Transcriptional control of lysosome biogenesis is an important mechanism underlying cellular adaptation to stress. It is largely unclear how cell surface changes or signals induce alteration in lysosome numbers. By developing a Caenorhabditis elegans-based heterologous TFE3 activation system, we here identify the non-receptor tyrosine kinases SRC-1/-2 (C. elegans) and FGR (mammals) as critical regulators of lysosome biogenesis. In C. elegans, inactivation of src-1/-2 leads to nuclear enrichment of ectopically expressed TFE3 and increased intensity of lysosomal markers. In mammalian cells, FGR inhibition or deficiency similarly results in TFEB/TFE3-dependent lysosomal increase. FGR acts through AKT2 by promoting the activation of the latter. FGR associates with the plasma membrane but is internalized onto endosomes and reaches lysosomes along the endosome-lysosome pathway following endocytosis. Lysosomal FGR promotes AKT2 recruitment to lysosomes, where it phosphorylates TFEB/TFE3 to prevent their activation. Together, these findings reveal a plasma membrane-to-lysosome signaling axis that is required for endocytosis-associated lysosome homeostasis.

Dysregulation of the autophagy–lysosome pathway is implicated in neurodegenerative disorders. To extend the chemical diversity of jatrophane diterpenoids from Euphorbia peplus and evaluate lysosome-associated phenotypes, phytochemical isolation and a LysoTracker Green assay in HeLa cells were performed. Five previously undescribed jatrophane diterpenoids, Euphjatrophanes M–Q (1–5), together with three known analogues (6–8), were isolated. The structures of 1–5 were assigned from one- and two-dimensional NMR, IR, and UV and the available HR-ESI-MS data, together with a comparison with reported compounds. At 10 and 40 μM, compounds 1, 2, and 4–8 produced higher LysoTracker-associated fluorescence than the DMSO vehicle control, whereas compound 3 showed a small numerical increase at 10 μM and a decrease at 40 μM. Compound 1 gave the largest numerical response (1.76-fold and 3.01-fold at 10 and 40 μM, respectively). These findings provide preliminary cell-based phenotypic evidence of lysosome-modulating activity and require orthogonal validation.

Mitochondrial Ca2+ uptake couples cellular Ca2+ signals to mitochondrial metabolism and stress responses and is primarily mediated by the mitochondrial Ca2+ uniporter (MCU). Here, we identify an ATAD-3-dependent mechanism that promotes mitochondrial Ca2+ uptake following plasma membrane injury in C. elegans. Living imaging reveals robust, albeit delayed, mitochondrial Ca2+ influx following injury in mcu-1 mutants, indicating the existence of a compensatory Ca2+ uptake mechanism. RNAi screen identifies the conserved AAA+ ATPase ATAD-3 as an essential regulator of this pathway, whereas MICU-3 acts as a negative regulator. Wounding induces ATAD-3 oligomerization and mitochondrial enrichment, which are enhanced in mcu-1 mutants. Disrupting ATAD-3 oligomerization or expression of disease-associated ATAD-3 variants abolishes wounding-induced mitochondrial Ca2+ uptake. Structure modeling predicts that oligomeric ATAD-3 adopts a channel-like architecture. These findings uncover ATAD-3 as a context-dependent regulator of mitochondrial Ca2+ homeostasis during cellular stress and provide insight into how this process may be disrupted in disease. In this study, Xu et al. identify ATAD-3 as a context-dependent regulator of mitochondrial Ca²⁺ uptake that is activated by cellular injury through oligomerization and mitochondrial enrichment, independent of MCU-1.

Mitochondria form highly complex and dynamic networks to maintain their homeostasis. However, the underlying mechanisms remain elusive. Here we report a PI(3)P-dependent mechanism that regulates the mitochondrial dynamics required for formation of mitochondrial networks. Using genetic screening, we reveal that mutations of Caenorhabditis elegans EXC-5/FGD lead to formation of spherical and unconnected mitochondria. EXC-5 binds to endosomal PI(3)P generated by the PI 3-kinase VPS-34 and is recruited to endosome-mitochondrion contacts, where it acts as the guanine nucleotide exchange factor to activate the CDC-42 GTPase. Loss of exc-5 or vps-34 similarly disrupts mitochondrial and actin networks as well as mitochondrial recruitment of DRP-1, leading to failure of mitochondrial fission, branching, and elongation. In contrast, expression of constitutively activated CDC-42 ameliorates the defective mitochondrial networks in an actin-dependent manner. Together, these findings suggest a PI(3)P-EXC-5-CDC-42 axis that acts at endosome-mitochondrion contacts to regulate actin organization for maintenance of mitochondrial dynamics and networks.

