RESUMEN
We cloned and characterized two new coral fluorescent proteins: h2-3 and 1-41. h2-3 formed an obligate dimeric complex and exhibited bright green fluorescence. On the other hand, 1-41 formed a highly multimeric complex and exhibited dim red fluorescence. We engineered 1-41 into AzaleaB5, a practically useful red-emitting fluorescent protein for cellular labeling applications. We fused h2-3 and AzaleaB5 to the ubiquitination domains of human Geminin and Cdt1, respectively, to generate a new color variant of Fucci (Fluorescent Ubiquitination-based Cell-Cycle Indicator): Fucci5. We found Fucci5 provided more reliable nuclear labeling for monitoring cell-cycle progression than the 1st and 2nd generations that used mAG/mKO2 and mVenus/mCherry, respectively.Key words: fluorescent protein, cell cycle, time-lapse imaging, flow cytometry.
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Proteínas de Ciclo Celular , Colorantes , Humanos , Color , División Celular , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Microscopía Fluorescente , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismoRESUMEN
The ErbB-family receptors play pivotal roles in the proliferation, migration and survival of epithelial cells. Because our knowledge on the ErbB-family receptors has been largely obtained by the exogenous application of their ligands, it remains unknown to what extent each of the ErbB members contributes to these outputs. We here knocked out each ErbB gene, various combinations of ErbB genes or all ErbB genes in Madin-Darby canine kidney cells to delineate the contribution of each gene. ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) activation waves during collective cell migration were mediated primarily by ErbB1 and secondarily by the ErbB2 and ErbB3 heterodimer. Either ErbB1 or the ErbB2 and ErbB3 complex was sufficient for the G1/S progression. The saturation cell density was markedly reduced in cells deficient in all ErbB proteins, but not in cells retaining only ErbB2, which cannot bind to ligands. Thus, a ligand-independent ErbB2 activity is sufficient for preventing apoptosis at high cell density. In short, systematic knockout of ErbB-family genes has delineated the roles of each ErbB receptor.
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Receptor ErbB-2 , Transducción de Señal , Animales , Perros , Ligandos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Fosforilación , Genes erbB , Proliferación Celular/genética , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismoRESUMEN
Medullary thymic epithelial cells (mTECs) are critical for self-tolerance induction in T cells via promiscuous expression of tissue-specific antigens (TSAs), which are controlled by the transcriptional regulator, AIRE. Whereas AIRE-expressing (Aire+) mTECs undergo constant turnover in the adult thymus, mechanisms underlying differentiation of postnatal mTECs remain to be discovered. Integrative analysis of single-cell assays for transposase-accessible chromatin (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) suggested the presence of proliferating mTECs with a specific chromatin structure, which express high levels of Aire and co-stimulatory molecules, CD80 (Aire+CD80hi). Proliferating Aire+CD80hi mTECs detected using Fucci technology express a minimal number of Aire-dependent TSAs and are converted into quiescent Aire+CD80hi mTECs expressing high levels of TSAs after a transit amplification. These data provide evidence for the existence of transit-amplifying Aire+mTEC precursors during the Aire+mTEC differentiation process of the postnatal thymus.
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Cromatina , Análisis de la Célula Individual , Animales , Diferenciación Celular/genética , Cromatina/metabolismo , Células Epiteliales/metabolismo , Ratones , Ratones Endogámicos C57BL , Timo , Transposasas/metabolismoRESUMEN
Gravity determines shape of body tissue and affects the functions of life, both in plants and animals. The cellular response to gravity is an active process of mechanotransduction. Although plants and animals share some common mechanisms of gravity sensing in spite of their distant phylogenetic origin, each species has its own mechanism to sense and respond to gravity. In this review, we discuss current understanding regarding the mechanisms of cellular gravity sensing in plants and animals. Understanding gravisensing also contributes to life on Earth, e.g., understanding osteoporosis and muscle atrophy. Furthermore, in the current age of Mars exploration, understanding cellular responses to gravity will form the foundation of living in space.
