EMBO Reports最新文献

Sphingolipids govern diverse cellular processes; their dysregulation underlies numerous diseases. Despite extensive characterizations, understanding the orchestration of the sphingolipid network within living organisms remains challenging. We established a versatile genetic platform of CRISPR-engineered reporters of 52 sphingolipid regulators, recapitulating endogenous gene activity and protein distribution. This platform further allows conditional protein degradation for functional characterization. In addition, we developed the biosensor OlyA^w to detect ceramide phosphoethanolamine and visualize membrane raft dynamics in vivo. Using this platform, we established comprehensive profiles of the sphingolipid metabolic network in the brain at the transcriptional and translational levels. The highly heterogeneous patterns indicate extensive coordination between distinct cell types and regions, suggesting the brain functions as a coherent unit to execute specific steps of sphingolipid metabolism. As a proof-of-concept application, we showed cell type-specific requirements of sphingomyelinases, including CG6962/dSMPD4 and CG3376/aSMase, degrading distinct subcellular pools of ceramide phosphoethanolamine to maintain brain function. These findings establish a foundation for future studies on brain sphingolipid metabolism and showcase the utilization of this genetic platform in elucidating in vivo mechanisms of sphingolipid metabolism.

Brain organoids are a promising model for studying human neurodevelopment and disease. Despite the potential, their 3D structure exhibits high variability during differentiation across batches and cell lines, presenting a significant challenge for biomedical applications. During development, organoids are exposed to fluid flow shear stress (fFSS) generated by the flow of culture media over the developing tissue. This stress is thought to disrupt cellular integrity and morphogenesis, leading to variation in organoids architecture, ultimately affecting reproducibility. Understanding the interplay between tissue morphology, cell identity and organoid development is therefore essential for advancing the use of brain organoids. Here, we demonstrate that reducing fFSS, by employing a vertically rotating chamber during neuronal induction, a critical phase for organoid morphogenesis, along with an extended cell aggregation phase to minimize fusions, significantly improves the reproducibility of brain organoids. Remarkably, reducing fFSS minimizes morphological structure variation and preserves transcriptional signature fidelity across differentiation batches and cell lines. This approach could enhance the reliability of brain organoid models, with important implications for neurodevelopmental research and preclinical studies.

Detecting temperature is crucial for the survival of living organisms. Although the temperature sensitive Transient Receptor Potential Melastatin 8 (TRPM8) channel has been identified as the prototypical cold sensor, the mechanisms by which it detects temperature remain elusive. In this study, we first identify groups of clustered residues that undergo conformational rearrangements between buried and exposed states during cold activation by hydroxyl radical footprinting-mass spectrometry (HRF-MS). By systematically perturbing water-protein interactions at these residues with point mutations that change side chain hydrophobicity (SCH), we achieve rational tuning of temperature sensitivity in this channel. Specifically, mutations with the clearest impacts on TRPM8 cold sensitivity are clustered in the MHR1-3 domains, where the protein of isolated MHR1-3 domains also exhibits clear conformational rearrangements in response to cold. Guided by this mechanism, we rationally edit the Trpm8 gene in mice, introducing a single point mutation to render them insensitive to coldness.

The impact of early-stage tumors on gene expression in adjacent tissues remains uncertain, despite the known influence of the tumor microenvironment on tumor progression. Here, we systematically analyze early-stage lung adenocarcinoma (LUAD) and surrounding tissues across multiple distinct regions, from the tumor core to distant tissues. DNA methylation profiling in a 12-patient cohort reveals two distinct patterns of methylation changes. Steep changes occurring at the tumor boundary and shallow changes showing a gradual shift over increasing distance to the tumor. Approximately 17,000 CpG sites demonstrate shallow changing trends without clear boundaries, potentially affecting 2655 genes. In half of the patients, tissues within 10 mm beyond the tumor show methylation patterns similar to tumors. We test mRNA expression of key genes affected by these methylation patterns and observe that the protein expression pattern of WNT7B demonstrates no steep changes at the tumor boundary, supporting their regulatory role. Adding a 59-patient four-year-prognosis cohort allowed us to rigorously assess the clinical relevance of these methylation change trends. These shallow changes reflect tumor characteristics and have the potential for prognostic prediction in patients, warranting further investigation.

The histone-fold domain (HFD) is a conserved protein interaction module that requires stabilization through a handshake interaction with an HFD partner. All HFD proteins known to date form obligate dimers to shield the extensive hydrophobic residues along the HFD. Here, we find that the lepidopteran kinetochore protein CENP-T is soluble as a monomer. We attribute this stability to a structural rearrangement, which leads to the repositioning of the HFD helix α3. This brings a conserved two-helical extension closer to the histone fold, where it takes over the position and function of the CENP-T partner CENP-W. This change has no effect on the DNA-binding ability of the lepidopteran CENP-T. Our analysis suggests that the monomeric HFD originated in the last common ancestor of insects, with a possible second independent origin in Acariformes, both of which lack CENP-W. Our study highlights an unexpected structural variation in a protein module as conserved and optimized as the HFD, providing a unique perspective on the evolution of protein structure and the forces driving it.