Mitochondria catabolize nutrients by generating sequentially-ordered organic acid intermediates that are oxidized through the tricarboxylic acid cycle. Pathogenic accumulation of metabolic organic acids manifests as devastating organic acidemias/acidurias and other severe diseases, but the underlying mechanisms are largely unknown. Using unbiased C. elegans genetic screening, we here reveal that mutations in the phosphoenolpyruvate carboxykinases PCK-1 and PCK-2 cause buildup of oxaloacetate, a key tricarboxylic acid cycle intermediate, leading to severe mitochondrial damage. Depletion of mitochondrial GOT-2.1 or GOT-2.2, which catalyze oxaloacetate conversion to aspartate, also causes oxaloacetate accumulation and defective mitochondria with disrupted cristae. We demonstrate that oxaloacetate binds the MICOS complex subunit CHCH-3/MIC19 and inhibits its function of promoting IMMT-1/MIC60-dependent membrane shaping and remodeling. In mammalian cells, aberrant OAA buildup similarly causes mitochondrial impairment through MIC19 and MIC60. These findings not only provide important mechanistic insights into mitochondrial damage in the context of defective oxaloacetate metabolism, but also suggest therapeutic strategies for oxaloacetate-related mitochondriopathies.

Maintenance of mitochondrial integrity is fundamental for cellular survival, yet how cells recognize catastrophic mitochondrial membrane damage remains unknown. Here, we identify MAI-1 as the first genetically encoded reporter of severe mitochondrial membrane damage. MAI-1 is a Caenorhabditis elegans homolog of the ATP synthase inhibitor IF1 that lacks a mitochondrial targeting sequence, resides in the cytosol under basal conditions, but rapidly and irreversibly translocates to severely damaged mitochondria within milliseconds. We validate MAI-1 across diverse injury paradigms and demonstrate that cytosolic IF1 variants from other species exhibit conserved damage-induced recruitment. Mechanistically, MAI-1 recruitment requires the presence of an intact ATP synthase complex. Using MAI-1 as a sensor, we uncover that these severely damaged mitochondria are cleared through the LGG-1-mediated, PINK1/PARKIN-independent lysosomal pathway. Together, our findings establish a powerful tool for visualizing severe mitochondrial membrane damage and reveal a surveillance mechanism dedicated to structural integrity control.

Lysosomes are membranous organelles that are crucial for cell function and organ physiology. Serving as the terminal stations of the endocytic pathway, lysosomes have fundamental roles in the degradation of endogenous and exogenous macromolecules and particles as well as damaged or superfluous organelles. Moreover, the lysosomal membrane is a docking and activation platform for several signalling components, including mTOR complex 1 (mTORC1), which orchestrates metabolic signalling in the cell. The integrity of their membrane is crucial for lysosomes to function as hubs for the regulation of cell metabolism. Various agents, including pathogens, nanoparticles and drugs, can compromise lysosomal membrane integrity. Membrane permeabilization causes leakage of proteases and cations into the cytosol, which can induce cell death pathways and innate immunity signalling. Multiple pathways repair damaged lysosomes, and severely damaged lysosomes are degraded by an autophagic process, lysophagy. Moreover, lysosome damage activates transcriptional programmes that orchestrate lysosome biogenesis to replenish the cellular lysosome pool. In this Review, we discuss recent insights into the mechanisms that ensure the maintenance of lysosomal membrane homeostasis, including novel mechanisms of lysosomal membrane repair and the interplay between lysosome damage, repair, lysophagy and lysosome biogenesis. We highlight the importance of lysosomal membrane homeostasis in cell function, physiology, disease and ageing, and discuss the potential for therapeutic exploitation of lysosomal membrane permeabilization. Lysosomes degrade cellular components, and their membrane is an important signalling hub. Recent insights into the mechanisms that maintain lysosomal membrane homeostasis — including the interplay between membrane damage, repair, lysophagy and lysosome biogenesis — highlight their importance in physiology, in disease and during ageing.