RESUMEN
Space travel has advanced significantly over the last six decades with astronauts spending up to 6 months at the International Space Station. Nonetheless, the living environment while in outer space is extremely challenging to astronauts. In particular, exposure to space radiation represents a serious potential long-term threat to the health of astronauts because the amount of radiation exposure accumulates during their time in space. Therefore, health risks associated with exposure to space radiation are an important topic in space travel, and characterizing space radiation in detail is essential for improving the safety of space missions. In the first part of this review, we provide an overview of the space radiation environment and briefly present current and future endeavors that monitor different space radiation environments. We then present research evaluating adverse biological effects caused by exposure to various space radiation environments and how these can be reduced. We especially consider the deleterious effects on cellular DNA and how cells activate DNA repair mechanisms. The latest technologies being developed, e.g., a fluorescent ubiquitination-based cell cycle indicator, to measure real-time cell cycle progression and DNA damage caused by exposure to ultraviolet radiation are presented. Progress in examining the combined effects of microgravity and radiation to animals and plants are summarized, and our current understanding of the relationship between psychological stress and radiation is presented. Finally, we provide details about protective agents and the study of organisms that are highly resistant to radiation and how their biological mechanisms may aid developing novel technologies that alleviate biological damage caused by radiation. Future research that furthers our understanding of the effects of space radiation on human health will facilitate risk-mitigating strategies to enable long-term space and planetary exploration.
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Radiación Cósmica/efectos adversos , Vuelo Espacial , Rayos Ultravioleta , Animales , Astronautas , Carcinogénesis/efectos de la radiación , Sistema Nervioso Central/efectos de la radiación , Aberraciones Cromosómicas/efectos de la radiación , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Medio Ambiente Extraterrestre , Inestabilidad Genómica/efectos de la radiación , Humanos , Micronúcleos con Defecto Cromosómico/efectos de la radiación , Sustancias Protectoras/farmacología , Dosis de Radiación , Exposición a la Radiación/efectos adversos , Exposición a la Radiación/prevención & control , Estrés Psicológico , IngravidezRESUMEN
Epigenetic information (epigenome) on chromatin is crucial for the determination of cellular identity and for the expression of cell type-specific biological functions. The cell type-specific epigenome is maintained beyond replication and cell division. Nucleosomes of chromatin just after DNA replication are a mixture of old histones with the parental epigenome and newly synthesized histones without such information. The diluted epigenome is mostly restored within one cell cycle using the epigenome on the parental DNA and nucleosomes as replication templates. However, many important questions about the epigenome replication process remain to be clarified. In this study, we investigated the model system comprising of dimethylated histone H3 lysine 9 (H3K9me2) and its regulation by the lysine methyltransferase G9a. Using this epigenome model system, we addressed whether H3K9me2 can be induced in specific cell cycle stages, especially G1. Using cell cycle-specific degrons, we achieved G1 or late G1-to M phases specific accumulation of exogenous G9a in G9a deficient cells. Importantly, global levels of H3K9me2 were significantly recovered by both cell types. These data indicate that H3K9me2 may be plastic and inducible, even in the long-living, terminally-differentiated, post-mitotic, G0-G1 cell population in vivo. This knowledge is valuable in designing epigenome-manipulation-based treatments for diseases.
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Fase G1 , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Animales , Línea Celular , Femenino , Geminina/metabolismo , Humanos , Lisina/metabolismo , Metilación , Ratones Noqueados , MitosisRESUMEN
The cilia and cell cycles are inextricably linked. Centrioles in the basal body of cilia nucleate the ciliary axoneme and sequester pericentriolar matrix (PCM) at the centrosome to organize the mitotic spindle. Cilia themselves respond to growth signals, prompting cilia resorption and cell cycle re-entry. We describe a fluorescent cilia and cell cycle biosensor allowing live imaging of cell cycle progression and cilia assembly and disassembly kinetics in cells and inducible mice. We define assembly and disassembly in relation to cell cycle stage with single-cell resolution and explore the intercellular heterogeneity in cilia kinetics. In all cells and tissues analyzed, we observed cilia that persist through the G1/S transition and into S/G2/M-phase. We conclude that persistence of cilia after the G1/S transition is a general property. This resource will shed light at an individual cell level on the interplay between the cilia and cell cycles in development, regeneration, and disease.
