Nature Cell Biology

Restoration of organellar membrane integrity is critical for maintaining cellular homeostasis. Lysosomal membrane damage activates local repair machineries and global stress responses, but how signaling lipid metabolism is engaged by damage sensors to support and mechanistically link these processes remains poorly understood. Here we show that the phosphoinositide 3-phosphatase MTMR14 is recruited to damaged lysosomes through calcium-dependent binding to sphingomyelin. At these sites, MTMR14 promotes local PI(3)P hydrolysis and supports PI(4)P accumulation, thereby facilitating formation of ER-lysosome contact sites associated with membrane repair, without affecting ESCRT recruitment. MTMR14-dependent lipid remodelling causes reduced mTORC1 signalling and a decrease in global protein synthesis, consistent with an acute proteostatic adaptation to lysosomal injury. Cells lacking MTMR14 display impaired damage-induced lipid remodelling, altered repair-associated structures, sustained protein synthesis, and increased sensitivity to lysosomal injury, all of which can be mitigated by mTORC1/S6K inhibition. Our findings identify damage-sensing recruitment of MTMR14 and local PI(3)P turnover on damaged lysosomes as a phosphoinositide module that promotes lysosomal membrane integrity and homeostasis while functionally linking nutrient signalling to proteostasis under membrane stress.

Lysosomes are essential for cell survival but are highly susceptible to diverse physical and pathological stressors. Thus, the ability to initiate an acute damage response and promote recovery after stressor resolution is critical for maintaining cellular homeostasis and viability. Although recent studies have advanced our understanding of acute responses to lysosomal injury, the molecular mechanisms governing the recovery stage and distinguishing it from the acute phase remain poorly defined. Here, we delineate a key difference between these two stages in translational regulation and uncover lysosomal recovery from acute damage as a novel trigger for processing body (PB) formation. PBs are membraneless biomolecular condensates involved in RNA metabolism and translational reprogramming. We provide the first evidence that PBs are critical for lysosomal quality control and cell survival during recovery. Mechanistically, PBs are induced selectively during the recovery phase, but not during the acute damage response, through interactions with stress granules (SGs), distinct membraneless biomolecular condensates formed upon acute injury to stabilize damaged lysosomal membranes for repair. Functional analyses reveal that PBs promote lysosomal quality control by collaborating with SG-mediated membrane stabilization, while independently recruiting released cathepsins, thereby collectively supporting cell survival. Together, these findings establish PBs as central effectors of the lysosomal recovery program and underscore the broader relevance of biomolecular condensates in cellular responses to lysosomal damage and related disease processes.

Actin assembly facilitates vesicle formation in several trafficking pathways. Clathrin-mediated endocytosis (CME) shows elevated actin assembly dependence under high membrane tension. Why actin assembly at CME sites occurs heterogeneously even within the same cell, and how assembly forces are harnessed, are not fully understood. Here, endocytic dynamics, actin presence, and geometry of CME proteins from three different functional modules, were analyzed using three-dimensional (3D) super-resolution microscopy, live-cell imaging, and machine-learning-based computation. When hundreds of CME events were compared, sites with actin assembly showed a distinct signature, a delay between completion of coat expansion and vesicle scission, indicating that actin assembly occurs preferentially at stalled CME sites. N-WASP is recruited to one side of CME sites where it is positioned to stimulate asymmetric actin assembly. We propose that asymmetric actin assembly rescues stalled CME sites by pulling vesicles into the cell much like a bottle opener pulls off a bottle cap.

Senescent cells influence their surroundings through the senescence-associated secretory phenotype (SASP), an assortment of secreted molecules and macromolecular complexes. Among SASPs intracellular drivers are cytoplasmic chromatin fragments (CCFs), nuclear-derived DNA that activates the pro-inflammatory cGAS/STING pathway. While autophagy contributes to CCFs degradation, the full repertoire of CCF fates and signaling functions remains unclear. Here, we show that senescent cells release CCF components, {gamma}H2AX and double-stranded DNA (dsDNA), into the extracellular space via an ESCRT-independent multivesicular body pathway. Secreted CCF components localize to extracellular particles exhibiting an unusual "popcorn"-like morphology, distinct from canonical small extracellular vesicles. Notably, inhibition of autophagy enhances secretion of CCF components and particles, suggesting an inverse relationship between intracellular clearance and extracellular release. A fraction of CCF-containing extracellular particles activates cGAS-STING signaling in non-senescent proliferating cells and is enriched in the circulation of aged mice, pointing to a previously unrecognized mode of extracellular signaling by senescent cells.