Membraneless organelles assembled by liquid–liquid phase separation interact with diverse membranous organelles to regulate distinct cellular processes. It remains unknown how membraneless organelles are engaged in mitochondrial homeostasis. Here we demonstrate that mitochondria-associated translation organelles (MATOs) mediate local synthesis of proteins required for structural and functional maintenance of mitochondria. In Caenorhabditis elegans, the RNA-binding protein LARP-1 (La-related protein 1) orchestrates coalescence of translation machinery and multiple RNA-binding proteins via liquid–liquid phase separation into MATOs that associate with mitochondria in a translocase of the outer membrane complex-dependent manner. LARP-1 deficiency markedly reduces mitochondrial protein levels, impairing cristae organization and ATP production. Specifically, we show that the membrane-shaping MICOS subunit IMMT-1(MIC60) and the ATP synthase β subunit ATP-2, both being important for cristae organization, are synthesized in LARP-1 MATOs. During aging and starvation, LARP-1 MATOs dissociate from mitochondria; however, mitochondrion-persistent LARP-1 MATOs protect mitochondrial health and greatly extend lifespan. These findings suggest an important mitochondrion-regulating mechanism in aging and stress. Membraneless organelles formed by liquid–liquid phase separation regulate cellular processes. Here Bai et al. show that mitochondria-associated translation organelles (MATOs) consisting of RNA-binding proteins and translation machinery mediate localized synthesis of mitochondrial proteins to promote mitochondrial health and extend lifespan in worms.

The activation of conventional (α) and novel (δ) protein kinase C (PKC) isoforms promotes lysosomal biogenesis, a critical process for clearance of pathogenic protein aggregates including β-amyloid (Aβ) and phosphorylated Tau (p-Tau) in neurodegenerative disorders. Notably, PKC activators HEP14/15, characterized by 20-methyl moiety, fail to establish classical C1B domain pharmacophore interactions, suggesting a non-canonical activation mechanism. In this study, structural diversification of 20-deoxyingenol through esterification and acetonide protection yielded 18 new derivatives ( 2–19 ). Systematic screening revealed their lysosome-promoting activities, with structure–activity relationship analysis identifying compounds 4 and 18 as superior autophagy inducers. At 20 μM, these derivatives enhanced autophagic flux by 2.45-fold and 2.31-fold versus vehicle control. Moreover, compounds 4 and 18 exhibited a dose-dependent increase in lysosome numbers, promoted TFEB nuclear translocation, and enhanced lysosome-mediated lipid droplet clearance. Western blot analysis further revealed that compounds 4/18 upregulated proteins associated with the autophagy-lysosome system, suggesting their potential as promising autophagy inducers. Mechanistically, molecular docking simulations indicated thier high-affinity binding to PKCδ, which may explain their autophagy-enhancing properties. Graphical Abstract

Lysosomes are degradation and signaling organelles central to metabolic homeostasis. It remains unclear whether and how harmful metabolites compromise lysosome function in the etiopathology of metabolic disorders. Combining Caenorhabditiselegans and mouse models, we demonstrate that homocysteine, an intermediate in methionine-cysteine metabolism and the cause of the life-threatening disease homocystinuria, disrupts lysosomal functions. In C. elegans, mutations in cystathionine β-synthase cause strong buildup of homocysteine and developmental arrest. We reveal that homocysteine binds to and homocysteinylates V-ATPase, causing its inhibition and consequently impairment of lysosomal degradative capacity. This leads to enormous enlargement of lysosomes with extensive cargo accumulation and lysosomal membrane damage in severe cases. Cbs-deficient mice similarly accumulate homocysteine, displaying abnormal or damaged lysosomes reminiscent of lysosomal storage diseases in multiple tissues. These findings not only uncover how a metabolite can damage lysosomes but also establish lysosomal impairment as a critical contributing factor to homocystinuria and homocysteine-related diseases.

Lysosomes are the degradation centers and signaling hubs in the cell. Lysosomes undergo adaptation to maintain cell homeostasis in response to a wide variety of cues. Dysfunction of lysosomes leads to aging and severe diseases including lysosomal storage diseases (LSDs), neurodegenerative disorders, and cancer. To understand the complexity of lysosome biology, many research approaches and tools have been developed to investigate lysosomal functions and regulatory mechanisms in diverse experimental systems. This review summarizes the current approaches and tools adopted for studying lysosomes, and aims to provide a methodological overview of lysosomal research and related fields.

Lysosomes are degradation and signalling centres crucial for homeostasis, development and ageing1. To meet diverse cellular demands, lysosomes remodel their morphology and function through constant fusion and fission2,3. Little is known about the molecular basis of fission. Here we identify HPO-27, a conserved HEAT repeat protein, as a lysosome scission factor in Caenorhabditis elegans. Loss of HPO-27 impairs lysosome fission and leads to an excessive tubular network that ultimately collapses. HPO-27 and its human homologue MROH1 are recruited to lysosomes by RAB-7 and enriched at scission sites. Super-resolution imaging, negative-staining electron microscopy and in vitro reconstitution assays reveal that HPO-27 and MROH1 self-assemble to mediate the constriction and scission of lysosomal tubules in worms and mammalian cells, respectively, and assemble to sever supported membrane tubes in vitro. Loss of HPO-27 affects lysosomal morphology, integrity and degradation activity, which impairs animal development and longevity. Thus, HPO-27 and MROH1 act as self-assembling scission factors to maintain lysosomal homeostasis and function. The conserved HEAT repeat protein HPO-27 is identified as a lysosome scission factor in Caenorhabditis elegans, and the human homologue MROH1 also serves the same function to maintain lysosomal homeostasis.

