RESUMO
While ultraviolet (UV) radiation damages DNA, eliciting the DNA damage response (DDR), it also damages RNA, triggering transcriptome-wide ribosomal collisions and eliciting a ribotoxic stress response (RSR). However, the relative contributions, timing, and regulation of these pathways in determining cell fate is unclear. Here we use time-resolved phosphoproteomic, chemical-genetic, single-cell imaging, and biochemical approaches to create a chronological atlas of signaling events activated in cells responding to UV damage. We discover that UV-induced apoptosis is mediated by the RSR kinase ZAK and not through the DDR. We identify two negative-feedback modules that regulate ZAK-mediated apoptosis: (1) GCN2 activation limits ribosomal collisions and attenuates ZAK-mediated RSR and (2) ZAK activity leads to phosphodegron autophosphorylation and its subsequent degradation. These events tune ZAK's activity to collision levels to establish regimes of homeostasis, tolerance, and death, revealing its key role as the cellular sentinel for nucleic acid damage.
Assuntos
Apoptose , Dano ao DNA , Raios Ultravioleta , Raios Ultravioleta/efeitos adversos , Apoptose/efeitos da radiação , Fosforilação/efeitos da radiação , Humanos , Transdução de Sinais/efeitos da radiação , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico/efeitos da radiação , Ribossomos/metabolismo , Morte Celular/efeitos da radiaçãoRESUMO
Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.
Assuntos
Ribossomos/metabolismo , Estresse Fisiológico , Transportadores de Cassetes de Ligação de ATP/metabolismo , Anisomicina/farmacologia , Apoptose/efeitos dos fármacos , Dano ao DNA/efeitos da radiação , Ativação Enzimática , Humanos , MAP Quinase Quinase Quinases/deficiência , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Fosforilação , Polirribossomos/metabolismo , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Raios Ultravioleta , eIF-2 Quinase/metabolismoRESUMO
N6-methyladenosine (m6A), installed by the Mettl3/Mettl14 methyltransferase complex, is the most prevalent internal mRNA modification. Whether m6A regulates mammalian brain development is unknown. Here, we show that m6A depletion by Mettl14 knockout in embryonic mouse brains prolongs the cell cycle of radial glia cells and extends cortical neurogenesis into postnatal stages. m6A depletion by Mettl3 knockdown also leads to a prolonged cell cycle and maintenance of radial glia cells. m6A sequencing of embryonic mouse cortex reveals enrichment of mRNAs related to transcription factors, neurogenesis, the cell cycle, and neuronal differentiation, and m6A tagging promotes their decay. Further analysis uncovers previously unappreciated transcriptional prepatterning in cortical neural stem cells. m6A signaling also regulates human cortical neurogenesis in forebrain organoids. Comparison of m6A-mRNA landscapes between mouse and human cortical neurogenesis reveals enrichment of human-specific m6A tagging of transcripts related to brain-disorder risk genes. Our study identifies an epitranscriptomic mechanism in heightened transcriptional coordination during mammalian cortical neurogenesis.
Assuntos
Neurogênese , Prosencéfalo/embriologia , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Animais , Ciclo Celular , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Neurais/metabolismo , Organoides/metabolismo , Prosencéfalo/citologia , Prosencéfalo/metabolismo , Estabilidade de RNARESUMO
Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.
Assuntos
Técnicas Biossensoriais/métodos , Fosfotransferases/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Quinases JNK Ativadas por Mitógeno/química , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Dados de Sequência Molecular , Alinhamento de Sequência , Análise de Célula ÚnicaRESUMO
Ongoing efforts within synthetic and systems biology have been directed towards the building of artificial computational devices using engineered biological units as basic building blocks. Such efforts, inspired in the standard design of electronic circuits, are limited by the difficulties arising from wiring the basic computational units (logic gates) through the appropriate connections, each one to be implemented by a different molecule. Here, we show that there is a logically different form of implementing complex Boolean logic computations that reduces wiring constraints thanks to a redundant distribution of the desired output among engineered cells. A practical implementation is presented using a library of engineered yeast cells, which can be combined in multiple ways. Each construct defines a logic function and combining cells and their connections allow building more complex synthetic devices. As a proof of principle, we have implemented many logic functions by using just a few engineered cells. Of note, small modifications and combination of those cells allowed for implementing more complex circuits such as a multiplexer or a 1-bit adder with carry, showing the great potential for re-utilization of small parts of the circuit. Our results support the approach of using cellular consortia as an efficient way of engineering complex tasks not easily solvable using single-cell implementations.