Selective autophagy is a lysosomal degradation pathway that is critical for maintaining cellular homeostasis by disposing of harmful cellular material. While the mechanisms by which soluble cargo receptors recruit the autophagy machinery are becoming increasingly clear, the principles governing how organelle-localized transmembrane cargo receptors initiate selective autophagy remain poorly understood. Here, we demonstrate that transmembrane cargo receptors can initiate autophagosome biogenesis not only by recruiting the upstream FIP200/ULK1 complex but also via a WIPI-ATG13 complex. This latter pathway is employed by the BNIP3/NIX receptors to trigger mitophagy. Additionally, other transmembrane mitophagy receptors, including FUNDC1 and BCL2L13, exclusively use the FIP200/ULK1 complex, while FKBP8 and the ER-phagy receptor TEX264 are capable of utilizing both pathways to initiate autophagy. Our study defines the molecular rules for initiation by transmembrane cargo receptors, revealing remarkable flexibility in the assembly and activation of the autophagy machinery, with significant implications for therapeutic interventions.

Damaged mitochondria can be subject to lysosomal degradation via mitophagy. However, whole-organelle degradation exhibits relatively slow kinetics and thus its impact may be limited in response to acute, fast-acting cellular stress. We previously reported that in Parkin-deficient cells endolysosomes directly target mitochondria when subjected to bioenergetic stress. Here, using high-resolution live cell imaging we reveal a striking level of dynamic targeting of Rab5+ early endosomes to stressed mitochondria, culminating in a switch-like accumulation in the entire mitochondrial population, independently of canonical autophagy. This process of rapid, largescale Rab5+ vesicle trafficking to mitochondria coincides with, and is mediated by, XIAP E3 ligase activated mitochondrial ubiquitylation and results in ultrastructural changes to, and degradation of, intra-mitochondrial components. Mitochondria-targeting vesicles include early endosomal subpopulations marked by Rab5 effector APPL1 and ubiquitin-binding endocytic adaptors OPTN, TAX1BP1 and Tollip, and Rab7-positive late endosomes/lysosomes. In Parkin expressing cells, XIAP- and Parkin-dependent mitochondrial targeting and resulting processing modes are competitively regulated. Together, our data suggest that XIAP-mediated targeting of endolysosomes to mitochondria functions as a stress-responsive, sub-organelle level mitochondrial processing mode that is distinct from, and competitive to, Parkin-mediated mitophagy.

Regionalized disease prevalence is a common feature of the gastrointestinal tract. Herein, we employed regionally resolved Smart-seq3 single-cell sequencing, generating a comprehensive cell atlas of the adult mouse oesophagus. Characterizing the oesophageal axis, we unveil non-uniform distribution of epithelial basal cells, fibroblasts and immune cells. In addition, we reveal a position-dependent, but cell subpopulation-independent, transcriptional signature, collectively generating a regionalized oesophageal landscape. Combining in vivo models with organoid co-cultures, we demonstrate that proximal and distal basal progenitor cell states are functionally distinct. We find that proximal fibroblasts are more permissive for organoid growth compared to distal fibroblasts and that the immune cell profile is regionalized in two dimensions, where proximal-distal and epithelial-stromal gradients impact epithelial maintenance. Finally, we predict and verify how WNT-, BMP-, IGF-and NRG-signalling are differentially engaged along the oesophageal axis. We establish a cellular and transcriptional framework for understanding oesophageal regionalization, providing a functional basis for epithelial disease susceptibility.

Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired, or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological and genetic approaches, we show that lysosome damage induces rapid Conjugation of ATG8s to Single Membranes (CASM). ATG8 proteins are recruited directly onto damaged membranes, independently of ATG13/WIPI2, and conjugated to PS, as well as PE, a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, and direct recruitment of ATG16L1, dependent on K490 (WD40-domain), and sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with the formation of dynamic LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics.