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Ciclo Celular/fisiología , Centriolos/metabolismo , Centrosoma/metabolismo , Cilios/metabolismo , Animales , Cuerpos Basales/metabolismo , Técnicas Biosensibles/métodos , Proteínas de Ciclo Celular/metabolismo , Cinética , Ratones , Microtúbulos/metabolismoRESUMEN
Glucose metabolism is regulated by insulin, which is produced from ß-cells in the pancreas. Because insulin is secreted into vessels in response to blood glucose, vascular structures of the pancreas, especially the relationship between vessels and ß-cells, are important for physiological and pathological glucose metabolism. Here, we developed a system to visualize vessels surrounding mature ß-cells expressing transcription factor MafA in a three-dimensional manner. Optical clearing of the pancreas prevented light scattering of fluorescence driven by the bacterial artificial chromosome (BAC)-mafA promoter in ß-cells. Reconstruction of confocal images demonstrated mature ß-cells and the glomerular-like structures of ß-cell vasculatures labeled with DyLight 488-conjugated lectin in normal mice as well as in low-dose streptozotocin-injected diabetes model mice with reduced ß-cell mass. This technological innovation of organ imaging can be used to investigate morphological changes in vascular structures during transplantation, regeneration and diabetes development.
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Diabetes Mellitus Experimental/diagnóstico por imagen , Células Secretoras de Insulina/metabolismo , Imagen Óptica/métodos , Páncreas/diagnóstico por imagen , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones , Páncreas/metabolismoRESUMEN
Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.
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Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas Cullin/metabolismo , Células Madre Embrionarias/fisiología , Colorantes Fluorescentes/metabolismo , Proteínas Luminiscentes/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteínas Cullin/genética , Células Madre Embrionarias/citología , Genes Reporteros , Células HeLa , Humanos , Proteínas Luminiscentes/genética , RatonesRESUMEN
Protein-protein interactions (PPIs) are essential components of cellular function. Current fluorescence-based technologies to measure PPIs have limited dynamic range and quantitative reproducibility. Here, we describe a genetically-encoded PPI visualization system that harnesses the dynamics of condensed liquid-phase transitions to analyze protein interactions in living cells. The fluorescent protein Azami-Green and p62-PB1 domain when fused to PPI partners triggered a rapid concatenation/oligomerization process that drove the condensation of liquid-phase droplets for real-time analysis of the interaction with unlimited dynamic range in the fluorescence signal. Proof-of-principle studies revealed novel insights on the live cell dynamics of XIAP-Smac and ERK2-dimer interactions. A photoconvertible variant allowed time-resolved optical highlighting for PPI kinetic analysis. Our system, called Fluoppi, demonstrates the unique signal amplification properties of liquid-phase condensation to detect PPIs. The findings introduce a general method for discovery of novel PPIs and modulators of established PPIs.
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Colorantes Fluorescentes/química , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Proteínas Reguladoras de la Apoptosis , Sitios de Unión , Fenómenos Biofísicos , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Dominios Proteicos , Proteínas/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/química , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismoRESUMEN
While implicated in therapeutic resistance, malignant progenitor cell cycle kinetics have been difficult to quantify in real-time. We developed an efficient lentiviral bicistronic fluorescent, ubiquitination-based cell cycle indicator reporter (Fucci2BL) to image live single progenitors on a defined niche coupled with cell cycle gene expression analysis. We have identified key differences in cell cycle regulatory gene expression and transit times between normal and chronic myeloid leukemia progenitors that may inform cancer stem cell eradication strategies.
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Perfilación de la Expresión Génica/métodos , Leucemia Mielógena Crónica BCR-ABL Positiva/diagnóstico por imagen , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Células Madre Neoplásicas/metabolismo , Análisis de Secuencia de ARN/métodos , Nicho de Células Madre , Ciclo Celular , Línea Celular , Técnicas de Cocultivo , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Humanos , Cinética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Microscopía Confocal , Imagen de Lapso de TiempoRESUMEN
Osteoclasts are specialized polyploid cells that resorb bone. Upon stimulation with receptor activator of nuclear factor-κB ligand (RANKL), myeloid precursors commit to becoming polyploid, largely via cell fusion. Polyploidization of osteoclasts is necessary for their bone-resorbing activity, but the mechanisms by which polyploidization is controlled remain to be determined. Here, we demonstrated that in addition to cell fusion, incomplete cytokinesis also plays a role in osteoclast polyploidization. In in vitro cultured osteoclasts derived from mice expressing the fluorescent ubiquitin-based cell cycle indicator (Fucci), RANKL induced polyploidy by incomplete cytokinesis as well as cell fusion. Polyploid cells generated by incomplete cytokinesis had the potential to subsequently undergo cell fusion. Nuclear polyploidy was also observed in osteoclasts in vivo, suggesting the involvement of incomplete cytokinesis in physiological polyploidization. Furthermore, RANKL-induced incomplete cytokinesis was reduced by inhibition of Akt, resulting in impaired multinucleated osteoclast formation. Taken together, these results reveal that RANKL-induced incomplete cytokinesis contributes to polyploidization of osteoclasts via Akt activation.
