RESUMEN
Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.
Asunto(s)
Receptores de Trombopoyetina , Trombopoyetina , Animales , Humanos , Ratones , Ciclo Celular , Microscopía por Crioelectrón , Receptores de Trombopoyetina/genética , Trombopoyesis , Metilación de ADNRESUMEN
Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The histone H3-H4 modification landscape is restored by parental histone recycling and modification of new histones. How DNA replication impacts on histone H2A-H2B is currently unknown. Here, we measure H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub1, H2BK120ub1, and H2A.Z are recycled accurately during DNA replication. Modified H2A-H2B are segregated symmetrically to daughter strands via POLA1 on the lagging strand, but independent of H3-H4 recycling. Post-replication, H2A-H2B modification and variant landscapes are quickly restored, and H2AK119ub1 guides accurate restoration of H3K27me3. This work reveals epigenetic transmission of parental H2A-H2B during DNA replication and identifies cross talk between H3-H4 and H2A-H2B modifications in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates restoration of stable H3-H4 chromatin states.
Asunto(s)
Cromatina , Memoria a Corto Plazo , Ciclo Celular , Replicación del ADN , Histonas/metabolismo , Nucleosomas , Animales , Ratones , ConejosRESUMEN
CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 in Cdk2-/- mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhibition, cells exhibit a rapid loss of substrate phosphorylation that rebounds within several hours. CDK4/6 activity backstops inhibition of CDK2 and sustains the proliferative program by maintaining Rb1 hyperphosphorylation, active E2F transcription, and cyclin A2 expression, enabling re-activation of CDK2 in the presence of drug. Our results augment our understanding of CDK plasticity and indicate that co-inhibition of CDK2 and CDK4/6 may be required to suppress adaptation to CDK2 inhibitors currently under clinical assessment.
Asunto(s)
Proteínas de Ciclo Celular , Quinasas Ciclina-Dependientes , Animales , Ratones , Quinasas Ciclina-Dependientes/metabolismo , Ciclo Celular/fisiología , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fosforilación , División CelularRESUMEN
DNA replication in eukaryotic cells initiates from large numbers of sites called replication origins. Initiation of replication from these origins must be tightly controlled to ensure the entire genome is precisely duplicated in each cell cycle. This is accomplished through the regulation of the first two steps in replication: loading and activation of the replicative DNA helicase. Here we describe what is known about the mechanism and regulation of these two reactions from a genetic, biochemical, and structural perspective, focusing on recent progress using proteins from budding yeast.
Asunto(s)
Eucariontes , Células Eucariotas , Ciclo Celular/genética , Replicación del ADN , Eucariontes/genética , Células Eucariotas/metabolismo , Origen de RéplicaRESUMEN
We present a whole-cell fully dynamical kinetic model (WCM) of JCVI-syn3A, a minimal cell with a reduced genome of 493 genes that has retained few regulatory proteins or small RNAs. Cryo-electron tomograms provide the cell geometry and ribosome distributions. Time-dependent behaviors of concentrations and reaction fluxes from stochastic-deterministic simulations over a cell cycle reveal how the cell balances demands of its metabolism, genetic information processes, and growth, and offer insight into the principles of life for this minimal cell. The energy economy of each process including active transport of amino acids, nucleosides, and ions is analyzed. WCM reveals how emergent imbalances lead to slowdowns in the rates of transcription and translation. Integration of experimental data is critical in building a kinetic model from which emerges a genome-wide distribution of mRNA half-lives, multiple DNA replication events that can be compared to qPCR results, and the experimentally observed doubling behavior.
Asunto(s)
Células/citología , Simulación por Computador , Adenosina Trifosfato/metabolismo , Ciclo Celular/genética , Proliferación Celular/genética , Células/metabolismo , Replicación del ADN/genética , Regulación de la Expresión Génica , Imagenología Tridimensional , Cinética , Lípidos/química , Redes y Vías Metabólicas , Metaboloma , Anotación de Secuencia Molecular , Nucleótidos/metabolismo , Termodinámica , Factores de TiempoRESUMEN
Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. We interrogated the composition of BrM niches, molecularly defined the blood-tumor interface, and revealed stromal immunosuppressive states enriched with infiltrated T cells and macrophages. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our resource provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits.
