EMBO Reports

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.

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.

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.

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.

Robo receptors are conserved across bilaterians and best known for their ability to mediate axonal repulsion in response to Slit family ligands. In mammals, this applies to Robo1 and Robo2, but mammalian Robo3 binds NELL proteins instead of Slits. The evolutionary origin of NELL-Robo interactions and the possible existence of dual-ligand responsiveness across species remain unknown. Here, we systematically analyzed Robo and NELL homologs across bilaterians and found that NELL-Robo binding is conserved among chordate Robos, but not in protostomes. We show that cephalochordate Robo and NELL can mediate axon repulsion in vitro, suggesting conserved functionality. We observed that conformational masking of the NELL-binding site is prevalent among chordate Robos, modulating NELL-Robo affinity. We also demonstrate that NELL-Robo complexes undergo liquid-liquid phase separation in vitro, a property preserved from cephalochordates to mammals. Our findings support a model in which an ancestral chordate Robo receptor was dual-responsive to Slit and NELL, still the case for some extant Robos, and vertebrate paralogs subfunctionalized, with full ligand specialization emerging in mammals.

Neuronal pruning refines neural circuits by eliminating unnecessary connections to ensure proper nervous system function. In Drosophila mushroom body gamma-neuron remodeling, the chemokine-like protein Orion is required for astrocyte infiltration and phagocytosis. This study investigated whether glial responses depend on how Orion is presented. We found that Orion interacts with glycosaminoglycans (GAG) associated to proteoglycans, which are essential for proper axon pruning. Tethering Orion to the gamma-neuron plasma membrane, mimicking glycosaminoglycan binding, was sufficient to induce astrocyte infiltration into axon bundles. However, membrane-bound Orion alone could not efficiently support phagocytosis, which additionally requires a soluble and mobile Orion pool. We also identified exosomes carrying GAG-bound Orion as potential mediators of neuronal remodeling. These findings provide the first evidence that glial infiltration can be uncoupled from phagocytosis, demonstrating that the mode of Orion presentation regulates distinct glial mechanisms during neuronal remodeling.

Embryonic ovarian development requires high levels of WNT/B-catenin signaling yet the molecular basis of this requirement remains unclear. Mutations in the WNT potentiator RSPO1 impair ovarian development in both mouse and human, leading to gonadal masculinization in XX individuals. Here, we show that RSPO1 acts within a restricted developmental window at the onset of ovarian differentiation and that its absence triggers precocious androgen signaling in the developing ovaries. Moreover, androgen receptor inhibition rescues ovarian development in Rspo1 mutant mice. Our findings identify RSPO1/WNT/B-catenin signaling as an essential pathway that suppresses androgen signaling to ensure proper ovarian development in mouse.

During apoptosis, the permeabilization of the mitochondrial inner membrane (MIM) through still-unclear mechanisms releases mtDNA into the cytosol, triggering the inflammatory cGAS/STING pathway under low caspase activity. Here, we report that, in apoptosis, the active form of the pore-forming protein Gasdermin E (GSDME-N) damages mitochondria before plasma membrane disruption. We visualize GSDME-N pore-like nano-assemblies in the MIM of apoptotic cells and of isolated mitochondria, which we bridge with the cryo-EM structure of the GSDME-N pore in mitochondria-like membranes. Deep membrane insertion of the anchor domain, which acts as a determinant of GSDME-N cardiolipin binding preferences, supports a role in pore formation. Notably, GSDME depletion results in a reduction in cristae swelling, MIM extrusion and mtDNA release during apoptosis. Subsequently, this decreases STING activation and inflammatory responses under caspase inhibition. Our findings reveal that GSDME mediates MIM permeabilization and mtDNA release during apoptosis and define the mechanism of GSDME-mediated membrane damage.

The B cell receptor (BCR) drives the differentiation of naive B cells into activated plasma cells that produce antibodies. The BCR consists of a plasma membrane-bound surface immunoglobulin (sIg) that binds antigens, and two coreceptor subunits CD79A and CD79B. The intracellular domains of CD79A and CD79B initiate immune signaling and endocytosis of the receptor in healthy B cells. In diffuse large B cell lymphoma (DLBCL), an aggressive form of human blood cancer, the activated B cell like (ABC) subtype exhibits constitutive signaling that drives survival and proliferation. Here, in a model of human ABC DLBCL, we determine the localization of BCR components relative to plasma membrane structures using correlative super-resolution light and platinum replica transmission electron microscopy. We find that spontaneous clusters of the surface Immunoglobulin common to ABC lymphoma localize to smooth raised membrane domains. These structures are involved in the endocytosis of large receptor clusters. Surprisingly, activated phosphorylated CD79A (pCD79A) shows little colocalization with clathrin on or off smooth raised membranes. Furthermore, pCD79A spatially segregates away from both the slg and CD79B subunits of the BCR complex. A similar distribution was found for downstream phosphorylated Src family kinases. We propose that a disengagement of pCD79A away from sIg and CD79B allows the receptor to evade down-regulation through endocytosis and maintains signaling at the plasma membrane. This mechanism could drive aberrant signaling and the proliferation of lymphomas leading to disease.

