RESUMEN
Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical in vivo genomic technologies that capture cellular dynamics are currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24 hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48 hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies.
Asunto(s)
Glioblastoma , Humanos , Perfilación de la Expresión Génica , Glioblastoma/patología , Inmunoterapia , Células Asesinas Naturales , Macrófagos , Microambiente Tumoral , Análisis de la Célula IndividualRESUMEN
Aging is the key risk factor for cognitive decline, yet the molecular changes underlying brain aging remain poorly understood. Here, we conducted spatiotemporal RNA sequencing of the mouse brain, profiling 1,076 samples from 15 regions across 7 ages and 2 rejuvenation interventions. Our analysis identified a brain-wide gene signature of aging in glial cells, which exhibited spatially defined changes in magnitude. By integrating spatial and single-nucleus transcriptomics, we found that glial aging was particularly accelerated in white matter compared with cortical regions, whereas specialized neuronal populations showed region-specific expression changes. Rejuvenation interventions, including young plasma injection and dietary restriction, exhibited distinct effects on gene expression in specific brain regions. Furthermore, we discovered differential gene expression patterns associated with three human neurodegenerative diseases, highlighting the importance of regional aging as a potential modulator of disease. Our findings identify molecular foci of brain aging, providing a foundation to target age-related cognitive decline.
Asunto(s)
Envejecimiento , Disfunción Cognitiva , Sustancia Blanca , Animales , Humanos , Ratones , Disfunción Cognitiva/genética , Perfilación de la Expresión Génica , Núcleo Solitario , Sustancia Blanca/patología , Análisis de Expresión Génica de una Sola Célula , Encéfalo/patologíaRESUMEN
Axial development of mammals involves coordinated morphogenetic events, including axial elongation, somitogenesis, and neural tube formation. To gain insight into the signals controlling the dynamics of human axial morphogenesis, we generated axially elongating organoids by inducing anteroposterior symmetry breaking of spatially coupled epithelial cysts derived from human pluripotent stem cells. Each organoid was composed of a neural tube flanked by presomitic mesoderm sequentially segmented into somites. Periodic activation of the somite differentiation gene MESP2 coincided in space and time with anteriorly traveling segmentation clock waves in the presomitic mesoderm of the organoids, recapitulating critical aspects of somitogenesis. Timed perturbations demonstrated that FGF and WNT signaling play distinct roles in axial elongation and somitogenesis, and that FGF signaling gradients drive segmentation clock waves. By generating and perturbing organoids that robustly recapitulate the architecture of multiple axial tissues in human embryos, this work offers a means to dissect mechanisms underlying human embryogenesis.
Asunto(s)
Desarrollo Embrionario , Mesodermo , Somitos , Animales , Humanos , Tipificación del Cuerpo , Regulación del Desarrollo de la Expresión Génica , Mamíferos/genética , Mesodermo/fisiología , Morfogénesis , Vía de Señalización Wnt , Organoides/metabolismoRESUMEN
A fundamental feature of cellular growth is that total protein and RNA amounts increase with cell size to keep concentrations approximately constant. A key component of this is that global transcription rates increase in larger cells. Here, we identify RNA polymerase II (RNAPII) as the limiting factor scaling mRNA transcription with cell size in budding yeast, as transcription is highly sensitive to the dosage of RNAPII but not to other components of the transcriptional machinery. Our experiments support a dynamic equilibrium model where global RNAPII transcription at a given size is set by the mass action recruitment kinetics of unengaged nucleoplasmic RNAPII to the genome. However, this only drives a sub-linear increase in transcription with size, which is then partially compensated for by a decrease in mRNA decay rates as cells enlarge. Thus, limiting RNAPII and feedback on mRNA stability work in concert to scale mRNA amounts with cell size.
