EMBO Reports

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

Cancers maintain their telomeres through two telomere maintenance mechanisms: 85-90% of cancers rely on telomerase (TEL+), while 10-15% of cancers adopt the Alternative Lengthening of Telomeres (ALT) pathway. The Break-Induced Replication (BIR) pathway plays a critical role in maintaining telomere length in the ALT+ cells. In both yeast and human, PIF1, a 5' to 3' helicase, is required for the robust activity of BIR. However, the extent of human PIF1 (hPIF1) involvement in the ALT pathway remains unknown. Here we showed that hPIF1 can be recruited to damaged telomeres in ALT+ cells. In addition, we demonstrated that inhibition of hPIF1 induced DNA damage and G quadruplex (G4) accumulation at ALT telomeres, leading to a moderate reduction of the mean telomere length. Most interestingly, we demonstrated that inhibition of hPIF1 also attenuates checkpoint activation, BLM recruitment, single-stranded DNA (ssDNA) formation, DNA damage, and G4s at telomeres in the FANCM deficient ALT+ cells. Finally, we showed that inactivation of hPIF1 affects the viability of both ALT+ and TEL+ cancers, suggesting that hPIF1 is a potential drug target for cancer therapy.

Adult neural stem cells (NSCs) in the mouse brain are predominantly quiescent, with activation tightly regulated to balance neurogenesis and stem cell maintenance. Circadian clocks temporally organize core cellular processes, potentially gating NSC activation. We examined adult NSC temporal dynamics across the day and observed rhythmic expression of core circadian clock components BMAL1 and PER2 in both mammalian niches, the subgranular zone (SGZ) of the dentate gyrus and the subventricular zone (SVZ). While cell cultures derived from both niches show BMAL1 and PER2 protein, only SGZ-derived NSCs exhibit synchronized self-sustained core clock oscillations across the population. Comparative analyses of WT and Per2 knockout NSCs revealed state-specific, circadian clock-dependent oscillatory transcriptional programs. This identified ASCL1 and CCND1 as candidate regulators of cell-cycle coordination, with daytime accumulation preceding a nighttime S-phase peak. The temporal control of S-phase entry in NSCs was abolished in Per2 knockout mice. Together, our findings reveal state-specific, PER2-dependent circadian regulation of adult NSCs in both neurogenic niches, and a specific dependence on external stimuli for synchronization in the SVZ.

Previous studies have demonstrated that Telomerase Reverse Transcriptase (TERT) is localized inside mitochondria, where it associates with mitochondrial DNA (mtDNA), thereby influencing replication and transcription. Using a combination of quantitative fluorescence microscopy and biochemistry, we demonstrate that TERT binds indirectly to mtDNA through the interaction with mitochondrial transcription factor A (TFAM) to induce mitochondrial fragmentation. These findings reveal a novel mitochondrial interaction involving TERT and highlight a role for TERT and TFAM in the regulation of auto/mitophagy. This interaction is an important factor for mitochondrial function and contributes to the remodelling of mitochondrial respiratory capacity.

Life on earth has always been exposed to the fluctuating Earth's magnetic field, but a magnetic sense affecting behavior has been debated for mammals. We here report that mice, kept under constant laboratory conditions, showed fluctuations in spontaneous behavioral activity with a periodicity of ~14 and ~28 days. This was confirmed in nine cohorts from four facilities on two continents, covering 3 to 41 months. Such oscillations were also maintained in brain Bmal1 knockout mice lacking circadian rhythms, suggesting independence of the circadian clock. The behavioral activity peaked around full and new Moon, and showed a strong alignment with the periodic geomagnetic fluctuations originating in the Earth's iono- and magnetosphere that are modulated by solar rotation and the orbital motion of the Moon. In the ultradian range, this alignment persisted in CRY1/2 knockout mice, suggesting that solar-lunar-driven geomagnetic fluctuations can modulate behavior rhythms independently of CRY1/2.

