Speir M, Tye H, Gottschalk TA
… +18 more, Simpson DS, Djajawi TM, Deo P, Ambrose RL, Conos SA, Emery J, Abraham G, Pascoe A, Hughes SA, Weir A, Hawkins ED, Kong I, Herold MJ, Pearson JS, Lalaoui N, Naderer T, Vince JE, Lawlor KE
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 s...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.
Missense mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, are common in several developmental disorders and cancers. While many mutations disrupt auto-inhibition and hyperactivate SHP2, several d...Missense mutations in PTPN11, which encodes the protein tyrosine phosphatase SHP2, are common in several developmental disorders and cancers. While many mutations disrupt auto-inhibition and hyperactivate SHP2, several do not enhance catalytic activity. Both activating and non-activating mutations could potentially drive pathogenic signaling by altering SHP2 interactions or localization. We employed proximity-labeling proteomics to map the interaction networks of wild-type SHP2, ten clinically relevant mutants, and SHP2 bound to an inhibitor that stabilizes its auto-inhibited state. Our analyses reveal mutation- and inhibitor-dependent alterations in the SHP2 interactome, with several mutations also changing localization. Some mutants show increased mitochondrial localization and impact mitochondrial function. This study provides a resource for exploring SHP2 signaling and offers new insights into the molecular basis of SHP2-driven diseases. Furthermore, this work highlights the capacity for proximity-labeling proteomics to detect missense-mutation-dependent changes in protein interactions and localization.
The article discusses recent progress with developing tools for organellar genome editing, highlights current and potential future applications, and analyzes benefits and limitations of genome editing technologies compar...The article discusses recent progress with developing tools for organellar genome editing, highlights current and potential future applications, and analyzes benefits and limitations of genome editing technologies compared to organellar genome transformation. [Image: see text]
Endosymbiosis between dinoflagellate algae and cnidaria is fundamental for coral reef health. Appropriate symbiont selection is required for sufficient host nutrient acquisition and could be tailored to increase cnidaria...Endosymbiosis between dinoflagellate algae and cnidaria is fundamental for coral reef health. Appropriate symbiont selection is required for sufficient host nutrient acquisition and could be tailored to increase cnidarian stress tolerance. Previous research suggested glycan-lectin interactions facilitate symbiont uptake; however, blockage of such interactions does not fully inhibit symbiosis establishment, suggesting other receptors are at play. Here, we use a combination of cnidarian model systems and human cell lines to determine if phagocytic integrins facilitate symbiont recognition and uptake. Integrins are highly expressed in the gastrodermal tissue of the host, where symbiosis takes place, and symbiont uptake alters the expression of integrins and downstream signaling molecules. Blockage of integrin binding sites with competitor peptides reduces symbiont uptake, while uptake of non-symbiotic algae, or uptake in a non-symbiotic cnidarian, is unaffected. Finally, overexpression of phagocytic integrins in human cells increases symbiont uptake, and mutation of the active binding site abolishes uptake. Our findings reveal integrins as important receptors for symbiosis establishment and shed light on the evolutionary functions of integrins during phagocytosis.
Solo papers (SPs) have experienced a sustained decline over the last century, which has turned them into a residual publication type. However, SPs have traditionally been highly influential and disruptive, and efforts to...Solo papers (SPs) have experienced a sustained decline over the last century, which has turned them into a residual publication type. However, SPs have traditionally been highly influential and disruptive, and efforts to protect them from extinction are worthwhile. [Image: see text]
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...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.
Eukaryotic cells are highly compartmentalized, enabling sophisticated division of labour. For example, genetic information is stored in the nucleus while energy is produced in mitochondria. Despite this clear specialisat...Eukaryotic cells are highly compartmentalized, enabling sophisticated division of labour. For example, genetic information is stored in the nucleus while energy is produced in mitochondria. Despite this clear specialisation, compartments depend on intensive communication, including the exchange of metabolites and macromolecules. This is achieved through intracellular trafficking with membranous carriers such as endosomes, which constitute versatile transport vehicles. Key cargos include mRNAs and ribosomes that hitchhike on endosomes, linking RNA and membrane biology. In this review, we summarize recent advances showing how mRNAs are mechanistically attached to membranes of endosomes and lysosomal vesicles and how cargos are identified for transport. The encoded proteins illuminate the biological processes that rely on such spatiotemporal control. This is particularly true for the regulation of subcellular mitochondrial homeostasis, disclosing intensive multi-organelle networking. As a general concept, the underlying protein/protein and protein/RNA interactions exhibit significant redundancy yet are organized in a strict hierarchy with distinct core and accessory functions. This ensures both the robustness and specificity of mRNA hitchhiking.