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Núcleo Celular/metabolismo , Citocinesis , Células Progenitoras Mieloides/metabolismo , Osteoclastos/metabolismo , Osteólisis/metabolismo , Poliploidía , Ligando RANK/metabolismo , Animales , Bencimidazoles/farmacología , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Fusión Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/patología , Células Cultivadas , Cruzamientos Genéticos , Citocinesis/efectos de los fármacos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones Transgénicos , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/efectos de los fármacos , Células Progenitoras Mieloides/patología , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Osteogénesis/efectos de los fármacos , Osteólisis/patología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/agonistas , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinoxalinas/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Although it is widely recognized that the osteoclast differentiation induced by RANKL is linked to the anti-proliferative activity of the cytokine, we report here that RANKL in the presence of M-CSF actually stimulates DNA synthesis and cell proliferation during the early proliferative phase (0-48 h) of osteoclastogenesis ex vivo, while the same cytokine exerts an anti-proliferative activity in the latter half (48-96 h). A tracing of the individual cells using Fucci cell cycle indicators showed that waves of active DNA synthesis in the S phase during the period 0-48 h are followed by cell-cycle arrest and cell fusion after 48 h. Inhibition of DNA synthesis with hydroxyurea (HU) during the first half almost completely inhibited osteoclastogenesis; however, the same HU-treated cells, when re-plated at 48 h at increasing cell densities, exhibited restored osteoclast formation, suggesting that a sufficient number of cells, rather than prior DNA synthesis, is the most critical requirement for osteoclast formation. In addition, varying either the number of bone marrow macrophages at the start of osteoclastogenic cultures or pre-osteoclasts halfway through the process had a substantial impact on the number of osteoclasts that finally formed, as well as the timing of the peak of osteoclast formation. Thus, caution should be exerted in the performance of any manipulative procedure, whether pharmacological or genetic, that affects the cell number prior to cell fusion. Such procedures can have a profound effect on the number of osteoclasts that form, the final outcome of "differentiation", leading to misinterpretation of the results.
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Diferenciación Celular , Osteoclastos/citología , Ligando RANK/metabolismo , Animales , Células de la Médula Ósea/citología , Resorción Ósea , Ciclo Celular , Proliferación Celular , Citocinas/metabolismo , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Hematopoyesis , Hidroxiurea/química , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Osteoclastos/metabolismo , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
The precise assemblage of several types of cardiac precursors controls heart organogenesis. The cardiac precursors show dynamic movement during early development and then form the complicated heart structure. However, cardiomyocyte movements inside the newly organized mammalian heart remain unclear. We previously established the method of ex vivo time-lapse imaging of the murine heart to study cardiomyocyte behavior by using the Fucci (fluorescent ubiquitination-based cell cycle indicator) system, which can effectively label individual G1, S/G2/M, and G1/S-transition phase nuclei in living cardiomyocytes as red, green, and yellow, respectively. Global analysis of gene expression in Fucci green positive ventricular cardiomyocytes confirmed that cell cycle regulatory genes expressed in G1/S, S, G2/M, and M phase transitions were upregulated. Interestingly, pathway analysis revealed that many genes related to the cell cycle were significantly upregulated in the Fucci green positive ventricular cardiomyocytes, while only a small number of genes related to cell motility were upregulated. Time-lapse imaging showed that murine proliferating cardiomyocytes did not exhibit dynamic movement inside the heart, but stayed on site after entering the cell cycle.