Macroautophagy is a conserved catabolic process that facilitates the degradation of cellular material by capturing it in double membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, selective macroautophagy is initiated by the scaffolding protein Atg11. Atg11 recruits the transmembrane protein Atg9, which resides in small vesicles, to autophagic cargo. Atg9 vesicles then fuse, forming the initial membrane sheet that expands into the autophagosomal membrane. While it is known that Atg9 interacts with Atg11 via a set of hydrophobic amino acids in the disordered N-terminus of Atg9, it is unclear how Atg11 mediates this interaction. To gain insight into this unknown aspect of autophagy initiation we utilized a combination of biochemical, structural, and cellular approaches. We demonstrate that the N-terminal domain (NTD) of Atg11 is the primary interaction site for Atg9, but the NTD requires clustering by the C-terminal region of Atg11 for its complete interaction with Atg9. We investigated the structure of the Atg11-NTD using cryo-EM which, in combination with AlphaFold modeling, revealed a positively charged binding pocket within the Atg11-NTD that is essential for Atg9 binding. Mutation of this conserved binding pocket leads to a loss of Atg9 binding in yeast and a reduction in the selective autophagy of mitochondria. Taken together, our results demonstrate the mechanism by which Atg11 recruits Atg9 to autophagy initiation sites.

Animal cells respond to hypoxic stress through cell-autonomous stabilization of HIF-1, a conserved transcription factor that mediates adaptation to low-oxygen environments. Although HIF-1 is essential for viability under hypoxia, its persistent activation during development causes miswiring defects in the nervous system, raising the question of how developing neurons balance the pro-survival benefits of HIF-1 stabilization against these associated risks. Here we show that C. elegans neurons address this challenge by mobilizing an internal promoter within the hif-1 locus to generate a dominant-negative isoform, HIF-1c, which acts in the nucleus to limit HIF-1/AHA-1 heterodimer formation. This buffering mechanism supports proper neuronal migration, axon guidance, and circuit formation and its loss phenocopies the defects seen when the ubiquitous HIF-1 degradation pathway is disrupted, confirming that HIF-1c serves as a critical layer of protection for the developing nervous system against hypoxic-induced errors. Lastly, we identify the retinoblastoma protein LIN-35 as a modulator of HIF-1 signaling in worms via control of hif-1c expression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/745244v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@3ad347org.highwire.dtl.DTLVardef@4fd4forg.highwire.dtl.DTLVardef@1928804org.highwire.dtl.DTLVardef@11fd1e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Faithful inheritance of DNA methylation contributes to the memory of epigenetic states and protects against disease. While the mechanisms underlying DNA methylation maintenance at replication forks are well characterized, whether and how DNA methylation is altered or maintained at sites of DNA damage repair is still poorly understood. Here, by exploiting sequencing, imaging and proteomic approaches in mammalian cells exposed to UV radiation, we show that the majority of DNA methylation marks are maintained during UV damage repair and we dissect the molecular machinery involved in DNA methylation control. We detect the recruitment to sites of repair synthesis of the DNMT1 and DNMT3A DNA methylating enzymes, driven by the DNMT1 cofactor UHRF1 and by UV damage repair endonucleases. We also uncover a crosstalk with histone dynamics, whereby newly deposited H3.3 histones at UV damage sites promote the recruitment of DNMT1. Functionally, we reveal the importance of the DNA methylation maintenance machinery for the transcriptional response to UV damage and sustained cell proliferation. This work provides a comprehensive picture of DNA methylation control mechanisms following DNA damage, with important implications for our understanding of human diseases with an altered methylome.