Cell surface glycoproteins Basigin or embigin form heterodimers with monocarboxylate transporters (MCTs), enhancing their membrane trafficking and modulating their transport functions. Cancer cells often reprogram their...Cell surface glycoproteins Basigin or embigin form heterodimers with monocarboxylate transporters (MCTs), enhancing their membrane trafficking and modulating their transport functions. Cancer cells often reprogram their metabolism and depend on proton-coupled lactate transport mediated by MCTs to sustain their glycolytic state and to maintain intracellular pH. A deeper understanding of MCTs regulation may open avenues for the development of novel inhibitors, potentially applicable in clinical settings. Here, we determine the cryo-EM structures of the human MCT2-embigin complex in both apo and AR-C155858-bound states and observe that embigin engages in extensive interactions with MCT2, facilitating its localization to the plasma membrane and substrate transport. Given the high structural conservation among MCTs, we conduct virtual screening based on MCT1/2 structures and identify Tucatinib as an effective inhibitor of pyruvate transport mediated by both MCT1 and MCT2. We show that Tucatinib potently inhibits the proliferation and migration of cervical tumor cells in vitro and tumor growth in a mouse xenograft model, while exhibiting excellent biological safety. These findings offer molecular insights into the structural and functional mechanism of MCT2 and identify Tucatinib as novel dual inhibitor of both transporters.
The Arctic ground squirrel (AGS, Urocitellus parryii), an extreme hibernator, exhibits remarkable resilience to stressors like hypoxia and hypothermia, making it an ideal model for studying cellular metabolic adaptation....The Arctic ground squirrel (AGS, Urocitellus parryii), an extreme hibernator, exhibits remarkable resilience to stressors like hypoxia and hypothermia, making it an ideal model for studying cellular metabolic adaptation. The underlying mechanisms of AGS resilience are largely unknown. Here, we use lipidomic and metabolomic profiling to discover specific downregulation of triglyceride lipids and upregulation of the lipid biosynthetic precursor malonic acid in AGS neural stem cells (NSC) versus murine NSCs. Inhibiting lipid biosynthesis recapitulates hypoxic resilience of squirrel NSCs. Extending this model, we find that acute exposure to hypoxia downregulates key lipid biosynthetic enzymes in C. elegans, while inhibiting lipid biosynthesis reduces mitochondrial fission and facilitates hypoxic survival. Moreover, inhibiting lipid biosynthesis protects against APOE4-induced pathologies and aging trajectories in C. elegans. These findings suggest triglyceride downregulation as a conserved metabolic resilience mechanism, offering insights into protective strategies for neural tissues under hypoxic or ischemic conditions, APOE4-induced pathologies and aging.
AI-assisted polygenic embryo screening is a new technology to predict embryonic implantation, disease risk and non-clinical traits for IVF embryos. To highlight its social and ethical implications, we locate it within th...AI-assisted polygenic embryo screening is a new technology to predict embryonic implantation, disease risk and non-clinical traits for IVF embryos. To highlight its social and ethical implications, we locate it within the national landscapes of Germany’s legal caution, Israel’s techno-enthusiasm and US market liberalism. [Image: see text]
Even before the advent of multicellular life, unicellular creatures would communicate with their neighbours to coordinate their behaviours. Multicellular organisms have the particular challenge of orchestrating the diffe...Even before the advent of multicellular life, unicellular creatures would communicate with their neighbours to coordinate their behaviours. Multicellular organisms have the particular challenge of orchestrating the differentiation of stem and progenitor cells to generate and maintain coherent functional tissues. However, stem and progenitor cells face a problem: their differentiation response can be buffeted by oscillations or stochastic fluctuations in intrinsic regulators. This generates cell-to-cell variability, which can be further compounded when extrinsic cues don't provide clear unambiguous instructions. So, left to their own devices, cells may differentiate at different rates or different directions even in response to the same cues. Fortunately, cells in multicellular organisms are not left to their own devices: they continually sense and respond to the behaviours of their neighbours. Here I discuss when, where, and how stem and progenitor cells communicate to synchronise their response to differentiation cues. I highlight technical challenges in identifying such synchronisation mechanisms, and survey emerging technologies that may help overcome these challenges.
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 mediate...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.