P-bodies are cytoplasmic membraneless organelles involved in mRNA storage, yet their role in cellular stress responses remains poorly understood. Here, we demonstrate that P-bodies are rapidly and selectively remodeled during the early response to endoplasmic reticulum (ER) stress in D. melanogaster oogenesis, positioning them as key early stress responders. Notably, this remodeling occurs within minutes of stress induction and precedes stress granule formation. This early remodeling is characterized by changes in P-body morphology and internal organization and promotes selective mRNA regulation. Specifically, ER stress leads to the recruitment and stabilization of maternal mRNAs and those encoding P-body components, while transcripts not associated with P-bodies are degraded. These observations indicate that P-body remodeling is not merely structural but functionally linked to the selective preservation of mRNA populations during stress. Mechanistically, we find that this process is driven by transcriptional upregulation of the RNA-binding protein, Bruno 1, downstream of ATF4-dependent stress signaling, thereby establishing a direct connection between the unfolded protein response and condensate regulation. Consistent with this model, loss of Bruno 1 abolishes, whereas its overexpression enhances P-body remodeling, demonstrating that stress-induced changes in RNA binding protein levels can actively reprogram condensate properties. Together, our findings reveal that P-bodies function as dynamic, stress-responsive hubs that integrate transcriptional signaling with post-transcriptional control, enabling the selective preservation of essential mRNAs during ER stress. More broadly, this work uncovers a previously unrecognized mechanism by which stress signaling pathways reorganize cytoplasmic architecture to shape mRNA fate.

Glycans are ubiquitous sugar polymers with major biological functions that are assembled by glyco-enzymes onto cargo molecules during their transport through the Golgi complex. How the Golgi determines glycan assembly is poorly understood. By relying on the Golgi cisternal maturation model and using the glyco-enzyme adaptor and oncoprotein GOLPH3 as a molecular tool, we define the first example of how the Golgi controls glycosylation and associated cell functions. GOLPH3, acting as a component of the cisternal maturation mechanism, selectively binds and recycles a subset of glyco-enzymes of the glycosphingolipid synthetic pathway, hinders their escape to the lysosomes and hence increases their levels through a novel lysosomal degradation-regulated mechanism. This enhances the production of specific growth-inducing glycosphingolipids and reprograms the glycosphingolipid pathway to potentiate mitogenic signaling and cell proliferation. These findings unravel unforeseen organizing principles of Golgi-dependent glycosylation and delineate a paradigm for glycan assembly by the Golgi transport mechanisms. Moreover, they indicate a new role of cisternal maturation as a regulator of glycosylation, and outline a novel mechanism of action for GOLPH3-induced proliferation.

Endoplasmic reticulum (ER) stress triggers activation of the ER surveillance (ERSU) pathway-- a critical protective mechanism that transiently halts cortical ER inheritance to daughter cells and arrests cytokinesis by septin ring subunit Shs1 re-localization to the bud scar in response to ER stress. Once ER functional homeostasis is re-established, cells resume normal cell cycle progression; however, the molecular circuitry linking ER integrity to cell cycle regulation has remained largely unresolved. Here, we show that ER stress selectively disperse Bud2, a GAP for Bud1/Rsr1, severing its canonical role in cell polarity while integrating it into ER homeostasis signaling. Bud2 dispersion results in accelerated spindle pole body (SPB) duplication, spindle misorientation, defects in nuclear migration, and genome segregation errors under ER stress. Strikingly, a C-terminal truncation of Shs1 (shs1-{Delta}CTD) recapitulated the ER stress-induced dispersion of Bud2 phenotype even in the absence of ER stress, and delayed cell-cycle re-entry after ER homeostasis was regained--despite normal occurrence of typical ERSU hallmark events. Notably, Bud2 overexpression rescued the growth defects of shs1-{Delta}CTD mutants after ER homeostasis was re-established. Collectively, our findings reveal a new mechanistic axis whereby ER integrity coordinates organelle inheritance, cytoskeletal organization, and nuclear division via selective control of Bud2 and Shs1, establishing a direct regulatory bridge between ER status and mitotic fidelity.

Intracellular organelles support cellular physiology in diverse conditions. In the skin, epidermal keratinocytes undergo differentiation with gradual changes in cellular physiology, accompanying remodeling in lysosomes and the Golgi apparatus. However, the functional significance and the molecular link between lysosome and Golgi remodeling were unknown. Here, we show that in differentiated keratinocytes, the Golgi apparatus redistributes as ministacks leading to a significant increase in total protein secretion. The Golgi ministacks establish contact with lysosomes facilitated by Golgi tethering protein GRASP65, the depletion of which is associated with the loss of Golgi-lysosome contact and malformation of lysosomes defined by their aberrant morphology, size, and function. Strikingly, these lysosomes receive secretory Golgi cargoes, contribute to the protein secretion from the Golgi, and are critically maintained by the secretory function of the Golgi apparatus. We uncovered a novel mechanism of lysosome specialization through unique Golgi-lysosome contact that likely supports high secretion from differentiated keratinocytes. Key pointsO_LICalcium induced differentiation of human keratinocytes accompanies the dispersal of functional Golgi stacks and lysosomes. C_LIO_LIDispersed Golgi stacks establish contact/physical apposition with the lysosomes. C_LIO_LIGolgi tether GRASP65 surrounds keratinocyte lysosomes and facilitates Golgi-lysosome apposition. C_LIO_LIGRASP65 depletion abolishes Golgi-lysosome apposition and accumulates morphologically altered non-degradative lysosomes. C_LIO_LILysosomes of differentiated keratinocytes receive secretory Golgi cargo and contribute to the protein secretion from the Golgi. C_LIO_LISpecialized lysosomes are maintained by the secretory function of the Golgi apparatus. C_LI