Vacuolar ATPases (V-ATPases) are highly conserved multi-subunit proton pumps that drive the acidification of intracellular vesicles, especially endosomes and lysosomes. By regulating progressive acidification of the endolysosomal pathway, V-ATPase activity impacts signaling transduction pathways both positively (e.g., internalization and activation of receptor-ligand complexes in endosomes) and negatively (e.g., degradation of pathway mediators in lysosomes). While the role of V-ATPases in human neurodegenerative diseases and cancer has been extensively studied, the requirement for these proteins in intestinal restitution remains poorly understood. Here, we use Drosophila to study the role of V-ATPases in regulating intestinal-injury and repair driven by excessive oxidative stress. We find that RNAi driven depletion of multiple subunits of the V-ATPase complex suppressed oxidative stress-induced lethality. By contrast, depletion of the main lysosomal catabolic enzyme in Drosophila (Cathepsin-D) had no effect. On a cellular level, these effects map to absorptive enterocytes (ECs) of the Drosophila intestine. Molecular analysis of intestines following injury by oxidative stress compared to uninjured controls reveals increased cell death, increased JNK-pathway activity, and increased IMD/NF-{kappa}B pathway signaling reporter expression compared to uninjured controls. Depletion of Vha44 (subunit C of the V1 complex) was sufficient to suppress the increased cell death, JNK pathway, and IMD/NF-{kappa}B pathway markers induced by oxidative stress in the intestine. Furthermore, overexpression of the MAP3K TAK1 enhanced death, JNK pathway and IMD/NF-{kappa}B pathway activation in a Vha44 dependent manner. These findings suggest that inhibition of V-ATPase activity can protect against intestinal injury caused by excessive oxidative stress. On a molecular level, we find that attenuation of endolysosomal acidification dampens pro-apoptotic JNK and IMD/NF-{kappa}B pathways, highlighting endosomal acidification as a potential amplifier of excessive oxidative stress.

In response to tissue damage, the epidermal barrier must be restored immediately to prevent excessive blood loss and infections. How cells coordinate actin-driven protrusions and membrane transport during reepithelialization is poorly understood. Here, we identified Rab35 as an important driver of epidermal wound closure in Drosophila. Endogenous Rab35 positive vesicles are directed to the wound edge and in protruding lamellipodia. Suppression of Rab35 function results in striking lamellipodia defects and an accumulation of PIP2-positive vesicles. Depletion of Fascin, a known Rab35 effector phenocopies rab35 knockdown. Upstream of Rab35, we identified CG18659 as potential Rab35-GEF protein, which physically interacts and colocalizes with Rab35. Suppression of Rab35 GEF CG18659 or overexpression of Rab35 GAP Whacked results in strong wound healing defects. We propose model in which Rab35 drives epidermal wound closure by promoting Fascin-stabilized actin-driven protrusions and vesicle transport required for cell membrane expansion and local PIP2 delivery at the wound site.

Through mitochondrial outer membrane permeabilization (MOMP), mitochondria are central to apoptosis. MOMP is pro-inflammatory, impacting diverse processes including anti-tumor immunity and cellular senescence. Despite this, how damaged mitochondria activate inflammation remains unclear. Recent studies have shown that following MOMP, mitochondria are extensively ubiquitinated activating pro-inflammatory NF-{kappa}B signaling. We identify the E3 ligase, RNF213, as essential for pro-inflammatory mitochondrial ubiquitination following MOMP. RNF213 has an established role in pro- inflammatory, cell-autonomous immunity to bacteria and other pathogens. We discover that RNF213 is required for mitochondrial M1-linked (linear) ubiquitination. Interestingly, this was found to be independent of the canonical M1-ubiquitin ligase complex LUBAC. RNF213 can directly catalyze M1-linked ubiquitination. By promoting mitochondrial M1-linked ubiquitination, RNF213 initiates the recruitment of the essential NF{kappa}B adaptor molecule, NEMO, and subsequently activates pro-inflammatory NF-{kappa}B signaling. Collectively, our findings highlight striking similarities between how cells detect damaged mitochondria and intracellular pathogens, leading to inflammation.

