EMBO Reports最新文献

The lymphocyte immune response begins with antigen recognition on antigen-presenting cells, leading to the formation of the immunological synapse-a specialized interface for biochemical and biophysical exchange. At the synapse, most antigen-engaged receptor microclusters move inward toward the central supramolecular activation cluster (cSMAC) via retrograde F-actin flow, eventually clearing from the cell surface. This retrograde movement and receptor downregulation maintain antigen receptor homeostasis, critical for adaptive immunity, though its regulation remains unclear. Using live T cells, we identify a significant pool of antigen-engaged microclusters moving anterogradely toward the cell periphery, rather than the cSMAC. This movement is driven by actin waves propagating outward and coupling to microclusters through the Wiskott-Aldrich Syndrome Protein. These findings reveal a previously unrecognized mode of actin dynamics-anterograde actin waves-that co-exist with retrograde flow and direct microclusters away from the downregulation zone. This dual actin behavior underscores the complex cytoskeletal mechanisms T cells employ to regulate receptor distribution and maintain signaling homeostasis during immune activation.

Hematopoietic stem and progenitor cells (HSPCs) emerge from arterial endothelial cells (ECs) through a process termed endothelial-to-hematopoietic-transition (EHT), a process induced by paracrine signals and driven by a transcriptional cascade. Despite inductive signals being broadly received by ECs in the dorsal aorta (DA), only a subset of ECs undergoes EHT, while others maintain their vascular identity. The molecular mechanisms that determine this selective fate decision remain poorly understood. Here, we discover Apelin signaling as a critical regulator of cell fates in the DA, acting as a molecular switch to balance vascular and hematopoietic identities. We show that Apelin receptor (Aplnr)-expressing ECs retain their arterial identity, while Aplnr non-expressing ECs are primed to become hemogenic endothelial cells (HECs) and transition into HSPCs. Loss of Apelin signaling leads to excessive EC-to-HEC conversion and increased HSPC numbers. Conversely, forced Aplnr expression abolishes HSPC formation by maintaining EC identity. These findings reveal that Apelin signaling regulates HSPC formation by preserving endothelial identity. In summary, our findings establish Apelin signaling as a critical regulator for balancing endothelial and hematopoietic fates.

Motile cilia are evolutionarily conserved protrusions critical for motility and homeostasis. Their rhythmic movements require the central pair microtubules (CP-MTs). While the initial CP-MT assembly in mammals is mediated by WDR47 and microtubule minus-end-binding CAMSAPs, the mechanism by which CP-MTs are stabilized remains unclear. Here, we demonstrate that WDR47 coordinates JHY and SPEF1 to maintain the stability of mammalian CP-MTs. By generating a proximity interactome of WDR47, we identify a group of CP-MT-associated proteins, including SPEF1 and JHY. WDR47 enriches JHY and SPEF1 to the central lumen and tip of nascent cilia, whereas SPEF1 recruits WDR47 and JHY to CP-MTs through direct interactions. Jhy deficiency in mice preferentially disrupts distal CP-MTs, resulting in rotatory ciliary beats. Phylogenetic analyses suggest conserved functions of WDR47 and SPEF1 in protozoa and metazoans, as well as a role for JHY in animals with radial or bilateral body symmetry. We propose that JHY emerges to further reinforce CP-MTs, enabling the transition from switchable to fixed ciliary waveforms in metazoan evolution.

Cryptorchidism is a common congenital abnormality that increases infertility and testicular cancer risk in adulthood. However, a few mammals exhibit naturally undescended testes while maintaining normal reproduction. The mechanisms underlying this natural cryptorchidism remain unclear. Here, we found evolutionary relaxation in INSL3 and RXFP2 of cryptorchid mammals, with the highest dN/dS ratio observed in cetaceans. Cellular experiments demonstrated that cetacean INSL3 downregulated the cAMP-PKA-CREB pathway, thereby reducing gubernacular cell proliferation and contraction. Cetacean INSL3 knock-in mice exhibited groin-located testes, nearly perfectly mimicking cryptorchid phenotypes in cetaceans and other mammals with incompletely descended testes. Collagen and muscle fibers in the gubernaculum of transgenic mice were reduced, with differentially expressed genes enriched in muscle development and contraction pathways. Additionally, the knock-in mice displayed male sterility, impaired testicular development, and upregulated inflammatory pathways in the testes. Our findings reveal how evolutionary changes in the INSL3/RXFP2 pathway contribute to natural cryptorchidism in mammals and provide insights for investigating reproductive health and cancer resistance in cryptorchid species.

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.