellular protein quality control, which appears to be lost in the context of CMT2A-associated MFN2 variants.
Identifying mechanisms underlying chemoresistance is essential for improving the efficacy of chemotherapeutic drugs. Previously, we showed that cancer cells respond to gemcitabine by activating protective signals dependent on the master regulator of autophagy and lysosomal biogenesis, transcription factor EB (TFEB). However, how gemcitabine triggers these protective responses remains elusive. While gemcitabine primarily aims at disrupting DNA replication, it is also suspected to induce nucleolar stress. In this study, we aimed to examine the effect of gemcitabine on nucleolar stress and investigate whether nucleolar stress inducers could trigger TFEB-dependent protective signals. Besides gemcitabine causing nucleolar stress, the anticancer agent CX-5461, primarily designed to induce nucleolar stress, promoted TFEB nuclear accumulation. Interfering with TFEB improved the sensitivity of cancer cells to both CX-5461 and gemcitabine. Our findings suggest that TFEB provides broad protection against the stress caused by chemotherapeutic drugs, representing a promising target for intercepting chemoresistance and improving the efficacy of anticancer agents.
In PINK1 (PTEN induced kinase 1)/PRKN (Parkin)-mediated mitophagy, the rupture of the outer mitochondrial membrane (OMM) emerges as a crucial event required for efficient mitochondrial clearance. Mechanistically, OMM rupture exposes inner mitochondrial membrane (IMM) mitophagy receptors, facilitating subsequent autophagic removal. Despite the important role of OMM rupture in mitophagy, the underlying mechanism remains elusive and technically difficult to monitor. In a recent study, we developed a novel fluorescent biosensor to directly visualize OMM rupture. This technique enables temporal and spatial characterization of OMM rupture and provides a powerful platform to dissect the underlying mechanism. Using this tool, we revealed that VCP (valosin containing protein) and its recruitment factors are required for OMM rupture, suggesting that VCP-dependent remodeling of the OMM proteome primes the rupture of OMM during mitophagy.Abbreviations: ARIH1, Ariadne RBR E3 ubiquitin protein Ligase 1; AMFR, autocrine motility factor receptor; ANKRD13A, ankyrin repeat domain-containing protein 13 A; FUNDC1, FUN14 domain containing 1; OA, oligomycin and antimycin; CID, chemical-induced dimerization; IMM, nner mitochondrial membrane; LC3, microtubule-associated protein 1 light chain 3; MUL1, mitochondrial E3 ubiquitin protein ligase 1; NIX, BCL2 interacting protein 3 like; OMM, outer mitochondrial membrane; UBXN1, ubiquitin regulatory X domain-containing protein 1; UBXN6, ubiquitin regulatory X domain-containing protein 6; VCP, valosin-containing protein; WIPI2, WD repeat domain phosphoinositide interacting protein 2.
Autophagy is a cellular process that maintains kidney physiology by recycling intracellular components to preserve homeostasis. In the kidney, autophagy supports energy metabolism and integrity across multiple cell types. Its regulation is tightly governed by nutrient availability, hormonal cues, and oxygen levels, primarily through signaling pathways such as mechanistic target of rapamycin kinase (mTOR), AMP-activated protein kinase (AMPK), and transcription factor EB (TFEB). Under physiological conditions, autophagy is dynamically regulated to meet metabolic demands. However, aging, obesity, and metabolic stress impair lysosomal function, leading to a pathological state termed autophagic stagnation, in which autophagosomes accumulate but degradative flux is compromised. Rather than being uniformly protective, this stagnation promotes cellular damage and contributes to kidney disease progression. Notably, autophagic stagnation in proximal tubular epithelial cells (PTECs) contributes to acute kidney injury (AKI)-to-chronic kidney disease (CKD) transition and exacerbates lipotoxicity in obesity-related kidney disease. Recent studies highlight the importance of transcriptional regulators - including TFEB and MondoA - in maintaining autophagic activity and mitochondrial homeostasis. Therapeutic strategies aimed at restoring autophagic flux - pharmacologically or through lifestyle interventions such as caloric restriction - hold promise for preserving kidney function. Deeper understanding of cell type - specific autophagy regulation will be critical for developing targeted and context-specific therapies.
Macroautophagy/Autophagy is a highly conserved mechanism that targets cytoplasmic cargo for degradation and recycling. At present, 45 autophagy-related (ATG) genes have been identified in fungi. Due to this complexity, the autophagy pathway must be strictly regulated at multiple levels (transcriptional, post-transcriptional, translational, and post-translational). Dysregulation of autophagy can have detrimental effects on cell health and survival. Therefore, investigation into the mechanisms regulating autophagy is critical. The nonsense-mediated mRNA decay (NMD) pathway targets transcripts with premature translation termination codons (PTCs), although NMD also regulates normal transcripts. NMD requires conserved factors in yeast - Upf1, Upf2, and Upf3. Here, we demonstrate that autophagy activity increases in upf1∆ upf2∆ upf3∆ cells. We also show that autophagy is enhanced in upf3∆ cells through multiple assays. UPF3/Upf3 expression decreases during starvation and autophagy induction. Loss of UPF3 results in the upregulation of ATG16/Atg16, which is required for autophagosome formation. Furthermore, ATG16 is likely targeted by NMD. These findings provide insight into how yeast cells may modulate autophagy through the mRNA decay factor Upf3.