Phagocytosis is a conserved effector process of innate immunity that enables specialized immune cells to detect, engulf, and degrade invading microbes as well as dying cells and cellular debris. Soluble opsonins enhance this process by coating target surfaces and promoting their recognition and uptake by phagocytic cells. In Drosophila, many phagocytic receptors of the Nimrod family have been characterized, but opsonins remain comparatively poorly understood. Here, we identified the secreted Nimrod protein NimB2 as an insect opsonin that promotes the clearance of Staphylococcus aureus. NimB2 is produced by the fat body, secreted into the hemolymph, and required for resistance to S. aureus infection. NimB2 null mutants showed reduced phagocytosis of S. aureus while maintaining Toll dependent antimicrobial peptide expression. NimB2 promotes S. aureus binding to plasmatocytes (Drosophila macrophages). Using binding assays and bacterial cell wall mutants, we found that NimB2 recognizes a lipoteichoic acid (LTA) dependent determinant on the S. aureus surface and coats the bacterium. We further show that this coating enables efficient recognition by hemocytes via the phagocytic receptor Eater, which is necessary for NimB2 dependent binding. Moreover, Eater overexpression enhances NimB2 mediated association of S. aureus with hemocytes. Together, our results establish NimB2 as an insect opsonin that links a specific bacterial ligand to a phagocytic receptor.

Telomeres, the ends of linear chromosomes, form a cap composed of specific telomeric DNA associated with specialized proteins that ensures efficient protection against chromosome degradation and, consequently, overall genome instability. Three main complexes have been implicated in chromosome end protection, namely telomerase, shelterin, and the CST complex (CTC1/Cdc13-STN1-TEN1). In a recent study, we uncovered mutants of the S. cerevisiae CST in which damage was sensed both by the two major spindle checkpoints and the major DNA damage checkpoint. In the best-studied of these mutants, stn1-sz2, the stability and organization of the mitotic tubulin spindle was profoundly affected, as were the organization of the centrosomes and the kinetochores. Here, we find that the stn1-sz2 mutant is hypersensitive to deletion mutants of two members of the kinetochores, Ctf19 and Mcm21, which correlated with mis-localization of their associated subunit in the COMA complex, Okp1, during anaphase. Defects in interactions between the kinetochores and the tubulin spindle are entirely sensed by the SAC (spindle assembly checkpoint). Here, we find that some of the stn1-sz2 damage is recognized by the Mad2 checkpoint protein independently from the SAC. The data presented here contribute to a better understanding of the damage in stn1-sz2, the prototype of novel mutants of telomeric CST characterized by mitotic-spindle-damage in addition to the classical telomeric-DNA-damage.

Rubicon is a negative regulator of autophagy and the endolysosomal network (ELN) and an antagonist of the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Inhibition of Rubicon is considered a potential means to therapeutically upregulate autophagy and the ELN to treat Parkinsons disease and other conditions characterized by autophagic and ELN dysfunction. Rubicon is specific for the UVRAG-containing PI3KC3-C2 over the purely autophagic ATG14- containing PI3KC3-C1 complex. Here, we determined the high-resolution cryo-electron microscopy structure of PI3KC3-C2 in complex with the PI3KC3-binding domain (PIKBD) of Rubicon and compared it to cryo-EM structures of unbound PI3KC3-C2 and PI3KC3-C1. Rubicon binds directly to PI3KC3-C2 only via the BARA domain of the BECN1 subunit, which is common to both C1 and C2. The selectivity of Rubicon for the PI3KC3-C2 complex over the PI3KC3-C1 complex is attributed to a conformation of the BECN1BARA domain induced by UVRAG, rather than to direct contact with UVRAG or direct antagonism by the ATG14 subunit of PI3KC3-C1. Targeted disruption of the Rubicon:PI3K3-C2 structural interface by site-directed mutations enhances mitophagic activity in human epithelial cells to levels comparable to those observed in Rubicon knockout (KO) cells. Similarly, disruption of the interaction in Rubicon-overexpressing hippocampal neurons restored lysosomal flux to wild-type levels. These data show that suppressing the function of PI3K3- C2 can fully account for the negative regulatory effects of Rubicon in the autophagy and ELN pathways. Significance StatementEndolysosome maturation and autophagosome-lysosome fusion require the production of phosphatidylinositol 3-phosphate (PI(3)P) by the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Rubicon is a key negative regulator of endolysosomes and autophagy that suppresses PI3KC3-C2 activity. Here, we reveal in atomistic detail how Rubicon selectively recognizes PI3KC3-C2. Disrupting the Rubicon-PI3KC3-C2 interaction restores mitophagy and enhances lysosomal activity to the same extent as Rubicon gene deletion, establishing that PI3KC3-C2 inhibition fully accounts for the biological regulatory effects of Rubicon in the autophagy and lysosome pathways.