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Corazón Fetal/citología , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Animales , Puntos de Control del Ciclo Celular/genética , Movimiento Celular , Proliferación Celular , Femenino , Corazón Fetal/embriología , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Corazón/crecimiento & desarrollo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , EmbarazoRESUMEN
The precise pathways of memory T-cell differentiation are incompletely understood. Here we exploit transgenic mice expressing fluorescent cell cycle indicators to longitudinally track the division dynamics of individual CD8(+) T cells. During influenza virus infection in vivo, naive T cells enter a CD62L(intermediate) state of fast proliferation, which continues for at least nine generations. At the peak of the anti-viral immune response, a subpopulation of these cells markedly reduces their cycling speed and acquires a CD62L(hi) central memory cell phenotype. Construction of T-cell family division trees in vitro reveals two patterns of proliferation dynamics. While cells initially divide rapidly with moderate stochastic variations of cycling times after each generation, a slow-cycling subpopulation displaying a CD62L(hi) memory phenotype appears after eight divisions. Phenotype and cell cycle duration are inherited by the progeny of slow cyclers. We propose that memory precursors cell-intrinsically modulate their proliferative activity to diversify differentiation pathways.
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Linfocitos T CD8-positivos/citología , Ciclo Celular , Diferenciación Celular , Animales , Linfocitos T CD8-positivos/fisiología , Perfilación de la Expresión Génica , Genes Reporteros , Ratones Endogámicos C57BL , Ratones Transgénicos , TranscriptomaRESUMEN
Fluorescent ubiquitination-based cell cycle indicator (Fucci) technology utilizing the cell cycle-dependent proteolysis of ubiquitin oscillators enables visualization of cell cycle progression in living cells. The Fucci probe consists of two chimeric fluorescent proteins, FucciS/G2/M and FucciG1, which label the nuclei of cells in S/G2/M phase green and those in G1 phase red, respectively. In this study, we generated Fucci transgenic mice and analyzed transgene expression in hematopoietic cells using flow cytometry. The FucciS/G2/M-#474 and FucciG1-#639 mouse lines exhibited high-level transgene expression in most hematopoietic cell populations. The FucciG1-#610 line expressed the transgene at high levels predominantly in the hematopoietic stem cell (HSC) population. Analysis of the HSC (CD34(-)KSL: CD34(-/low)c-Kit(+)Sca-1(+)lineage marker(-)) population in the transgenic mice expressing both FucciS/G2/M and FucciG1 (#474/#610) confirmed that more than 95% of the cells were in G0/G1 phase, although the FucciG1(red) intensity was heterogeneous. An in vivo competitive repopulation assay revealed that repopulating activity resided largely in the FucciG1(red)(high) fraction of CD34(-)KSL cells. Thus, the CD34(-)KSL HSC population can be further purified on the basis of the Fucci intensity.
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Ciclo Celular , Separación Celular/métodos , Colorantes Fluorescentes/metabolismo , Células Madre Hematopoyéticas/citología , Ubiquitinación , Animales , Citometría de Flujo , Fluorescencia , Células Madre Hematopoyéticas/metabolismo , Ratones Transgénicos , TransgenesRESUMEN
In multicellular organism development, a stochastic cellular response is observed, even when a population of cells is exposed to the same environmental conditions. Retrieving the spatiotemporal regulatory mode hidden in the heterogeneous cellular behavior is a challenging task. The G1/S transition observed in cell cycle progression is a highly stochastic process. By taking advantage of a fluorescence cell cycle indicator, Fucci technology, we aimed to unveil a hidden regulatory mode of cell cycle progression in developing zebrafish. Fluorescence live imaging of Cecyil, a zebrafish line genetically expressing Fucci, demonstrated that newly formed notochordal cells from the posterior tip of the embryonic mesoderm exhibited the red (G1) fluorescence signal in the developing notochord. Prior to their initial vacuolation, these cells showed a fluorescence color switch from red to green, indicating G1/S transitions. This G1/S transition did not occur in a synchronous manner, but rather exhibited a stochastic process, since a mixed population of red and green cells was always inserted between newly formed red (G1) notochordal cells and vacuolating green cells. We termed this mixed population of notochordal cells, the G1/S transition window. We first performed quantitative analyses of live imaging data and a numerical estimation of the probability of the G1/S transition, which demonstrated the existence of a posteriorly traveling regulatory wave of the G1/S transition window. To obtain a better understanding of this regulatory mode, we constructed a mathematical model and performed a model selection by comparing the results obtained from the models with those from the experimental data. Our analyses demonstrated that the stochastic G1/S transition window in the notochord travels posteriorly in a periodic fashion, with doubled the periodicity of the neighboring paraxial mesoderm segmentation. This approach may have implications for the characterization of the pathophysiological tissue growth mode.