Spatiotemporal allocation and dynamic redistribution of centrosomal proteins are critical for coordinating the structural and molecular events that ensure successful cell division. While depletion of the multifunctional protein Optineurin (OPTN) impairs cell-cycle progression, our previous report of the presence of OPTN at key cell-division assemblies raised the question of how its trafficking is coordinated during cell division. In this study, using cell-cycle synchronisation coupled with flow cytometric analysis and confocal microscopy, we report that OPTN progressively dissociates from centrosomes during mitosis, redistributes to cytokinetic structures, accumulates at the spindle midzone, and then concentrates at the midbody during cytokinesis. Moreover, following this transition, OPTN progressively re-emerges at centrosomes during G1 of the subsequent cell cycle. Further, microtubule perturbation and regrowth assays provide evidence that OPTN recruitment and retention at the pericentriolar matrix (PCM) can occur independently of microtubules, whereas its exit is microtubule-dependent. Collectively, this study depicts, for the first time, a detailed movement trail of OPTN, highlighting its centrosome-linked redistribution and microtubule dependency. This context-dependent localisation and trafficking of OPTN across the centrosome-midbody axis establish a definitive spatiotemporal roadmap that can be strategically utilised to study its functional role in cell division.

Cell cycle transitions are driven by cyclins in complex with cyclin-dependent kinases (CDK). Here, we demonstrate that cyclin B3, an evolutionarily divergent B-type cyclin previously thought to function exclusively in female meiosis, is expressed in mitotic cells and interacts with both CDK1 and CDK2. We show that cyclin B3-CDK1/2 is essential for timely mitotic progression by ensuring proper chromosome alignment and stable kinetochore-microtubule interactions, while its overexpression accelerates mitosis and improves chromosome alignment kinetics. Through phospho-mass spectrometry, we identify several kinetochore and mitotic spindle components as substrates of cyclin B3-CDK1/2 in mitosis. Notably, cyclin B3 depletion disrupts mitotic progression in most cancer cell lines tested - including colorectal, osteosarcoma, and breast cancer models - while non-transformed cells are largely unaffected. These findings identify cyclin B3-CDK1/2 as a novel driver of mitotic progression and reveal a preferential dependency in a subset of cancer cells that may represent a potential therapeutic vulnerability.

As the main microtubule network organisers of animal cells, centrosomes orchestrate diverse cellular processes. Centrosomes have been linked to cancer when numerically or structurally aberrant, yet their precise roles at different cancer stages remains to be defined. Here, we study centrosome function in the epithelial-to-mesenchymal transition (EMT), a key driver of cancer initiation, progression and metastasis. We first depleted centrosomes in a breast cancer mesenchymal-like model, resulting in insignificant cellular or nuclear rearrangements. We then investigated non-carcinogenic breast cells undergoing the EMT via TGF{beta} signalling, and removed the centrosomes before triggering this transition. Stimulated epithelial cells acquired a mesenchymal phenotype and rearranged their cytoskeleton. By contrast, stimulated cells lacking centrosomes retained epithelial morphology, showed little cytoskeletal remodelling, yet still altered nuclear shape and upregulated key EMT markers. Transcriptome analysis revealed reduced expression of important genes involved in extracellular matrix organisation and a downregulation of cell junction components when lacking centrosomes; further analysis revealed paxillin showing reduced colocalisation with actin focal adhesions. Centrosomes thus prove essential for cytoskeletal remodelling in the EMT of breast cancer cells.

WAVE and WASH polymerize Arp2/3-mediated branched actin at the leading edge of migrating cells and on the surface of endosomes, respectively. These two proteins are regulated within similar multiprotein complexes, the assembly mechanism of which remains poorly understood. Here we found using mass spectrometry that the two smallest subunits, BRK1 of the WAVE complex and CCDC53 of the WASH complex, interact with each other and with the assembly factor HSBP1. HSBP1 promotes WAVE and WASH assemblies and their respective activities, migration persistence and endosomal branched actin. Reduced levels of WAVE and WASH complexes caused by HSBP1 depletion can be corrected by providing cells with an excess of BRK1 or CCDC53 to assemble their specific complex. Unexpectedly, endogenous levels of BRK1 cross-regulate WASH assembly, whereas those of CCDC53 cross-regulate WAVE assembly. We found that various oligomers comprising BRK1, CCDC53 and HSBP1, including a heterotrimer containing one molecule of each, are formed through their promiscuous coiled coils. These oligomers play a coordinating role in the assembly of WAVE and WASH complexes.