MTCH2 (mitochondrial carrier homolog 2) is a noncanonical member of the solute carrier family with five transmembrane (TM) helices, localized to the mitochondrial outer membrane. MTCH2's atypical topology creates a membrane-accessible hydrophilic groove, predicted to be necessary for its protein insertase and lipid scramblase activities. MTCH2 is linked to lipid metabolism and obesity and is required for starvation-induced mitochondrial hyperfusion. Here, we show that MTCH2 is a stable component of a complex containing the Armadillo (ARM) repeat-containing protein, ARMC1, and the DnaJ/Hsp40 chaperone protein, DNAJC11. Protein crosslinking, protein structural modeling, and molecular dynamics simulations demonstrate that the ARMC1 alpha-helical C-terminal domain (CTD) inserts into and stably interacts with the MTCH2 hydrophilic groove and blocks its lipid scramblase activity. We observe that starvation-induced mitochondrial fatty acid oxidation (FAO) is negatively regulated by the ARMC1-MTCH2 interaction. In ARMC1-deficient cells, FAO is stimulated compared to wild-type cells and lipid droplet abundance is significantly reduced. The altered lipid phenotype of ARMC1-/- cells is strictly dependent on MTCH2 and is reversed by ARMC1 expression in a manner dependent on its CTD. Beyond this metabolic axis, we also identify a function for ARMC1 in regulating lysosomal distribution and autophagic flux that is independent of its CTD and interaction with MTCH2. Thus, our data support a model in which the MTCH2-ARMC1 interaction functions as a metabolic switch during starvation to regulate the balance between fat storage and fat burning.

Polyphosphate (polyP), a linear polymer of orthophosphate residues, is enriched in secretory granules in specialised mammalian cell types including platelets and mast cells. Although polyP released during activation and degranulation of these cells has been shown to promote blood clotting and inflammation, little is known about the mechanisms governing polyP synthesis in these granules. In mice, the loss of IP6K1, an enzyme that catalyses the production of 5-InsP7, has been shown to result in depletion of platelet polyP and impaired hemostasis. Here, we use the rat mast cell line RBL-2H3 as a model to study the regulation of polyP synthesis in secretory granules. By monitoring real-time polyP synthesis in isolated mast cell granules, we demonstrate that ATP is the substrate fuelling granule polyP production. By the use of inhibitors, we show that accumulation of polyP in granules requires an intact transmembrane proton gradient maintained by vacuolar H+ATPase (V-ATPase). In RBL-2H3 cells, depletion of IP6K1 led to a substantial reduction in cellular polyP levels and defective accumulation of polyP, serotonin, and tryptase inside granules. Cells with reduced IP6K1 showed a profound loss of granule acidification, correlating with downregulated levels of V1 subunits of V-ATPase. Adding back active or catalytically inactive IP6K1 rescued the expression of V-ATPase V1 subunits, reversed granule deacidification, and restored polyP levels in IP6K1-depleted cells. Together, these data unveil a role for IP6K1 in maintaining granule pH and thereby supporting polyP synthesis in mammals.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with presequences which serve as targeting signals for the mitochondrial matrix, where they are cleaved by the mitochondrial processing peptidase (MPP). In this study, we comprehensively elucidated the role of the presequence and the mature part of mitochondrial precursors in the cytosol, by use of a cytosol-targeted MPP which prematurely processed mitochondrial precursors. Over time, cytoMPP resulted in mitochondrial depletion. However, the cellular response to cytoMPP was surprisingly different to that observed for other models of mitochondrial import inhibition. Cytosolic maturation rendered many proteins stable in the cytosol, indicating that their mature parts lack ubiquitination signals. Accordingly, cytoMPP did not induce the upregulation of the proteasome, which normally is a hallmark of mitochondrial dysfunction. Instead, cytoMPP elicited a heat shock response and impaired the sequestration of precursors in the cytosol. Our observations demonstrate that mitochondrial presequences are more than just address labels. Rather, they play an important role in quality control and orchestrate the cellular response to defects in mitochondrial protein import.