Niehaus I, Wilsch-Bräuninger M, Mora-Bermúdez F
… +17 more, Rost F, Bobic-Rasonja M, Radosevic V, Milkovic-Perisa M, Wimberger P, Severino M, Haase A, Martin U, Kuenzel K, Guan K, Neumann K, Walker N, Schröck E, Jovanov-Milosevic N, Huttner WB, Di Donato N, Heide M
Actins are cytoskeletal proteins that are essential for multiple cellular processes. Mutations in the ACTB and ACTG1 genes, encoding the ubiquitous beta- and gamma-cytoskeletal actin isoforms, respectively, cause a broad...Actins are cytoskeletal proteins that are essential for multiple cellular processes. Mutations in the ACTB and ACTG1 genes, encoding the ubiquitous beta- and gamma-cytoskeletal actin isoforms, respectively, cause a broad spectrum of neurodevelopmental disorders, with microcephaly as the most frequent one. To investigate the pathogenesis underlying this cortical malformation, we studied patient-derived cerebral organoids from induced pluripotent stem cells of individuals with the Baraitser-Winter-CerebroFrontoFacial syndrome (BWCFF-S) carrying an ACTB/ACTG1 missense mutation. These organoids were reduced in size, showing a thinner ventricular zone (VZ) due to reduced VZ progenitor abundance. Strikingly, VZ progenitors in BWCFF-S cerebral organoids displayed a shift in the orientation of their cleavage plane from a predominantly vertical to a majoritarian horizontal orientation. The latter cleavage plane orientation is incompatible with increasing VZ progenitor abundance and instead promotes basal progenitor generation. Various cytoskeletal and morphological irregularities of BWCFF-S VZ progenitors, notably in the apical region, seemingly contribute to this change in cleavage plane orientation. Our results provide insight into the cell biological basis of the microcephaly associated with BWCFF-S caused by actin mutations.
Mouse embryonic stem cells (mESCs), in addition to differentiating into the three germ layers, can reverse typical developmental trajectories, as exemplified by their ability to de-differentiate into 2-cell-like cells (2...Mouse embryonic stem cells (mESCs), in addition to differentiating into the three germ layers, can reverse typical developmental trajectories, as exemplified by their ability to de-differentiate into 2-cell-like cells (2CLCs) that resemble the mammalian embryo during zygotic genome activation (ZGA). This unique property offers the opportunity to elucidate the molecular principles that govern the pre-implantation stages of mammalian development. Here, we dissect the functions of the chromatin repressor EHMT2, a candidate antagonist of the mESC-to-2CLC transition, by leveraging a multipurpose allele for acute protein depletion and efficient immunoprecipitation. Our experiments revealed distinct principles of EHMT2-mediated gene repression in mESCs based on specific chromatin binding patterns and protein co-factors. Most notably, EHMT2 directly represses large clusters of co-regulated gene loci that comprise a significant fraction of the 2CLC-specific transcriptome by initiating H3K9me2 spreading from distal LINE-1 elements. EHMT2 counteracts the recruitment of the activator DPPA2/4 to promoter-proximal endogenous retroviral elements (ERVs) at 2CLC genes. EHMT2 depletion enhances the expression of ZGA-associated transcripts in 2CLCs and synergizes with spliceosome inhibition and retinoic acid signaling to facilitate the mESC-to-2CLC transition. In contrast to ZGA-associated genes, the repression of germ layer-associated transcripts by EHMT2 occurs outside of gene clusters, in collaboration with ZFP462, and involves binding to non-repetitive candidate enhancers. Our observations provide novel mechanistic insight into how pluripotent cells achieve attenuation of their bidirectional differentiation potential and reveal unique transcriptional features of murine totipotent cells.
Argonautes are ancient proteins with well-characterised functions in cell-autonomous gene regulation and genome defence, but less clear roles in non-cell-autonomous processes. Extracellular Argonautes have been reported...Argonautes are ancient proteins with well-characterised functions in cell-autonomous gene regulation and genome defence, but less clear roles in non-cell-autonomous processes. Extracellular Argonautes have been reported across plants, animals and protozoa, yet their biochemical and functional properties remain elusive. Here, we demonstrate that an extracellular Argonaute (exWAGO) released by the rodent-infective nematode Heligmosomoides bakeri is detectable inside mouse cells during the natural infection. We show that exWAGO is released from H. bakeri in both vesicular and non-vesicular forms that have different resistances to proteolysis, different accessibilities to antibodies and associate with different subsets of secondary siRNAs. Using recombinant exWAGO protein, we demonstrate that non-vesicular exWAGO is internalised by mouse cells in vitro and that immunisation of mice with exWAGO confers partial protection against subsequent H. bakeri infection and generates antibodies that block exWAGO uptake into cells. Finally, we show that properties of exWAGO are conserved across Clade V nematodes that infect humans and livestock. Together, this work expands the context in which Argonautes function and illuminates an RNA-binding protein as a vaccine target for parasitic nematodes.