Epithelial tissues undergo dynamic transitions between fluid-like collective motion and mechanically jammed states during development, injury repair, and disease progression. However, the cellular programs that drive these transitions and regulate collective behavior remain unclear. Using a controlled crowding model integrated with live-cell imaging and time-resolved multi-omics, we demonstrate that epithelial crowding triggers early metabolic changes characterized by increased mitochondrial pyruvate anaplerosis that precedes the jamming transition. Functional inhibition of mitochondrial pyruvate import is sufficient to sustain collective cell motility, impeding jamming transition in crowded cells. This unjammed state is driven by enhanced cytoskeletal remodeling and requires RhoA-myosin II activity. Mechanistically, we show that elevated cytoskeletal signaling promotes macropinocytic uptake, which serves as a required feedback loop to maintain motility. These findings identify mitochondrial pyruvate utilization as a key regulator that links metabolic remodeling to the endocytic control of epithelial fluidity.

Etxtracellular vesicles (EVs) support cell-to-cell communication, both in physiological and pathological contexts and emerge as new biomarkers and potential therapeutics. Yet, despite EVs holding huge promises, our understanding of core mechanisms governing EV signaling remains significantly underdeveloped. Our previous work indicated that syndecans and their cytosolic adaptor syntenin control the biogenesis of a major subset of small EVs (sEVs). Here we show that syndecans control the accumulation of ADAM10 into sEVs. ADAM10 promotes the formation of sEVs enriched in cleaved receptors (reducing sEV corona), supports the sorting of proteins with intracellular functions, and tailors sEVs for the delivery of their internal content, e.g. syntenin, into the cytosol of recipient cells. Conversely, inhibition of ADAM10 favors the production of sEVs bearing full-length, signaling-competent receptors/ligands and enhances contact-dependent signaling. These findings uncover a protease-regulated switch that tailors sEV composition and signaling modality, providing important new mechanistic insights into the core molecular pathways supporting EV-mediated communication.

Chronic inflammation disrupts epithelial regeneration, yet how immune signaling reprograms progenitor fate remains unclear. Using the Drosophila airway as a model of epithelial remodeling, we identify the c-Jun N-terminal kinase (JNK) pathway as a central integrator of immune and stress cues that balances apoptosis and compensatory proliferation in airway progenitors. Persistent activation of the innate immune IMD pathway induces simultaneous cell death and proliferation through a non-canonical route that bypasses NF-{kappa}B/Relish and instead engages JNK. Downstream, distinct transcriptional modules orchestrate these divergent fates: Foxo and AP-1 drive apoptosis, whereas the ETS factor Ets21C mediates proliferation. Genetic inhibition of JNK or its effectors restores progenitor homeostasis, while constitutive activation recapitulates inflammation-induced tissue remodeling. These responses are cell-autonomous, revealing that airway progenitors actively interpret immune and stress signals to determine their fate. Collectively, our findings uncover a modular signaling architecture that links inflammation to regeneration and highlight conserved JNK-dependent transcriptional programs as potential therapeutic targets to prevent progenitor exhaustion in chronic airway disease.

The Golgi apparatus expands during differentiation and high secretory demand, yet the transcriptional control of its biogenesis remains poorly defined. Here, we developed a targeted enzymatic ablation method to eliminate the Golgi and trigger de novo organelle formation. Single-cell RNA-seq of cells rebuilding Golgi revealed an orchestrated induction of a broad Golgi gene network coinciding with structural and functional organelle maturation. This gene set spans all Golgi sub-compartments and functions, including glycosylation, trafficking, and ion transport, thus supporting the concept of a unified Golgi regulon, enabling the simultaneous expression of components required for the organelle structural and functional integrity. Through promoter analysis and RNAi screening, we identified CREB3L1 as a key transcriptional regulator critical for Golgi gene activation and organelle reformation. These findings indicate that CREB3L1-dependent transcriptional mechanisms orchestrate a complete Golgi biogenesis program that may be essential for organelle regeneration and for secretory pathway plasticity during physiological remodeling.

