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Preserving cells in a functional, non-senescent state is a major goal for extending human healthspans. Model organisms reveal that longevity and senescence are genetically controlled, but how genes control longevity in different mammalian tissues is unknown. Here, we report a new human genetic disease that causes cell senescence, liver and immune dysfunction, and early mortality that results from deficiency of GIMAP5, an evolutionarily conserved GTPase selectively expressed in lymphocytes and endothelial cells. We show that GIMAP5 restricts the pathological accumulation of long-chain ceramides (CERs), thereby regulating longevity. GIMAP5 controls CER abundance by interacting with protein kinase CK2 (CK2), attenuating its ability to activate CER synthases. Inhibition of CK2 and CER synthase rescues GIMAP5-deficient T cells by preventing CER overaccumulation and cell deterioration. Thus, GIMAP5 controls longevity assurance pathways crucial for immune function and healthspan in mammals.

Vascular morphogenesis requires a delicate gradient of Notch signaling controlled, in part, by the distribution of ligands (Dll4 and Jagged1). How Jagged1 (JAG1) expression is compartmentalized in the vascular plexus remains unclear. Here, we show that Jag1 mRNA is a direct target of zinc-finger protein 36 (ZFP36), an RNA-binding protein involved in mRNA decay that we find robustly induced by vascular endothelial growth factor (VEGF). Endothelial cells lacking ZFP36 display high levels of JAG1 and increase angiogenic sprouting in vitro. Furthermore, mice lacking Zfp36 in endothelial cells display mispatterned and increased levels of JAG1 in the developing retinal vascular plexus. Abnormal levels of JAG1 at the sprouting front alters NOTCH1 signaling, increasing the number of tip cells, a phenotype that is rescued by imposing haploinsufficiency of Jag1. Our findings reveal an important feedforward loop whereby VEGF stimulates ZFP36, consequently suppressing Jag1 to enable adequate levels of Notch signaling during sprouting angiogenesis.

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. , Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating β2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and β2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

How individual T cells compete for and respond to IL-2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high-affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high-affinity CD8 T cells. As part of an incoherent feed-forward loop, ZFP36L1 has a nonredundant role in suppressing multiple negative regulators of cytokine signaling and mediating a selection mechanism based on competition for IL-2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes the dominance of high-affinity T cells by installing a hierarchical response to IL-2.

EIF4A1 and cofactors EIF4B and EIF4H have been well characterised in cancers, including B cell malignancies, for their ability to promote the translation of oncogenes with structured 5' untranslated regions. However, very little is known of their roles in nonmalignant cells. Using mouse models to delete , or in B cells, we show that EIF4A1, but not EIF4B or EIF4H, is essential for B cell development and the germinal centre response. After B cell activation in vitro, EIF4A1 facilitates an increased rate of protein synthesis, MYC expression, and expression of cell cycle regulators. However, EIF4A1-deficient cells remain viable, whereas inhibition of EIF4A1 and EIF4A2 by Hippuristanol treatment induces cell death.

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Early organogenesis represents a key step in animal development, where pluripotent cells diversify to initiate organ formation. Here, we sampled 300,000 single cell transcriptomes from mouse embryos between E8.5 and E9.5 in 6-hour intervals and combined this new dataset with our previous atlas (E6.5-E8.5) to produce a densely sampled time course of >400,000 cells from early gastrulation to organogenesis. Computational lineage reconstruction identified complex waves of blood and endothelial development, including a new programme for somite-derived endothelium. We also dissected the E7.5 primitive streak into four adjacent regions, performed scRNA-Seq and predicted cell fates computationally. Finally, we defined developmental state/fate relationships by combining orthotopic grafting, microscopic analysis and scRNA-Seq to transcriptionally determine cell fates of grafted primitive streak regions after 24h of in vitro embryo culture. Experimentally determined fate outcomes were in good agreement with computationally predicted fates, demonstrating how classical grafting experiments can be revisited to establish high-resolution cell state/fate relationships. Such interdisciplinary approaches will benefit future studies in developmental biology and guide the in vitro production of cells for organ regeneration and repair.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

Extraction protocols and liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) methods for the measurement of four lipid categories, namely glycerophospholipids, glycerolipids, sphingolipids and sterol lipids in human plasma, are described here. Step-by-step instructions are provided for the liquid-liquid extraction methods, including solution preparation and the non-endogenous lipid internal standards used. All instrumental conditions, chromatography columns and solutions required for the LC-MS and LC-MS/MS methods are also provided in detail.