Asunto(s)
Tamaño de la Célula , ARN Polimerasa II , Transcripción Genética , Retroalimentación , ARN Polimerasa II/metabolismo , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The T cell receptor (TCR) expressed by T lymphocytes initiates protective immune responses to pathogens and tumors. To explore the structural basis of how TCR signaling is initiated when the receptor binds to peptide-loaded major histocompatibility complex (pMHC) molecules, we used cryogenic electron microscopy to determine the structure of a tumor-reactive TCRαß/CD3δγε2ζ2 complex bound to a melanoma-specific human class I pMHC at 3.08 Å resolution. The antigen-bound complex comprises 11 subunits stabilized by multivalent interactions across three structural layers, with clustered membrane-proximal cystines stabilizing the CD3-εδ and CD3-εγ heterodimers. Extra density sandwiched between transmembrane helices reveals the involvement of sterol lipids in TCR assembly. The geometry of the pMHC/TCR complex suggests that efficient TCR scanning of pMHC requires accurate pre-positioning of T cell and antigen-presenting cell membranes. Comparisons of the ligand-bound and unliganded receptors, along with molecular dynamics simulations, indicate that TCRs can be triggered in the absence of spontaneous structural rearrangements.
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Neoplasias , Receptores de Antígenos de Linfocitos T , Humanos , Complejo Mayor de Histocompatibilidad , Péptidos/química , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismoRESUMEN
A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes to predict the function of poorly characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena-from RNA processing to differentiation. We leverage this ability to systematically identify genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene and cellular function.
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Genómica , Análisis de la Célula Individual , Sistemas CRISPR-Cas/genética , Mapeo Cromosómico , Genotipo , Fenotipo , Análisis de la Célula Individual/métodosRESUMEN
Exosomes are small, single-membrane, secreted organelles of â¼30 to â¼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.
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Exosomas/metabolismo , Animales , Transporte Biológico , Exosomas/inmunología , Exosomas/fisiología , Exosomas/ultraestructura , Matriz Extracelular/metabolismo , Humanos , Neoplasias , Enfermedades Neurodegenerativas , Multimerización de Proteína , Transducción de SeñalRESUMEN
During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.
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Proteínas Bacterianas/química , Citidina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Motivos de Nucleótidos , Unión ProteicaRESUMEN
Neutrophils are critical to innate immunity, including host defense against bacterial and fungal infections. They achieve their host defense role by phagocytosing pathogens, secreting their granules full of cytotoxic enzymes, or expelling neutrophil extracellular traps (NETs) during the process of NETosis. NETs are weblike DNA structures decorated with histones and antimicrobial proteins released by activated neutrophils. Initially described as a means for neutrophils to neutralize pathogens, NET release also occurs in sterile inflammation, promotes thrombosis, and can mediate tissue damage. To effectively manipulate this double-edged sword to fight a particular disease, researchers must work toward understanding the mechanisms driving NETosis. Such understanding would allow the generation of new drugs to promote or prevent NETosis as needed. While knowledge regarding the (patho)physiological roles of NETosis is accumulating, little is known about the cellular and biophysical bases of this process. In this review, we describe and discuss our current knowledge of the molecular, cellular, and biophysical mechanisms mediating NET release as well as open questions in the field.
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Trampas Extracelulares/metabolismo , Animales , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Citosol/metabolismo , ADN/metabolismo , HumanosRESUMEN
The metabolic state of a cell is influenced by cell-extrinsic factors, including nutrient availability and growth factor signaling. Here, we present extracellular matrix (ECM) remodeling as another fundamental node of cell-extrinsic metabolic regulation. Unbiased analysis of glycolytic drivers identified the hyaluronan-mediated motility receptor as being among the most highly correlated with glycolysis in cancer. Confirming a mechanistic link between the ECM component hyaluronan and metabolism, treatment of cells and xenografts with hyaluronidase triggers a robust increase in glycolysis. This is largely achieved through rapid receptor tyrosine kinase-mediated induction of the mRNA decay factor ZFP36, which targets TXNIP transcripts for degradation. Because TXNIP promotes internalization of the glucose transporter GLUT1, its acute decline enriches GLUT1 at the plasma membrane. Functionally, induction of glycolysis by hyaluronidase is required for concomitant acceleration of cell migration. This interconnection between ECM remodeling and metabolism is exhibited in dynamic tissue states, including tumorigenesis and embryogenesis.
Asunto(s)
Proteínas Portadoras/fisiología , Matriz Extracelular/metabolismo , Matriz Extracelular/fisiología , Metabolismo de los Hidratos de Carbono/fisiología , Proteínas Portadoras/metabolismo , Línea Celular Tumoral , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1 , Glucólisis/fisiología , Humanos , Ácido Hialurónico/fisiología , Hialuronoglucosaminidasa/farmacología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Transducción de Señal , Tristetraprolina/metabolismo , Tristetraprolina/fisiologíaRESUMEN
ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.