Cancer cells adapt to nutrient stress by remodeling the repertoire of proteins on their surface, enabling survival and progression under starvation conditions. However, the molecular mechanisms by which nutrient cues reshape the cell surface proteome to influence cell behavior remain largely unresolved. Here, we show that acute glucose starvation, but not amino acid deprivation or mTOR inhibition, selectively impairs ER-to-Golgi export of specific cargoes, such as E-cadherin, in a SEC24C-dependent manner. Quantitative cell surface proteomics reveal that glucose deprivation remodels the cell surface proteome, notably reducing surface expression of key adhesion molecules. This nutrient-sensitive reprogramming enhances cell migration in vitro and promotes metastasis in vivo. Mechanistically, we show that AMPK and ULK1 signaling orchestrate this process independent of autophagy, with ULK1-mediated phosphorylation of SEC31A driving SEC24C-dependent COPII reorganization. These findings establish ER-to-Golgi trafficking as a nutrient-sensitive regulatory node that links metabolic stress to cell surface remodeling and metastatic potential.

Autophagy, a conserved recycling process, manages intracellular quality control to mitigate stress. To determine the rapid effects of autophagy perturbation, we developed the first optogenetic tool to rapidly inhibit autophagy, termed ASAP. Our approach selectively inhibits autophagy within 5 minutes, providing a precise and dynamic approach to study autophagy regulation. Proteomic profiling with ASAP revealed the most tightly regulated autophagy substrates along with novel, previously unidentified substrates, including nuclear pore complex (NPC) proteins. Interestingly, autophagy regulates quality control of incomplete NPCs still in the cytoplasm via specific LC3-interacting regions (LIRs), sparing NPCs embedded in the nuclear envelope. Upon rapid autophagy inhibition, incomplete NPCs accumulate and instead of undergoing autophagic degradation, cytoplasmic NPCs aggregate in processing bodies. Using ASAP, we demonstrate rapid and specific inhibition of autophagy, revealing that the nuclear pore complex is a tightly regulated autophagy substrate.

Cell-cell communication in tissues is influenced by contact geometry and molecular signalling. Here, we demonstrate that epithelial intercellular filopodia can penetrate neighbouring cells to form micrometre-long, double-membrane "impact sites" and, in some instances, initiate trans-endocytosis. Using super-resolution live imaging and three-dimensional electron microscopy in cell models, xenografts, and patient-derived tissues, we observe widespread intercellular filopodia and extensive interdigitation at the cell-cell interface. Filopodia impact sites in the recipient cell recruit PACSIN2 into dynamic "PACSIN2 fingers" and are specifically enriched for caveolin-1 and dynamin-2, while clathrin and CLIC pathway markers are not enriched. Most contacts are transient and resolved by retraction, but some undergo scission, with recipient cells internalising filopodial tips across both homotypic and heterotypic interactions, including cancer-endothelium contacts. Correlative light and FIB-SEM analysis reveals that internalised tips are often double-membraned, closely associated with endoplasmic reticulum tubules, and can traffic to lysosomes. These findings define a protrusion-driven, curvature-dependent uptake pathway at cell-cell interfaces and identify filopodia as exchange organelles in epithelial collectives.

Lysosomes are essential in maintaining cellular health. Endocytic lysosome reformation (ELR) regenerates functional lysosomes following degradation of endocytic cargo, yet the mechanisms driving this process remain largely unknown. Here, we define the molecular machinery underlying ELR. We find that unlike autophagic lysosome reformation (ALR), ELR proceeds independently of mTOR and dynamin 2, but requires the mitochondrial fission GTPase DRP1. DRP1 mediates scission of endolysosomal tubules at contact sites with the endoplasmic reticulum (ER) and mitochondria. Disruption of DRP1 function or ER-endolysosome contact results in elongated tubules, indicating defective lysosome reformation. Moreover, mitochondrial activity is essential for tubule initiation, and Ca2+ transfer from endolysosomes to mitochondria is crucial for ELR onset. Our findings reveal a dual role for mitochondria in ELR: first in ELR initiation and second in DRP1-dependent tubule fission at ER-mitochondria-endolysosome tripartite contact sites, uncovering the previously unappreciated role of mitochondria in endolysosome remodeling and fission.