Norbin is an adaptor protein that binds numerous G protein-coupled receptors (GPCRs), is highly expressed in neurons, and is essential for a functioning nervous system in rodent models. Yet, beyond its control of neurite outgrowth and synaptic plasticity, few cellular roles of Norbin have been investigated to date. Furthermore, while Norbin is known to regulate the steady-state cell surface levels of several GPCRs, only in one case has the protein been shown to control the agonist-induced receptor internalisation which serves to attenuate GPCR signalling. Here, we generated a Norbin-deficient PC12 cell line which enabled us to study both the cellular functions of Norbin and its roles in GPCR trafficking and signalling. We show that Norbin limits cell size and spreading, and is required for the growth, viability and cell cycle progression of PC12 cells. We also found that Norbin regulates both the steady-state surface level and agonist-induced internalisation of the GPCR sphingosine-1-phosphate receptor 1 (S1PR1) in these cells, suggesting that its role in agonist-dependent GPCR trafficking is more widespread than previously appreciated. Finally, we show that Norbin limits the S1P-stimulated activation of Akt and p38 Mapk, and is required for the activation of Erk in PC12 cells. Together, our findings provide a better understanding of the cellular functions of Norbin and its control of GPCR trafficking.

Recent advances in single-cell omics have transformed characterisation of cell types in challenging-to-study biological contexts. In contexts with limited single-cell samples, such as the early human embryo inference of transcription factor-gene regulatory network (GRN) interactions is especially difficult. Here, we assessed application of different linear or non-linear GRN predictions to single-cell simulated and human embryo transcriptome datasets. We also compared how expression normalisation impacts on GRN predictions, finding that transcripts per million reads outperformed alternative methods. GRN inferences were more reproducible using a non-linear method based on mutual information (MI) applied to single-cell transcriptome datasets refined with chromatin accessibility (CA) (called MICA), compared with alternative network prediction methods tested. MICA captures complex non-monotonic dependencies and feedback loops. Using MICA, we generated the first GRN inferences in early human development. MICA predicted co-localisation of the AP-1 transcription factor subunit proto-oncogene JUND and the TFAP2C transcription factor AP-2γ in early human embryos. Overall, our comparative analysis of GRN prediction methods defines a pipeline that can be applied to single-cell multi-omics datasets in especially challenging contexts to infer interactions between transcription factor expression and target gene regulation.

Studies of mammalian development have advanced our understanding of the genetic, epigenetic, and cellular processes that orchestrate embryogenesis and have uncovered new insights into the unique aspects of human embryogenesis. Recent studies have now produced the first epigenetic maps of early human embryogenesis, stimulating new ideas about epigenetic reprogramming, cell fate control, and the potential mechanisms underpinning developmental plasticity in human embryos. In this review, we discuss these new insights into the epigenetic regulation of early human development and the importance of these processes for safeguarding development. We also highlight unanswered questions and key challenges that remain to be addressed.

LIPID MAPS (LIPID Metabolites and Pathways Strategy), www.lipidmaps.org, provides a systematic and standardized approach to organizing lipid structural and biochemical data. Founded 20 years ago, the LIPID MAPS nomenclature and classification has become the accepted community standard. LIPID MAPS provides databases for cataloging and identifying lipids at varying levels of characterization in addition to numerous software tools and educational resources, and became an ELIXIR-UK data resource in 2020. This paper describes the expansion of existing databases in LIPID MAPS, including richer metadata with literature provenance, taxonomic data and improved interoperability to facilitate FAIR compliance. A joint project funded by ELIXIR-UK, in collaboration with WikiPathways, curates and hosts pathway data, and annotates lipids in the context of their biochemical pathways. Updated features of the search infrastructure are described along with implementation of programmatic access via API and SPARQL. New lipid-specific databases have been developed and provision of lipidomics tools to the community has been updated. Training and engagement have been expanded with webinars, podcasts and an online training school.

Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive protein folding and macromolecular interactions. Reciprocally, macromolecules restrict the movement of 'structured' water molecules within their hydration layers, reducing the available 'free' bulk solvent and therefore the total thermodynamic potential energy of water, or water potential. Here, within concentrated macromolecular solutions such as the cytosol, we found that modest changes in temperature greatly affect the water potential, and are counteracted by opposing changes in osmotic strength. This duality of temperature and osmotic strength enables simple manipulations of solvent thermodynamics to prevent cell death after extreme cold or heat shock. Physiologically, cells must sustain their activity against fluctuating temperature, pressure and osmotic strength, which impact water availability within seconds. Yet, established mechanisms of water homeostasis act over much slower timescales; we therefore postulated the existence of a rapid compensatory response. We find that this function is performed by water potential-driven changes in macromolecular assembly, particularly biomolecular condensation of intrinsically disordered proteins. The formation and dissolution of biomolecular condensates liberates and captures free water, respectively, quickly counteracting thermal or osmotic perturbations of water potential, which is consequently robustly buffered in the cytoplasm. Our results indicate that biomolecular condensation constitutes an intrinsic biophysical feedback response that rapidly compensates for intracellular osmotic and thermal fluctuations. We suggest that preserving water availability within the concentrated cytosol is an overlooked evolutionary driver of protein (dis)order and function.

With the increase in the number of parameters that can be detected at the single-cell level using flow and mass cytometry, there has been a paradigm shift when handling and analyzing data sets. Cytometry Shared Resource Laboratories (SRLs) already take on the responsibility of ensuring users have resources and training in experimental design and operation of instruments to promote high-quality data acquisition. However, the role of SRLs downstream, during data handling and analysis, is not as well defined and agreed upon. Best practices dictate a central role for SRLs in this process as they are in a pivotal position to support research in this context, but key considerations about how to effectively fill this role need to be addressed. Two surveys and one workshop at CYTO 2022 in Philadelphia, PA, were performed to gain insight into what strategies SRLs are successfully employing to support high-dimensional data analysis and where SRLs and their users see limitations and long-term challenges in this area. Recommendations for high-dimensional data analysis support provided by SRLs will be offered and discussed.

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 non-canonical autophagy and Conjugation of ATG8s to Single Membranes (CASM). Following damage, ATG8s are rapidly and directly conjugated onto lysosome membranes, independently of ATG13/WIPI2, lipidating to PS (and PE), a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, direct recruitment of ATG16L1 via its WD40-domain/K490A, and is sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with 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.

During mammalian embryo development, pluripotent epiblast cells diversify into the three primary germ layers, which will later give rise to all fetal and adult tissues. These processes involve profound transcriptional and epigenetic changes that require precise coordination. Peptidylarginine deiminase IV (PADI4) is a transcriptional regulator that is strongly associated with inflammation and carcinogenesis but whose physiological roles are less well understood. We previously found that expression is associated with pluripotency. Here, we examined the role of PADI4 in maintaining the multi-lineage differentiation potential of mouse embryonic stem (ES) cells. Using bulk and single-cell transcriptomic analyses of embryoid bodies (EBs) derived from knock-out () mouse ES cells, we find that PADI4 loss impairs mesoderm diversification and differentiation of cardimyocytes and endothelial cells. Additionally, deletion leads to concerted downregulation of genes associated with polarized growth, sterol metabolism and the extracellular matrix (ECM). This study indicates a requirement for in the specification of the mesodermal lineage and reports the associated transcriptome, providing a platform for understanding the physiological functions of in development and homeostasis. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

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Human pluripotent stem cells (hPSCs) are of fundamental relevance in regenerative medicine. Naïve hPSCs hold promise to overcome some of the limitations of conventional (primed) hPSCs, including recurrent epigenetic anomalies. Naïve-to-primed transition (capacitation) follows transcriptional dynamics of human embryonic epiblast and is necessary for somatic differentiation from naïve hPSCs. We found that capacitated hPSCs are transcriptionally closer to postimplantation epiblast than conventional hPSCs. This prompted us to comprehensively study epigenetic and related transcriptional changes during capacitation. Our results show that CpG islands, gene regulatory elements, and retrotransposons are hotspots of epigenetic dynamics during capacitation and indicate possible distinct roles of specific epigenetic modifications in gene expression control between naïve and primed hPSCs. Unexpectedly, PRC2 activity appeared to be dispensable for the capacitation. We find that capacitated hPSCs acquire an epigenetic state similar to conventional hPSCs. Significantly, however, the X chromosome erosion frequently observed in conventional female hPSCs is reversed by resetting and subsequent capacitation.