Asunto(s)
Antibacterianos/farmacología , Formas L/efectos de los fármacos , Muramidasa/metabolismo , beta-Lactamas/farmacología , Animales , Bacillus subtilis/efectos de los fármacos , Bacteriólisis/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Hidrolasas/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Viabilidad Microbiana/efectos de los fármacos , Osmorregulación/efectos de los fármacos , Penicilina G/farmacología , Proteínas de Unión a las Penicilinas , Peptidoglicano/metabolismo , Profagos/efectos de los fármacos , Células RAW 264.7RESUMEN
Microbial populations can maximize fitness in dynamic environments through bet hedging, a process wherein a subpopulation assumes a phenotype not optimally adapted to the present environment but well adapted to an environment likely to be encountered. Here, we show that oxygen induces fluctuating expression of the trimethylamine oxide (TMAO) respiratory system of Escherichia coli, diversifying the cell population and enabling a bet-hedging strategy that permits growth following oxygen loss. This regulation by oxygen affects the variance in gene expression but leaves the mean unchanged. We show that the oxygen-sensitive transcription factor IscR is the key regulator of variability. Oxygen causes IscR to repress expression of a TMAO-responsive signaling system, allowing stochastic effects to have a strong effect on the output of the system and resulting in heterogeneous expression of the TMAO reduction machinery. This work reveals a mechanism through which cells regulate molecular noise to enhance fitness.
Asunto(s)
Escherichia coli/metabolismo , Transducción de Señal , Aerobiosis , Anaerobiosis , Secuencia de Bases , Sitios de Unión , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Metilaminas/metabolismo , Metilaminas/farmacología , Oxígeno/metabolismo , Proteínas Periplasmáticas/química , Proteínas Periplasmáticas/genética , Proteínas Periplasmáticas/metabolismo , Fosfotransferasas/química , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética , Regulación hacia ArribaRESUMEN
The intricate network of the brain's neurons and synapses poses unparalleled challenges for research, distinct from other biological studies. This is particularly true when dissecting how neurons and their functional units work at a cell biological level. While traditional microscopy has been foundational, it was unable to reveal the deeper complexities of neural interactions. However, an imaging renaissance has transformed our capabilities. Advancements in light and electron microscopy, combined with correlative imaging, now achieve unprecedented resolutions, uncovering the most nuanced neural structures. Maximizing these tools requires more than just technical proficiency. It is crucial to align research aims, allocate resources wisely, and analyze data effectively. At the heart of this evolution is interdisciplinary collaboration, where various experts come together to translate detailed imagery into significant biological insights. This review navigates the latest developments in microscopy, underscoring both the promise of and prerequisites for bending this powerful tool set to understanding neuronal cell biology.
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Microscopía , Neuronas , Neuronas/fisiología , Animales , Humanos , Microscopía/métodos , Biología Celular , Encéfalo/fisiología , Sinapsis/fisiologíaRESUMEN
HIV infection persists during antiretroviral therapy (ART) due to a reservoir of latently infected cells that harbor replication-competent virus and evade immunity. Previous ex vivo studies suggested that CD8+ T cells from people with HIV may suppress HIV expression via non-cytolytic mechanisms, but the mechanisms responsible for this effect remain unclear. Here, we used a primary cell-based in vitro latency model and demonstrated that co-culture of autologous activated CD8+ T cells with HIV-infected memory CD4+ T cells promoted specific changes in metabolic and/or signaling pathways resulting in increased CD4+ T cell survival, quiescence, and stemness. Collectively, these pathways negatively regulated HIV expression and ultimately promoted the establishment of latency. As shown previously, we observed that macrophages, but not B cells, promoted latency in CD4+ T cells. The identification of CD8-specific mechanisms of pro-latency activity may favor the development of approaches to eliminate the viral reservoir in people with HIV.
Asunto(s)
Infecciones por VIH , Humanos , Linfocitos T CD8-positivos , Latencia del Virus , Linfocitos T CD4-Positivos , Replicación ViralRESUMEN
Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. The mechanism and function of this curvature were previously unknown. Here, we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm and thus can be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (quantitative analysis of sacculus architecture remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertions into the outer face than the inner face. Furthermore, crvA is quorum regulated, and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.