Assuntos
Bioengenharia , Lógica , Modelos Biológicos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Biologia de Sistemas/métodos , Candida albicans , Compartimento Celular , Contagem de Colônia Microbiana , Doxiciclina/farmacologia , Estradiol/farmacologia , Galactose/farmacologia , Fator de Acasalamento , Peptídeos/metabolismo , Peptídeos/farmacologia , Feromônios/metabolismo , Feromônios/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Cloreto de Sódio/farmacologiaRESUMO
To test the promise of whole-cell modeling to facilitate scientific inquiry, we compared growth rates simulated in a whole-cell model with experimental measurements for all viable single-gene disruption Mycoplasma genitalium strains. Discrepancies between simulations and experiments led to predictions about kinetic parameters of specific enzymes that we subsequently validated. These findings represent, to our knowledge, the first application of whole-cell modeling to accelerate biological discovery.
Assuntos
Biologia Computacional/métodos , Modelos Biológicos , Mycoplasma genitalium/genética , Mycoplasma genitalium/metabolismo , Biologia de Sistemas , Proteínas de Bactérias/metabolismo , Catálise , Simulação por Computador , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Genes Bacterianos/genética , Fenótipo , Análise de Regressão , Reprodutibilidade dos TestesRESUMO
The control of mRNA biogenesis is exerted at several steps. In response to extracellular stimuli, stress-activated protein kinases (SAPK) modulate gene expression to maximize cell survival. In yeast, the Hog1 SAPK plays a key role in reprogramming the gene expression pattern required for cell survival upon osmostress by acting during transcriptional initiation and elongation. Here, we genetically show that an intact nuclear pore complex is important for cell survival and maximal expression of stress-responsive genes. The Hog1 SAPK associates with nuclear pore complex components and directly phosphorylates the Nup1, Nup2, and Nup60 components of the inner nuclear basket. Mutation of those factors resulted in a deficient export of stress-responsive genes upon stress. Association of Nup1, Nup2, and Nup60 to stress-responsive promoters occurs upon stress depending on Hog1 activity. Accordingly, STL1 gene territory is maintained at the nuclear periphery upon osmostress in a Hog1-dependent manner. Cells containing non-phosphorylatable mutants in Nup1 or Nup2 display reduced expression of stress-responsive genes. Together, proper mRNA biogenesis of stress-responsive genes requires of the coordinate action of synthesis and export machineries by the Hog1 SAPK.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte de RNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Viabilidade Microbiana , Dados de Sequência Molecular , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Oxirredutases/genética , Oxirredutases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Tolerância ao Sal , Estresse FisiológicoRESUMO
Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G1) to synthesis (S) phase transition of the cell cycle by responding to mitogen signaling, promoting E2F transcription and inhibition of the anaphase-promoting complex. We found that this mechanism was still required in G2-arrested cells to prevent cell cycle exit after the S phase. This mechanism revealed a role for CDK4/6 in maintaining the G2 state, challenging the notion that the cell cycle is irreversible and that cells do not require mitogens after passing the restriction point. Exit from G2 occurred during ribotoxic stress and was actively mediated by stress-activated protein kinases. Upon relief of stress, a significant fraction of cells underwent a second round of DNA replication that led to whole-genome doubling.