WAVE is a major driver of lamellipodial protrusions. Previous studies suggested that Abelson (Abl)-dependent tyrosine phosphorylation of WAVE promotes actin polymerization and lamellipodia formation. Here, we provide evidence that Abl tyrosine kinase rather restricts lamellipodial localization of WAVE and inhibits lamellipodial cell spreading. Additionally, overexpression of a cytoplasmic PTP61F phosphatase increases WAVE localization at lamellipodia tips. PTP61F overexpression or pharmacological inhibition of Abl/Src kinases even protects cells from lamellipodia disruption induced by pervanadate, a potent protein tyrosine phosphatase inhibitor. By contrast, overexpression of a phosphatase-deficient PTP61F trapping mutant phenocopies loss of wave function. We identify the tyrosine Y234 as a prominent phosphorylation site in WAVE. Rescue experiments using CRISPR/Cas9-based KO wave mutant cells confirmed an important role of PTP61F-mediated WAVE dephosphorylation in lamellipodia formation. Our data suggest a model in which calpain-mediated proteolytic release of ER-bound PTP61F antagonizes Abl/Src-dependent WAVE phosphorylation and acts as a mechanism promoting re-localization of WAVE to lamellipodial tips.

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

Samp1 is an inner nuclear membrane protein required for myogenic differentiation and involved in chromatin organization at the nuclear periphery. Here, we investigated whether these functions are connected by studying the effects of Samp1 depletion during C2C12 myogenic differentiation using immunofluorescence microscopy, RNA sequencing, FRIC, and chromosome-positioning analysis. Samp1-depleted cells showed strongly reduced MyHC expression and virtually abrogated multinucleated fiber formation. Although cell-cycle withdrawal was not prevented, the transcriptional program driving differentiation was drastically perturbed, with reduced muscle-associated transcripts and incomplete repression of genes normally downregulated during myogenesis. Samp1 depletion also disrupted peripheral chromatin organization and prevented the accumulation of peripheral heterochromatin typically seen during differentiation. In addition, radial chromosome distribution was disrupted, evidenced by the failure of chromosome 8 to reposition to the nuclear periphery during differentiation. Together, these findings link the requirement for Samp1 in myogenic differentiation to its role in genome organization at the nuclear periphery.

Developmental genes often acquire accessible chromatin prior to their activation, yet the mechanisms regulating this permissive state remain poorly understood. During spermatogenesis, many meiotic gene promoters display this poised state in spermatogonia, characterized by accessible promoters and paused RNA Pol II occupancy before their activation during meiosis. Here, we identify the NURF chromatin remodeling complex, comprised of SMARCA5 and its accessory subunit BPTF, as a key regulator of this process. We demonstrate that the transcription factor NFYA interacts with SMARCA5 and is required for its occupancy at poised meiotic gene promoters. Approximately one-third of poised meiotic gene promoters is co-occupied by NFYA, SMARCA5, and BPTF. Germline deletion of Smarca5 disrupts chromatin accessibility at poised meiotic promoters, impairs transcriptional program required for mitosis-to-meiosis transition, resulting in developmental arrest at meiotic entry. These findings identify NFYA-NURF as a key regulatory axis that regulates accessible chromatin at developmentally poised meiotic genes and primes them for activation later during meiosis.

The rapid fertility decline in women over 35 years of age has been attributed to decreasing oocyte quality. Aging oocytes accumulate DNA damage, chromosome segregation defects and altered epigenetic modifications. Critically, the molecular mechanisms underlying these changes remain poorly understood. Here we uncovered that in the mouse, the HIRA driven H3.3 histone replacement is attenuated during oocyte aging leading to the loss of chromatin homeostasis. The loss of HIRA function is caused by its SUMOylation resulting in the disruption of HIRA-UBN1-CABIN1 complex and its association with chromatin. We further show that preventing HIRA SUMOylation restores H3.3 incorporation and chromatin integrity in aged mouse oocytes leading to improved oocyte maturation and preimplantation development rates. Importantly, similar chromatin homeostasis defect is observed in aging human oocytes pointing towards a conserved process. Our findings provide novel insights into the molecular mechanisms underlying age-related oocyte quality decline and pave the way for clinical interventions.

The mammalian BRG-/BRM-associated factor (BAF) chromatin remodeling complexes are crucial regulators of hematopoietic stem and progenitor cell (HSPC) fate. Expression of the ncBAF subcomponent, BRD9, is altered in both normal aging and myeloid malignancies. We find that in adult human HSPC, BRD9 regulates the differentiation of myeloid progenitors. Upon degradation of BRD9 in these cells, ncBAF is displaced from the chromatin, leading in turn to enhanced recruitment of cBAF to bona fide CEBPA target sites. With the shift from ncBAF to cBAF, HSPC differentiation results in increased generation of myeloid-derived suppressor cells (MDSC) that secrete elevated levels of IL-8. Soluble factors from these myeloid cells, which have lost BRD9, enhance the stemness of breast cancer cells in vitro. In contrast, bromodomain inhibition of BRD9 results in minimal alterations of ncBAF binding and myeloid differentiation, indicating that BRD9 acts as a barrier to human MDSC differentiation in a bromodomain-independent manner.