The mechanisms regulating gamete fusion and preventing polyspermy in mammalian fertilization remain incompletely understood. This study combines real-time imaging, confocal microscopy and statistical analysis to investig...The mechanisms regulating gamete fusion and preventing polyspermy in mammalian fertilization remain incompletely understood. This study combines real-time imaging, confocal microscopy and statistical analysis to investigate fertilization and polyspermy prevention dynamics in mice. By tracking the behavior of over one hundred spermatozoa entering the perivitelline space of oocytes, we dissect the respective contributions of oocyte structures (zona pellucida (ZP), perivitelline space (PVS), oolemma) and sperm components (head, flagellum) to fertilization and polyspermy prevention. We find that fertilization requires specific sperm head movements on the oolemma, driven by flagellar beating and facilitated by trapping the flagellum in the ZP, revealing a novel role for this structure. Our kinetic analysis characterizes a slow "penetration block" that gradually limits sperm entry into the PVS and a faster "fusion block" that prevents further fusion events. As the penetration block becomes significant after the fusion block is established, only the latter effectively prevents polyspermy in mice. We propose that it acts through neutralization of excess sperm in the PVS by oocyte-derived proteins CD9 and JUNO coating non-fertilizing spermatozoa.
The choice between somatic and germline fates is essential for species survival. This choice occurs in embryonic epiblast cells, as these cells are competent for both somatic and germline differentiation. The transcripti...The choice between somatic and germline fates is essential for species survival. This choice occurs in embryonic epiblast cells, as these cells are competent for both somatic and germline differentiation. The transcription factor OTX2 regulates this process, as Otx2-null epiblast-like cells (EpiLCs) form primordial germ cell-like cells (PGCLCs) with enhanced efficiency. Yet, how OTX2 achieves this function is not fully characterised. Here we show that OTX2 controls chromatin accessibility at specific chromatin loci to enable somatic differentiation. CUT&RUN for OTX2 and ATAC-seq in wild-type and Otx2-null embryonic stem cells and EpiLCs identifies regions where OTX2 binds and opens chromatin. Enforced OTX2 expression maintains accessibility at these regions and also induces opening of ~4000 somatic-associated regions in cells differentiating in the presence of PGC-inducing cytokines. Once cells have acquired germline identity, these additional regions no longer respond to OTX2 and remain closed. Our results indicate that OTX2 works in cells with dual competence for somatic and germline differentiation to increase accessibility of somatic regulatory regions and induce the somatic fate at the expense of the germline.
Human immune protection against bacteria critically depends on activation of the complement system. The direct bacteriolytic activity of complement molecules against Gram-negative bacteria acts via the formation of Membr...Human immune protection against bacteria critically depends on activation of the complement system. The direct bacteriolytic activity of complement molecules against Gram-negative bacteria acts via the formation of Membrane Attack Complex (MAC) pores. Bactericidal MAC pores damage the bacterial outer membrane, leading to destabilization of the inner membrane. Although it is well-established that inner membrane damage is crucial for bacterial cell death, the critical event causing MAC-mediated inner membrane damage remains elusive. Here we question whether the bacterial cell envelope possesses vulnerable spots for MAC pores to insert. By following the localization of MAC pores on E. coli over time using fluorescence microscopy, we elucidate that MAC deposition initiates at the new bacterial pole, which induces inner membrane damage and halts bacterial division. MAC components C8 and C9 preferentially localize at new bacterial poles, while C3b localizes randomly on the bacterial surface. This suggests that preferential MAC localization is determined by one of the initial steps of MAC formation. These findings provide valuable information about the interplay between immune components and the Gram-negative cell envelope.
Oocyte development is a fierce competitive process in which only a small fraction of germ cells survives massive waves of cell death to become a fertilizable egg. Using an ex vivo 4D imaging system, Zhang and colleagues...Oocyte development is a fierce competitive process in which only a small fraction of germ cells survives massive waves of cell death to become a fertilizable egg. Using an ex vivo 4D imaging system, Zhang and colleagues discovered that individual oocytes actively move, extend filopodia-like protrusions, and engulf the debris of their dying neighbors to fuel their own growth (Zhang et al, a). These findings reveal a previously unrecognized, phagocytosis-driven mechanism of oocyte selection that shapes the ovarian reserve.