Assuntos
Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Endorreduplicação , Pontos de Checagem da Fase G2 do Ciclo Celular , Estresse Fisiológico , Humanos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Fatores de Transcrição E2F/metabolismo , Fatores de Transcrição E2F/genética , Fase S , Linhagem CelularRESUMO
Whole-genome duplication (WGD) is a frequent event in cancer evolution that fuels chromosomal instability. WGD can result from mitotic errors or endoreduplication, yet the molecular mechanisms that drive WGD remain unclear. Here, we use live single-cell analysis to characterize cell-cycle dynamics upon aberrant Ras-ERK signaling. We find that sustained ERK signaling in human cells leads to reactivation of the APC/C in G2, resulting in tetraploid G0-like cells that are primed for WGD. This process is independent of DNA damage or p53 but dependent on p21. Transcriptomics analysis and live-cell imaging showed that constitutive ERK activity promotes p21 expression, which is necessary and sufficient to inhibit CDK activity and which prematurely activates the anaphase-promoting complex (APC/C). Finally, either loss of p53 or reduced ERK signaling allowed for endoreduplication, completing a WGD event. Thus, sustained ERK signaling-induced G2 cell cycle exit represents an alternative path to WGD.
Assuntos
Sistema de Sinalização das MAP Quinases , Proteína Supressora de Tumor p53 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Genoma Humano , Duplicação Gênica , Fase G2/genética , Dano ao DNA/genéticaRESUMO
Tissue regeneration and maintenance rely on coordinated stem cell behaviours. This orchestration can be impaired by oncogenic mutations leading to cancer. However, it is largely unclear how oncogenes perturb stem cells' orchestration to disrupt tissue. Here we used intravital imaging to investigate the mechanisms by which oncogenic Kras mutation causes tissue disruption in the hair follicle. Through longitudinally tracking hair follicles in live mice, we found that KrasG12D, a mutation that can lead to squamous cell carcinoma, induces epithelial tissue deformation in a spatiotemporally specific manner, linked with abnormal cell division and migration. Using a reporter mouse capture real-time ERK signal dynamics at the single-cell level, we discovered that KrasG12D, but not a closely related mutation HrasG12V, converts ERK signal in stem cells from pulsatile to sustained. Finally, we demonstrated that interrupting sustained ERK signal reverts KrasG12D-induced tissue deformation through modulating specific features of cell migration and division.
Assuntos
Movimento Celular , Folículo Piloso , Mutação , Proteínas Proto-Oncogênicas p21(ras) , Animais , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Camundongos , Folículo Piloso/metabolismo , Sistema de Sinalização das MAP Quinases/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Camundongos Transgênicos , Células-Tronco/metabolismo , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Humanos , Feminino , Ativação EnzimáticaRESUMO
The ability of stem cells to build and replenish tissues depends on support from their niche. Although niche architecture varies across organs, its functional importance is unclear. During hair follicle growth, multipotent epithelial progenitors build hair via crosstalk with their remodeling fibroblast niche, the dermal papilla, providing a powerful model to functionally interrogate niche architecture. Through mouse intravital imaging, we show that dermal papilla fibroblasts remodel individually and collectively to form a morphologically polarized, structurally robust niche. Asymmetric TGF-ß signaling precedes morphological niche polarity, and loss of TGF-ß signaling in dermal papilla fibroblasts leads them to progressively lose their stereotypic architecture, instead surrounding the epithelium. The reorganized niche induces the redistribution of multipotent progenitors but nevertheless supports their proliferation and differentiation. However, the differentiated lineages and hairs produced by progenitors are shorter. Overall, our results reveal that niche architecture optimizes organ efficiency but is not absolutely essential for organ function.
Assuntos
Folículo Piloso , Cabelo , Camundongos , Animais , Diferenciação Celular , Epitélio , Fator de Crescimento Transformador betaRESUMO
The classic network of mitogen-activated protein kinases (MAPKs) is highly interconnected and controls a diverse array of biological processes. In multicellular eukaryotes, the MAPKs ERK, JNK, and p38 control opposing cell behaviors but are often activated simultaneously, raising questions about how input-output specificity is achieved. Here, we use multiplexed MAPK activity biosensors to investigate how cell fate control emerges from the connectivity and dynamics of the MAPK network. Through chemical and genetic perturbation, we systematically explore the outputs and functions of all the MAP3 kinases encoded in the human genome and show that MAP3Ks control cell fate by triggering unique combinations of MAPK activity. We show that these MAPK activity combinations explain the paradoxical dual role of JNK signaling as pro-apoptotic or pro-proliferative kinase. Overall, our integrative analysis indicates that the MAPK network operates as a unit to control cell fate and shifts the focus from MAPKs to MAP3Ks to better understand signaling-mediated control of cell fate.