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Ciclo Celular/fisiología , Embrión no Mamífero/citología , Desarrollo Embrionario/fisiología , Modelos Biológicos , Animales , Biología Computacional , Simulación por Computador , Microscopía Fluorescente/métodos , Pez CebraRESUMEN
Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes-there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.
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Ciclo Celular , Expresión Génica , Genes Reporteros , Integrasas/metabolismo , Especificidad de Órganos , Células 3T3 , Animales , Proliferación Celular , Supervivencia Celular , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Fase G1 , Humanos , Riñón/embriología , Proteínas Luminiscentes/metabolismo , Pulmón/embriología , Ratones , Mitosis , Morfogénesis , Factores de Tiempo , Imagen de Lapso de Tiempo , Proteína Fluorescente RojaRESUMEN
The formation of vascular structures requires precisely controlled proliferation of endothelial cells (ECs), which occurs through strict regulation of the cell cycle. However, the mechanism by which EC proliferation is coordinated during vascular formation remains largely unknown, since a method of analyzing cell-cycle progression of ECs in living animals has been lacking. Thus, we devised a novel system allowing the cell-cycle progression of ECs to be visualized in vivo. To achieve this aim, we generated a transgenic zebrafish line that expresses zFucci (zebrafish fluorescent ubiquitination-based cell cycle indicator) specifically in ECs (an EC-zFucci Tg line). We first assessed whether this system works by labeling the S phase ECs with EdU, then performing time-lapse imaging analyses and, finally, examining the effects of cell-cycle inhibitors. Employing the EC-zFucci Tg line, we analyzed the cell-cycle progression of ECs during vascular development in different regions and at different time points and found that ECs proliferate actively in the developing vasculature. The proliferation of ECs also contributes to the elongation of newly formed blood vessels. While ECs divide during elongation in intersegmental vessels, ECs proliferate in the primordial hindbrain channel to serve as an EC reservoir and migrate into basilar and central arteries, thereby contributing to new blood vessel formation. Furthermore, while EC proliferation is not essential for the formation of the basic framework structures of intersegmental and caudal vessels, it appears to be required for full maturation of these vessels. In addition, venous ECs mainly proliferate in the late stage of vascular development, whereas arterial ECs become quiescent at this stage. Thus, we anticipate that the EC-zFucci Tg line can serve as a tool for detailed studies of the proliferation of ECs in various forms of vascular development in vivo.
Asunto(s)
Ciclo Celular/genética , Endotelio Vascular/fisiología , Neovascularización Fisiológica/genética , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente/embriología , Animales Modificados Genéticamente/genética , Ciclo Celular/fisiología , División Celular , Proliferación Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Endotelio Vascular/citología , Técnicas de Silenciamiento del Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Morfolinos/genética , Pez Cebra/genética , Proteínas de Pez Cebra/biosíntesisRESUMEN
The visualization of cell-cycle behavior of individual cells within complex tissues presents an irresistible challenge to biologists studying multicellular structures. However, the transition from G1 to S in the cell cycle is difficult to monitor despite the fact that the process involves the critical decision to initiate a new round of DNA replication. Here, we use ubiquitination oscillators that control cell-cycle transitions to develop genetically encoded fluorescent probes for cell-cycle progression. Fucci (fluorescent ubiquitination-based cell-cycle indicator) probes exploit the regulation of cell-cycle-dependent ubiquitination to effectively label individual nuclei in G1 phase red, and those in S/G2/M phases green. Cultured cells and transgenic mice constitutively expressing the probes have been generated, such that every cell nucleus shows either red or green fluorescence. This protocol details two experiments that use biological samples expressing Fucci probes. One experiment involves time-lapse imaging of cells stably expressing a Fucci derivative (Fucci2), which allows for the exploration of the spatiotemporal patterns of cell-cycle dynamics during structural and behavioral changes of cultured cells. The other experiment involves large-field, high-resolution imaging of fixed sections of Fucci transgenic mouse embryos, which provides maps that illustrate cell proliferation versus differentiation in various developing organs.