Asunto(s)
Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Adulto , Anciano , Animales , Biomarcadores de Tumor/metabolismo , Neoplasias Encefálicas/sangre , Neoplasias Encefálicas/inmunología , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Variación Genética , Humanos , Evasión Inmune , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Modelos Biológicos , Células Mieloides/patología , Análisis de Componente Principal , RNA-Seq , Análisis de la Célula Individual , Linfocitos T/inmunologíaRESUMEN
How early events in effector T cell (TEFF) subsets tune memory T cell (TMEM) responses remains incompletely understood. Here, we systematically investigated metabolic factors in fate determination of TEFF and TMEM cells using in vivo pooled CRISPR screening, focusing on negative regulators of TMEM responses. We found that amino acid transporters Slc7a1 and Slc38a2 dampened the magnitude of TMEM differentiation, in part through modulating mTORC1 signaling. By integrating genetic and systems approaches, we identified cellular and metabolic heterogeneity among TEFF cells, with terminal effector differentiation associated with establishment of metabolic quiescence and exit from the cell cycle. Importantly, Pofut1 (protein-O-fucosyltransferase-1) linked GDP-fucose availability to downstream Notch-Rbpj signaling, and perturbation of this nutrient signaling axis blocked terminal effector differentiation but drove context-dependent TEFF proliferation and TMEM development. Our study establishes that nutrient uptake and signaling are key determinants of T cell fate and shape the quantity and quality of TMEM responses.
Asunto(s)
Aminoácidos/metabolismo , Linfocitos T CD8-positivos/citología , Memoria Inmunológica , Transducción de Señal , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Sistemas CRISPR-Cas , Ciclo Celular , Diferenciación Celular , Modelos Animales de Enfermedad , Femenino , Técnicas de Sustitución del Gen , Coriomeningitis Linfocítica/inmunología , Masculino , Ratones , Ratones Transgénicos , Células Precursoras de Linfocitos T/citologíaRESUMEN
End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.
Asunto(s)
ARN Polimerasa III/metabolismo , Reparación del ADN por Recombinación , Ciclo Celular , Línea Celular Tumoral , Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/genética , Células HEK293 , Humanos , Proteína Homóloga de MRE11/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Hibridación de Ácido Nucleico , ARN/químicaRESUMEN
Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.
Asunto(s)
Citomegalovirus/fisiología , Macrófagos Alveolares/virología , Animales , Presentación de Antígeno , Efecto Espectador , Ciclo Celular , Línea Celular Transformada , Reprogramación Celular , Citomegalovirus/patogenicidad , Citomegalovirus/ultraestructura , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , Proteínas Fluorescentes Verdes/metabolismo , Pulmón/patología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/ultraestructura , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Células Madre/patología , Replicación Viral/fisiología , Vía de Señalización WntRESUMEN
Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.
Asunto(s)
Ciclo Celular , Reactivos de Enlaces Cruzados/química , ADN Viral/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/metabolismo , Plásmidos/metabolismo , Origen de Réplica , Replicación Viral/fisiología , Secuencia de Aminoácidos , Linfocitos B/metabolismo , Línea Celular , Aductos de ADN/metabolismo , Replicación del ADN , Endonucleasas/metabolismo , Antígenos Nucleares del Virus de Epstein-Barr/química , Antígenos Nucleares del Virus de Epstein-Barr/genética , Humanos , Mutación/genética , Unión Proteica , Recombinación Genética/genética , Tirosina/metabolismoRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) represents a large multisubunit E3-ubiquitin ligase complex that controls the unidirectional progression through the cell cycle by the ubiquitination of specific target proteins, marking them for proteasomal destruction. Although the APC/C's role is largely conserved among eukaryotes, its subunit composition and target spectrum appear to be species specific. In this review, we focus on the plant APC/C complex, whose activity correlates with different developmental processes, including polyploidization and gametogenesis. After an introduction into proteolytic control by ubiquitination, we discuss the composition of the plant APC/C and the essential nature of its core subunits for plant development. Subsequently, we describe the APC/C activator subunits and interactors, most being plant specific. Finally, we provide a comprehensive list of confirmed and suspected plant APC/C target proteins. Identification of growth-related targets might offer opportunities to increase crop yield and resilience of plants to climate change by manipulating APC/C activity.
Asunto(s)
Anafase , Plantas , Ciclosoma-Complejo Promotor de la Anafase/genética , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Plantas/genética , Plantas/metabolismo , Ubiquitinación , Ubiquitinas/metabolismoRESUMEN
Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.
Asunto(s)
Pirimidinas , Ciclo Celular , Diferenciación CelularRESUMEN
The accurate timing and execution of organelle biogenesis is crucial for cell physiology. Centriole biogenesis is regulated by Polo-like kinase 4 (Plk4) and initiates in S-phase when a daughter centriole grows from the side of a pre-existing mother. Here, we show that a Plk4 oscillation at the base of the growing centriole initiates and times centriole biogenesis to ensure that centrioles grow at the right time and to the right size. The Plk4 oscillation is normally entrained to the cell-cycle oscillator but can run autonomously of it-potentially explaining why centrioles can duplicate independently of cell-cycle progression. Mathematical modeling indicates that the Plk4 oscillation can be generated by a time-delayed negative feedback loop in which Plk4 inactivates the interaction with its centriolar receptor through multiple rounds of phosphorylation. We hypothesize that similar organelle-specific oscillations could regulate the timing and execution of organelle biogenesis more generally.