Mitochondrial DNA (mtDNA) serves as a potent activator for cellular innate immune responses. Topoisomerase 3α (TOP3α), a type IA topoisomerase, is canonically localized to mitochondria and nuclei, but its enigmatic cytosolic fraction-observed over two decades ago-has remained functionally undefined. Here, we uncover a critical role for cytosolic TOP3α in amplifying mtDNA-triggered innate immunity. We observe that aberrant TOP3α expression causes mtDNA clustering and release via mPTP-VDAC, stimulating cGAS-mediated inflammatory responses. Cytosolic TOP3α facilitates the sensing of released mtDNA by cGAS and amplifies downstream innate immune signaling. Using an in vitro cell-free system, we reveal that TOP3α directly augments mtDNA interaction with cGAS, which in turn competes with TOP3α for mtDNA binding. A rare mutation of a highly conserved residue (G250D) of TOP3α impairs the assembly of TOP3α polypeptides into protein complexes and its binding to mtDNA. Furthermore, mutant TOP3α hinders cGAS-mtDNA interaction and compromises cGAS-driven immunity. Our findings reveal a function for cytosolic TOP3α as a regulator for cGAS-driven inflammation.

The microbiome is increasingly recognized as playing a critical role in lung cancer prevention, diagnosis, and treatment. While bacteria are essential for tumor angiogenesis, the impact of fungi on this process remains largely unexplored. In this study, we investigate effects of Aspergillus fumigatus (A. fumigatus) on lung cancer. We show that inhalation of A. fumigatus increases tumor burden and angiogenesis in mouse models. Interestingly, A. fumigatus does not directly affect the proangiogenic abilities of tumor cells or endothelial cells. Instead, A. fumigatus promotes the accumulation of myeloid-derived suppressor cells (MDSCs), particularly G-MDSCs, in tumor tissues. A. fumigatus increases VEGF-A secretion from tumor-associated MDSCs, promoting tumor angiogenesis. Furthermore, we identify solute carrier family 7 member 11 (SLC7A11) as a key player in regulating this proangiogenic function through an interaction with High Mobility Group Box 1 (HMGB1) in MDSCs. Our results shed light on the mechanisms by which A. fumigatus influences MDSCs to promote angiogenesis and demonstrate that commensal fungi influence host immunity and support tumor progression.

Chromosome segregation errors in human oocytes increase dramatically as women age and premature loss of meiotic cohesion is one factor that contributes to a higher incidence of segregation errors in older oocytes. Here we show that knockdown of the NAD⁺-dependent deacetylase Sirt1 during meiotic prophase in Drosophila oocytes causes premature loss of arm cohesion and chromosome segregation errors. We demonstrate that acetylation of the Sirt1 substrate H4K16 increases significantly in sirt1 null and Sirt1 knockdown oocytes and use this as a marker for Sirt1 activity in vivo. When oocytes undergo aging, the H4K16ac signal increases significantly, consistent with an aging-dependent decline in Sirt1 deacetylase activity. However, if females are fed the Sirt1 activator SRT1720 as their oocytes age, the H4K16ac signal on oocyte DNA remains low in aged oocytes, consistent with preservation of Sirt1 activity during aging. Strikingly, age-dependent segregation errors are significantly reduced if mothers are fed SRT1720 while their oocytes age. Our data suggest that maintaining Sirt1 activity in aging oocytes may provide a viable therapeutic strategy to decrease age-dependent segregation errors.

Most eukaryotes share core meiosis-specific genes, suggesting meiosis evolved once in the last eukaryotic common ancestor (LECA). These genes are master regulators of meiotic recombination, ensuring genetically diverse lineages. However, meiosis in organisms outside the animal, plant, and yeast lineages remains poorly understood. Core meiotic genes were recently identified in the model brown alga Ectocarpus but remain uncharacterised. Here, we combine bioinformatic, structural, and biochemical approaches to characterise the axial-element orthologues meiotic Ectocarpus HORMA-domain protein (ecHOP1) and its interactor reductional division protein 1 (ecRED1), providing insight into meiotic-recombination regulation in brown algae. We define the chromatin-binding region of ecHOP1 and show that it binds double-stranded DNA, and we find that Ectocarpus assembles its axial element using evolutionarily conserved principles in a unique combination. Our work lays a foundation for further studies of meiosis in brown algae and broadens understanding of the diversity and conservation of meiotic mechanisms.

Telomeres are essential for chromosome protection and genomic stability, and telomerase function is critical for organ homeostasis. Zebrafish is a useful vertebrate model for understanding cellular and molecular mechanisms of regeneration. The regeneration capacity of the caudal fin of wild-type zebrafish is not affected by repetitive amputation, but the behaviour of telomeres during this process has not yet been studied. Here, we characterize the regeneration process in a telomerase-deficient zebrafish model, and study the regenerative capacity after repetitive amputations at different ages. We find that the regenerative efficiency decreases with aging in all genotypes but telomere length is maintained even in telomerase-deficient fish. Our data indicate that telomere length can be maintained by the regenerating cells through the recombination-mediated Alternative Lengthening of Telomeres (ALT) pathway, which likely supports high rates of cell proliferation during the caudal fin regeneration process.