WDR91 and SORF1, members of the WD repeat-containing protein 91 family, control phosphoinositide conversion by inhibiting phosphatidylinositol 3-kinase activity on endosomes, which promotes endosome maturation. Here, we report the crystal structure of the human WDR91 WD40 domain complexed with Rab7 that has an unusual interface at the C-terminus of the Rab7 switch II region. WDR91 is highly selective for Rab7 among the tested GTPases. A LIS1 homology (LisH) motif within the WDR91 N-terminal domain (NTD) mediates self-association and may contribute partly to the augmented interaction between full-length WDR91 and Rab7. Both the Rab7 binding site and the LisH motif are indispensable for WDR91 function in endocytic trafficking. For the WDR91 orthologue SORF1 lacking the C-terminal WD40 domain, a C-terminal amphipathic helix (AH) mediates strong interactions with liposomes containing acidic lipids. During evolution the human WDR91 ancestor gene might have acquired a WD40 domain to replace the AH for endosomal membrane targeting.

Daphmacrimines A - K ( 1 - 11 ) were isolated from the leaves and stems of Daphniphyllum macropodum Miq. Their structures and stereochemistries were determined by extensive techniques, including HRESIMS, NMR, ECD, IR, and single-crystal X-ray crystallography. Daphmacrimines A - D ( 1 - 4 ) are unprecedented Daphniphyllum alkaloids with a 2-oxazolidinone ring. Daphmacrimine I ( 9 ) contains a nitrile group, which is relatively rare in naturally occurring alkaloids. The abilities of daphmacrimines A - D and daphmacrimines G - K to enhance lysosomal biogenesis were evaluated through LysoTracker Red staining. Daphmacrimine K ( 11 ) can induce lysosomal biogenesis and promote autophagic flux.

Anthelmintics are drugs used for controlling pathogenic helminths in animals and plants. The natural compound betaine and the recently developed synthetic compound monepantel are both anthelmintics that target the acetylcholine receptor ACR-23 and its homologs in nematodes. Here, we present cryo-electron microscopy structures of ACR-23 in apo, betaine-bound, and betaine- and monepantel-bound states. We show that ACR-23 forms a homo-pentameric channel, similar to some other pentameric ligand-gated ion channels (pLGICs). While betaine molecules are bound to the classical neurotransmitter sites in the inter-subunit interfaces in the extracellular domain, monepantel molecules are bound to allosteric sites formed in the inter-subunit interfaces in the transmembrane domain of the receptor. Although the pore remains closed in betaine-bound state, monepantel binding results in an open channel by wedging into the cleft between the transmembrane domains of two neighboring subunits, which causes dilation of the ion conduction pore. By combining structural analyses with site-directed mutagenesis, electrophysiology and in vivo locomotion assays, we provide insights into the mechanism of action of the anthelmintics monepantel and betaine.

Lysosome-related organelles (LROs) are specialized lysosomes with cell type–specific roles in organismal homeostasis. Dysregulation of LROs leads to many human disorders, but the mechanisms underlying their biogenesis are not fully understood. Here, we identify a group of LYSMD proteins as evolutionarily conserved regulators of LROs. In Caenorhabditis elegans, mutations of LMD-2, a LysM domain–containing protein, reduce the levels of the Rab32 GTPase ortholog GLO-1 on intestine-specific LROs, the gut granules, leading to their abnormal enlargement and defective biogenesis. LMD-2 interacts with GLO-3, a subunit of GLO-1 guanine nucleotide exchange factor (GEF), thereby promoting GLO-1 activation. Mammalian homologs of LMD-2, LYSMD1, and LYSMD2 can functionally replace LMD-2 in C. elegans. In mammals, LYSMD1/2 physically interact with the HPS1 subunit of BLOC-3, the GEF of Rab32/38, thus promoting Rab32 activation. Inactivation of both LYSMD1 and LYSMD2 reduces Rab32 activation, causing melanosome enlargement and decreased melanin production in mouse melanoma cells. These findings provide important mechanistic insights into LRO biogenesis and functions.