Assuntos
Proteínas Quinases JNK Ativadas por Mitógeno , Sistema de Sinalização das MAP Quinases , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de SinaisRESUMO
Determining how signaling dynamics relate to gene expression and cell fate is essential to understanding multicellular development. We present a unified live imaging and lineage analysis method that allows integrated analysis of both techniques in the same mouse embryos. This protocol describes the embryo isolation, confocal imaging, immunofluorescence, and in silico alignment required to connect time-lapse and endpoint measurements. By utilizing different biosensors and fixed readouts, this method allows interrogation of signaling dynamics that specify cell fates in developing embryos. For complete details on the use and execution of this protocol, please refer to Pokrass et al. (2020).
Assuntos
Blastocisto , Microscopia Confocal/métodos , Técnicas de Sonda Molecular , Imagem com Lapso de Tempo/métodos , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Blastocisto/fisiologia , Células Cultivadas , Feminino , Imageamento Tridimensional , Masculino , Camundongos , Transdução de Sinais/fisiologiaRESUMO
To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.
Assuntos
Bactérias/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Lipopeptídeos/imunologia , NF-kappa B/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Bactérias/metabolismo , Linhagem Celular , Citocinas/metabolismo , Citocinas/farmacologia , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Flagelina/farmacologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Hibridização in Situ Fluorescente , Glândulas Mamárias Animais , Camundongos , Organoides/efeitos dos fármacos , Organoides/imunologia , Organoides/metabolismo , Regiões Promotoras Genéticas , RNA-Seq , Transdução de Sinais/imunologia , Análise de Célula Única , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismoRESUMO
Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.
Assuntos
Envelhecimento/fisiologia , Evolução Biológica , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vertebrados/fisiologia , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Feminino , Edição de Genes , Técnicas de Introdução de Genes , Masculino , Camundongos , Modelos Animais , Oxirredução , Filogenia , Aptidão Física/fisiologia , Mutação PuntualRESUMO
A large fraction of human cancers contain genetic alterations within the Mitogen Activated Protein Kinase (MAPK) signaling network that promote unpredictable phenotypes. Previous studies have shown that the temporal patterns of MAPK activity (i.e. signaling dynamics) differentially regulate cell behavior. However, the role of signaling dynamics in mediating the effects of cancer driving mutations has not been systematically explored. Here, we show that oncogene expression leads to either pulsatile or sustained ERK activity that correlate with opposing cellular behaviors (i.e. proliferation vs. cell cycle arrest, respectively). Moreover, sustained-but not pulsatile-ERK activity triggers ERK activity waves in unperturbed neighboring cells that depend on the membrane metalloprotease ADAM17 and EGFR activity. Interestingly, the ADAM17-EGFR signaling axis coordinates neighboring cell migration toward oncogenic cells and is required for oncogenic cell extrusion. Overall, our data suggests that the temporal patterns of MAPK activity differentially regulate cell autonomous and non-cell autonomous effects of oncogene expression.
In animals, the MAPK pathway is a network of genes that helps a cell to detect and then respond to an external signal by switching on or off a specific genetic program. In particular, cells use this pathway to communicate with each other. In an individual cell, the MAPK pathway shows fluctuations in activity over time. Mutations in the genes belonging to the MAPK pathway are often one of the first events that lead to the emergence of cancers. However, different mutations in the genes of the pathway can have diverse effects on a cell's behavior: some mutations cause the cell to divide while others make it migrate. Recent research has suggested that these effects may be caused by changes in the pattern of MAPK signaling activity over time. Here, Aikin et al. used fluorescent markers to document how different MAPK mutations influence the behavior of a human breast cell and its healthy neighbors. The experiments showed that cells with different MAPK mutations behaved in one of two ways: the signaling quickly pulsed between high and low levels of activity, or it remained at a sustained high level. In turn, these two signaling patterns altered cell behavior in different ways. Pulsed signaling led to more cell division, while sustained signaling stopped division and increased migration. Aikin et al. then examined the effect of the MAPK mutations on neighboring healthy cells. Sustained signaling from the cancerous cell caused a wave of signaling activity in the surrounding cells. This led the healthy cells to divide and migrate toward the cancerous cell, pushing it out of the tissue layer. It is not clear if these changes protect against or promote cancer progression in living tissue. However, these results explain why specific cancer mutations cause different behaviors in cells.
Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Neoplasias/genética , Oncogenes/genética , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Linhagem Celular Tumoral , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Neoplasias/metabolismoRESUMO
Despite the noisy nature of single cells, multicellular organisms robustly generate different cell types from one zygote. This process involves dynamic cross regulation between signaling and gene expression that is difficult to capture with fixed-cell approaches. To study signaling dynamics and fate specification during preimplantation development, we generated a transgenic mouse expressing the ERK kinase translocation reporter and measured ERK activity in single cells of live embryos. Our results show primarily active ERK in both the inner cell mass and trophectoderm cells due to fibroblast growth factor (FGF) signaling. Strikingly, a subset of mitotic events results in a short pulse of ERK inactivity in both daughter cells that correlates with elevated endpoint NANOG levels. Moreover, endogenous tagging of Nanog in embryonic stem cells reveals that ERK inhibition promotes enhanced stabilization of NANOG protein after mitosis. Our data show that cell cycle, signaling, and differentiation are coordinated during preimplantation development.
Assuntos
Blastocisto/citologia , Blastocisto/enzimologia , Ciclo Celular , Linhagem da Célula , Sistema de Sinalização das MAP Quinases , Mamíferos/embriologia , Animais , Camadas Germinativas/citologia , Humanos , Camundongos , Mitose , Modelos Biológicos , Proteína Homeobox Nanog/metabolismo , Estabilidade Proteica , Reprodutibilidade dos TestesRESUMO
Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood. Here, we developed a reporter system to simultaneously monitor CDK4/6 and CDK2 activities in single cells and found that CDK4/6 activity increases rapidly before CDK2 activity gradually increases, and that CDK4/6 activity can be active after mitosis or inactive for variable time periods. Markedly, stress signals in G1 can rapidly inactivate CDK4/6 to return cells to quiescence but with reduced probability as cells approach S phase. Together, our study reveals a regulation of G1 length by temporary inactivation of CDK4/6 activity after mitosis, and a progressively increasing persistence in CDK4/6 activity that restricts cells from returning to quiescence as cells approach S phase.
Assuntos
Quinase 2 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/genética , Fase G1/genética , Estresse Fisiológico , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Genes Reporter , Humanos , Mitose , Fase S/genética , Análise de Célula Única/métodosRESUMO
Over the last decade, multiple studies have shown that signaling proteins activated in different temporal patterns, such as oscillatory, transient, and sustained, can result in distinct gene expression patterns or cell fates. However, the molecular events that ensure appropriate stimulus- and dose-dependent dynamics are not often understood and are difficult to investigate. Here, we used single-cell analysis to dissect the mechanisms underlying the stimulus- and dose-encoding patterns in the innate immune signaling network. We found that Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling dynamics relied on a dose-dependent, autoinhibitory loop that rendered cells refractory to further stimulation. Using inducible gene expression and optogenetics to perturb the network at different levels, we identified IL-1R-associated kinase 1 (IRAK1) as the dose-sensing node responsible for limiting signal flow during the innate immune response. Although the kinase activity of IRAK1 was not required for signal propagation, it played a critical role in inhibiting the nucleocytoplasmic oscillations of the transcription factor NF-κB. Thus, protein activities that may be "dispensable" from a topological perspective can nevertheless be essential in shaping the dynamic response to the external environment.
Assuntos
NF-kappa B/metabolismo , Transdução de Sinais/fisiologia , Análise de Célula Única/métodos , Imagem com Lapso de Tempo/métodos , Animais , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Microscopia Confocal , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Células NIH 3T3 , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter's distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, we present a protocol to execute and analyze live-cell microscopy experiments using KTRs. We describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. We provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in â¼10 d.