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Vibrio cholerae/citología , Vibrio cholerae/patogenicidad , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Locomoción , Ratones , Peptidoglicano/metabolismo , Periplasma/metabolismo , Alineación de Secuencia , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , VirulenciaRESUMEN
Mitochondrial outer membrane âº-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing the targeting and insertion of these biophysically diverse proteins remain unknown. Here, we first defined the complement of required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct membrane proteins. Systematic analysis of nine identified factors across 21 diverse âº-helical substrates reveals that these components are organized into distinct targeting pathways that act on substrates based on their topology. NAC is required for the efficient targeting of polytopic proteins, whereas signal-anchored proteins require TTC1, a cytosolic chaperone that physically engages substrates. Biochemical and mutational studies reveal that TTC1 employs a conserved TPR domain and a hydrophobic groove in its C-terminal domain to support substrate solubilization and insertion into mitochondria. Thus, the targeting of diverse mitochondrial membrane proteins is achieved through topological triaging in the cytosol using principles with similarities to ER membrane protein biogenesis systems.
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Membranas Mitocondriales , Proteínas de Saccharomyces cerevisiae , Animales , Membranas Mitocondriales/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae/metabolismo , Mamíferos/metabolismoRESUMEN
The ciliate genus Paramecium served as one of the first model systems in microbial eukaryotic genetics, contributing much to the early understanding of phenomena as diverse as genome rearrangement, cryptic speciation, cytoplasmic inheritance, and endosymbiosis, as well as more recently to the evolution of mating types, introns, and roles of small RNAs in DNA processing. Substantial progress has recently been made in the area of comparative and population genomics. Paramecium species combine some of the lowest known mutation rates with some of the largest known effective populations, along with likely very high recombination rates, thereby harboring a population-genetic environment that promotes an exceptionally efficient capacity for selection. As a consequence, the genomes are extraordinarily streamlined, with very small intergenic regions combined with small numbers of tiny introns. The subject of the bulk of Paramecium research, the ancient Paramecium aurelia species complex, is descended from two whole-genome duplication events that retain high degrees of synteny, thereby providing an exceptional platform for studying the fates of duplicate genes. Despite having a common ancestor dating to several hundred million years ago, the known descendant species are morphologically indistinguishable, raising significant questions about the common view that gene duplications lead to the origins of evolutionary novelties.
Asunto(s)
Paramecium , Paramecium/genética , Evolución Molecular , Genómica , Genoma , Tasa de MutaciónRESUMEN
Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.
Asunto(s)
Infecciones por VIH , Ácidos Nucleicos , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Linfocitos T CD4-Positivos , Virus ADN , Terapia de Inmunosupresión , Macaca mulatta , Macrófagos , Virus de la Inmunodeficiencia de los Simios/fisiología , Carga ViralRESUMEN
Macromolecular phase separation is being recognized for its potential importance and relevance as a driver of spatial organization within cells. Here, we describe a framework based on synergies between networking (percolation or gelation) and density (phase separation) transitions. Accordingly, the phase transitions in question are referred to as phase separation coupled to percolation (PSCP). The condensates that result from PSCP are viscoelastic network fluids. Such systems have sequence-, composition-, and topology-specific internal network structures that give rise to time-dependent interplays between viscous and elastic properties. Unlike pure phase separation, the process of PSCP gives rise to sequence-, chemistry-, and structure-specific distributions of clusters that can form at concentrations that lie well below the threshold concentration for phase separation. PSCP, influenced by specific versus solubility-determining interactions, also provides a bridge between different observations and helps answer questions and address challenges that have arisen regarding the role of macromolecular phase separation in biology.
RESUMEN
Decades of work in developmental genetics has given us a deep mechanistic understanding of the fundamental signaling pathways underlying animal development. However, little is known about how these pathways emerged and changed over evolutionary time. Here, we review our current understanding of the evolutionary emergence of the Hippo pathway, a conserved signaling pathway that regulates tissue size in animals. This pathway has deep evolutionary roots, emerging piece by piece in the unicellular ancestors of animals, with a complete core pathway predating the origin of animals. Recent functional studies in close unicellular relatives of animals and early-branching animals suggest an ancestral function of the Hippo pathway in cytoskeletal regulation, which was subsequently co-opted to regulate proliferation and animal tissue size.