Asunto(s)
Relojes Biológicos/fisiología , Centriolos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Biogénesis de Organelos , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiologíaRESUMEN
Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
Asunto(s)
Alelos , Proteína 9 Asociada a CRISPR/metabolismo , Cromosomas Humanos/genética , Embrión de Mamíferos/metabolismo , Animales , Secuencia de Bases , Blastocisto/metabolismo , Ciclo Celular/genética , Línea Celular , Deleción Cromosómica , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Implantación del Embrión/genética , Proteínas del Ojo/genética , Fertilización , Edición Génica , Reordenamiento Génico/genética , Sitios Genéticos , Genoma Humano , Genotipo , Heterocigoto , Células Madre Embrionarias Humanas/metabolismo , Humanos , Mutación INDEL/genética , Ratones , Mitosis , Sistemas de Lectura Abierta/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.
Asunto(s)
Nervio Accesorio/fisiología , Folículo Piloso/citología , Cabello/crecimiento & desarrollo , Proteínas Hedgehog/metabolismo , Norepinefrina/metabolismo , Transducción de Señal/genética , Células Madre/metabolismo , Células Madre/fisiología , Nervio Accesorio/citología , Animales , Ciclo Celular/genética , Frío , Femenino , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Cabello/citología , Cabello/fisiología , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Piloerección , RNA-Seq , Receptores Adrenérgicos beta 2/deficiencia , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Nicho de Células Madre , Células Madre/citología , Sistema Nervioso Simpático/citología , Sistema Nervioso Simpático/fisiología , Sinapsis/fisiologíaRESUMEN
The centriole is an ancient microtubule-based organelle with a conserved nine-fold symmetry. Centrioles form the core of centrosomes, which organize the interphase microtubule cytoskeleton of most animal cells and form the poles of the mitotic spindle. Centrioles can also be modified to form basal bodies, which template the formation of cilia and play central roles in cellular signaling, fluid movement, and locomotion. In this review, we discuss developments in our understanding of the biogenesis of centrioles and cilia and the regulatory controls that govern their structure and number. We also discuss how defects in these processes contribute to a spectrum of human diseases and how new technologies have expanded our understanding of centriole and cilium biology, revealing exciting avenues for future exploration.
Asunto(s)
Centriolos/fisiología , Cilios/patología , Biogénesis de Organelos , Animales , Ciclo Celular , Centriolos/metabolismo , Centriolos/ultraestructura , Cilios/metabolismo , Cilios/ultraestructura , Ciliopatías , Eucariontes/citología , Eucariontes/fisiología , Humanos , Mitosis , Transducción de SeñalRESUMEN
Division of amoebas, fungi, and animal cells into two daughter cells at the end of the cell cycle depends on a common set of ancient proteins, principally actin filaments and myosin-II motors. Anillin, formins, IQGAPs, and many other proteins regulate the assembly of the actin filaments into a contractile ring positioned between the daughter nuclei by different mechanisms in fungi and animal cells. Interactions of myosin-II with actin filaments produce force to assemble and then constrict the contractile ring to form a cleavage furrow. Contractile rings disassemble as they constrict. In some cases, knowledge about the numbers of participating proteins and their biochemical mechanisms has made it possible to formulate molecularly explicit mathematical models that reproduce the observed physical events during cytokinesis by computer simulations.
Asunto(s)
Citocinesis , Eucariontes/fisiología , Huso Acromático/metabolismo , Actinas/metabolismo , Animales , Ciclo Celular , Eucariontes/metabolismo , Humanos , Modelos Biológicos , Miosinas/metabolismo , Transducción de Señal , Huso Acromático/fisiología , Levaduras/metabolismo , Levaduras/fisiologíaRESUMEN
Deneke et al. (2019) discover that dynamic interactions of cell cycle and actomyosin contractility systems synchronize nuclear cleavages, generating a cytoplasmic flow that results in a spatially uniform distribution of zygotic nuclei in the early Drosophila embryo. This work underscores the importance of self-organizing mechanisms before the onset of zygotic transcription.
Asunto(s)
Drosophila melanogaster , Drosophila , Animales , Ciclo Celular , Física , CigotoRESUMEN
Time-lapse imaging reveals a nuanced role for p21 in cancer cells challenged with chemotherapeutic drugs: cells with either high or low p21 are biased toward senescence, whereas intermediate p21 allows cells to re-enter the cell cycle after drug treatment.
Asunto(s)
Senescencia Celular , Ciclo Celular , Diferenciación Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la CiclinaRESUMEN
The 2019 Gairdner Prize will be given to John F.X. Diffley and Bruce Stillman for their groundbreaking work on the mechanisms and control of the initiation of eukaryotic DNA replication. No two people have contributed more extensively, or over a longer period of time, to enlighten us on how our genomes replicate themselves once and only once per cell cycle.