Affinity maturation, the progressive increase in serum Ab affinity after vaccination, is an essential process that contributes to an effective humoral response against vaccines and infections. Germinal centers are key for affinity maturation, because they are where B cells undergo somatic hypermutation of their Ig genes in the dark zone before going through positive selection in the light zone via interactions with T follicular helper cells and follicular dendritic cells. In aged mice, affinity maturation has been shown to be impaired after immunization, but whether B cell-intrinsic factors contribute to this defect remains unclear. In this study, we show that B cells from aged BCR transgenic mice are able to become germinal center B cells, which are capable of receiving positive selection signals to a similar extent as B cells from young adult mice. Consistent with this, aging also does not impact the ability of B cells to undergo somatic hypermutation and acquire affinity-enhancing mutations. By contrast, transfer of B cells from young adult BCR mice into aged recipients resulted in the impaired acquisition of affinity-enhancing mutations, demonstrating that the aged microenvironment causes altered affinity maturation.

During aging, proteostasis capacity declines and distinct proteins become unstable and can accumulate as protein aggregates inside and outside of cells. Both in disease and during aging, proteins selectively aggregate in certain tissues and not others. Yet, tissue-specific regulation of cytoplasmic protein aggregation remains poorly understood. Surprisingly, we found that the inhibition of 3 core protein quality control systems, namely chaperones, the proteasome, and macroautophagy, leads to lower levels of age-dependent protein aggregation in Caenorhabditis elegans pharyngeal muscles, but higher levels in body-wall muscles. We describe a novel safety mechanism that selectively targets newly synthesized proteins to suppress their aggregation and associated proteotoxicity. The safety mechanism relies on macroautophagy-independent lysosomal degradation and involves several previously uncharacterized components of the intracellular pathogen response (IPR). We propose that this protective mechanism engages an anti-aggregation machinery targeting aggregating proteins for lysosomal degradation.

To produce a diverse antibody repertoire, immunoglobulin heavy-chain (Igh) loci undergo large-scale alterations in structure to facilitate juxtaposition and recombination of spatially separated variable (V), diversity (D), and joining (J) genes. These chromosomal alterations are poorly understood. Uncovering their patterns shows how chromosome dynamics underpins antibody diversity. Using tiled Capture Hi-C, we produce a comprehensive map of chromatin interactions throughout the 2.8-Mb Igh locus in progenitor B cells. We find that the Igh locus folds into semi-rigid subdomains and undergoes flexible looping of the V genes to its 3' end, reconciling two views of locus organization. Deconvolution of single Igh locus conformations using polymer simulations identifies thousands of different structures. This heterogeneity may underpin the diversity of V(D)J recombination events. All three immunoglobulin loci also participate in a highly specific, developmentally regulated network of interchromosomal interactions with genes encoding B cell-lineage factors. This suggests a model of interchromosomal coordination of B cell development.

The massive accumulation of extrachromosomal ribosomal DNA circles (ERCs) in yeast mother cells has been long cited as the primary driver of replicative ageing. ERCs arise through ribosomal DNA (rDNA) recombination, and a wealth of genetic data connects rDNA instability events giving rise to ERCs with shortened life span and other ageing pathologies. However, we understand little about the molecular effects of ERC accumulation. Here, we studied ageing in the presence and absence of ERCs, and unexpectedly found no evidence of gene expression differences that might indicate stress responses or metabolic feedback caused by ERCs. Neither did we observe any global change in the widespread disruption of gene expression that accompanies yeast ageing, altogether suggesting that ERCs are largely inert. Much of the differential gene expression that accompanies ageing in yeast was actually associated with markers of the senescence entry point (SEP), showing that senescence, rather than age, underlies these changes. Cells passed the SEP irrespective of ERCs, but we found the SEP to be associated with copy number amplification of a region of chromosome XII between the rDNA and the telomere (ChrXIIr) forming linear fragments up to approximately 1.8 Mb size, which arise in aged cells due to rDNA instability but through a different mechanism to ERCs. Therefore, although rDNA copy number increases dramatically with age due to ERC accumulation, our findings implicate ChrXIIr, rather than ERCs, as the primary driver of senescence during budding yeast ageing.