Peroxisomes, along with mitochondria, coordinate and compartmentalize oxidative metabolism in eukaryotic cells. Rare genetic disorders caused by mutations in PEX genes impair peroxisome function and cause Peroxisome Biogenesis Disorders, which are characterized by liver and neurological dysfunction, hearing and vision loss, and metabolic abnormalities. The majority of Peroxisome Biogenesis Disorders (PBDs) are caused by mutations in the gene encoding PEX1, which together with PEX6 forms a hetero-hexameric AAA-ATPase complex that drives the import of enzymes into the peroxisome lumen. One particular destabilizing mutation - PEX1G843D - results in almost 30% of all cases. Here we show that deficiencies in PEX1 lead to increased levels of the oxygen-responsive transcription factor HIF1 in normoxia, as well as a HIF1 transcriptional signature. The increase in HIF1 protein was rescued by overexpression of PEX1WT, suggesting PEX1 deficiencies modulate HIF1 signaling. The increased levels of HIF1 were not explained by defects in the oxygen responsive degradation pathway of HIF1 that relies on proline hydroxylase domain enzyme-dependent hydroxylation and von Hippel Lindau tumor suppressor protein-dependent ubiquitination. Instead, we found that PEX1 deficiency alters HIF1 proteostasis by reducing degradation through a hydroxylation- and autophagy- dependent mechanism. Notably, enhancing autophagic capacity by ULK1 agonism was sufficient to reduce HIF1 levels in PEX1 deficient cells. Lastly, we demonstrate that upon hypoxia-reoxygenation, PEX1 deficient cells are slower to reset HIF1 levels. Our results suggest that PBD patients with PEX1 deficiency may be susceptible to dysregulation of the HIF1 pathway, particularly in tissues where oxygen gradients are physiological or developmentally required.

Reactive oxygen species (ROS) are early signals of tissue damage, but how they engage growth-promoting pathways in vivo is unclear. Here we show that ROS promote regeneration by inhibiting PTEN, thereby activating PI3K-Akt signaling in Drosophila melanogaster. We identify cysteine 79 as an essential residue for redox regulation of PTEN, demonstrating that a ROS-insensitive PTEN mutant uncouples Akt activation from oxidative stress. This disruption impairs regenerative growth in wing imaginal epithelia and stem cell proliferation in the adult gut. Mechanistically, Akt activation links ROS signaling to p38 MAPK-dependent regeneration responses. Together, our findings define a conserved ROS-PTEN-Akt signaling axis that integrates oxidative damage with regenerative growth, establishing PTEN as a key redox-sensitive regulator of tissue repair.

Endocytosis internalises nutrients, regulates extracellular signals, and recycles membrane components. Clathrin polymerises into a coat that shapes the endocytic vesicle from the plasma membrane. However, the role of clathrins dynamic assembly in the endocytic process remains unclear. We show, using two-colour fluorescence recovery after photobleaching assays in yeast, that the clathrin coat turns over rapidly in the early phase of endocytosis, dependent on the auxilin Swa2 and its ATPase. In the late phase the turnover is stopped by the coat protein Sla1. Regulated clathrin turnover is critical for the timing of endocytic progression and for controlling coat size. In the absence of this dynamic regulation the endocytic coats become abnormally large, resulting in the failure of the final actin-driven vesicle budding. These findings reveal that, in addition to its classic structural function, the dynamic properties of the clathrin lattice are critical for both the temporal and mechanical aspects of endocytosis.