Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
1.
J Cell Sci ; 135(13)2022 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-35662333

RESUMO

Cells exist in an astonishing range of volumes across and within species. However, our understanding of cell size control remains limited, owing in large part to the challenges associated with accurate determination of cell volume. Much of our comprehension of size regulation derives from yeast models, but even for these morphologically stereotypical cells, assessment of cell volume has mostly relied on proxies and extrapolations from two-dimensional measurements. Recently, the fluorescence exclusion method (FXm) was developed to evaluate the size of mammalian cells, but whether it could be applied to smaller cells remained unknown. Using specifically designed microfluidic chips and an improved data analysis pipeline, we show here that FXm reliably detects subtle differences in the volume of fission yeast cells, even for those with altered shapes. Moreover, it allows for the monitoring of dynamic volume changes at the single-cell level with high time resolution. Collectively, our work highlights how the coupling of FXm with yeast genetics will bring new insights into the complex biology of cell growth.


Assuntos
Saccharomyces cerevisiae , Schizosaccharomyces , Animais , Ciclo Celular , Tamanho Celular , Mamíferos , Microfluídica , Saccharomyces cerevisiae/genética
2.
Yeast ; 41(3): 87-94, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38099423

RESUMO

In poor nitrogen conditions, fission yeast cells mate, undergo meiosis and form spores that are resistant to deleterious environments. Natural isolates of Schizosaccharomyces pombe are homothallic. This allows them to naturally switch between the two h- and h+ mating types with a high frequency, thereby ensuring the presence of both mating partners in a population of cells. However, alteration of the mating type locus can abolish mating type switching or reduce it to a very low frequency. Such heterothallic strains have been isolated and are common in research laboratories due to the simplicity of their use for Mendelian genetics. In addition to the standard laboratory strains, a large collection of natural S. pombe isolates is now available, representing a powerful resource for investigating the genetic diversity and biology of fission yeast. However, most of these strains are homothallic, and only tedious or mutagenic strategies have been described to obtain heterothallic cells from a homothallic parent. Here, we describe a simple approach to generate heterothallic strains. It takes advantage of an alteration of the mating type locus that was previously identified in a mating type switching-deficient strain and the CRISPR-Cas9 editing tool, allowing for a one-step engineering of heterothallic cells with high efficiency.


Assuntos
Schizosaccharomyces , Schizosaccharomyces/genética , Reprodução/genética , Meiose/genética , Genes Fúngicos Tipo Acasalamento
3.
BMC Cell Biol ; 19(1): 8, 2018 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-29925307

RESUMO

BACKGROUND: Real-time monitoring of cellular responses to dynamic changes in their environment or to specific treatments has become central to cell biology. However, when coupled to live-cell imaging, such strategies are difficult to implement with precision and high time resolution, and the simultaneous alteration of multiple parameters is a major challenge. Recently, microfluidics has provided powerful solutions for such analyses, bringing an unprecedented level of control over the conditions and the medium in which cells under microscopic observation are grown. However, such technologies have remained under-exploited, largely as a result of the complexity associated with microfabrication procedures. RESULTS: In this study, we have developed simple but powerful microfluidic devices dedicated to live-cell imaging. These microsystems take advantage of a robust elastomer that is readily available to researchers and that presents excellent bonding properties, in particular to microscopy-grade glass coverslips. Importantly, the chips are easy-to-build without sophisticated equipment, and they are compatible with the integration of complex, customized fluidic networks as well as with the multiplexing of independent assays on a single device. We show that the chips are re-usable, a significant advantage for the popularization of microfluidics in cell biology. Moreover, we demonstrate that they allow for the dynamic, accurate and simultaneous control of multiple parameters of the cellular environment. CONCLUSIONS: While they do not possess all the features of the microdevices that are built using complex and costly procedures, the simplicity and versatility of the chips that we have developed make them an attractive alternative for a range of applications. The emergence of such devices, which can be fabricated and used by any laboratory, will provide the possibility for a larger number of research teams to take full advantage of these new methods for investigating cell biology.


Assuntos
Imageamento Tridimensional , Microfluídica/métodos , Sobrevivência Celular , Elastômeros/química , Fluorescência , Células HeLa , Humanos , Perfusão , Pressão , Reologia , Saccharomyces cerevisiae/metabolismo , Temperatura
4.
Yeast ; 34(8): 343-355, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28426144

RESUMO

Maintenance of long-term cultures of yeast cells is central to a broad range of investigations, from metabolic studies to laboratory evolution assays. However, repeated dilutions of batch cultures lead to variations in medium composition, with implications for cell physiology. In Saccharomyces cerevisiae, powerful miniaturized chemostat setups, or ministat arrays, have been shown to allow for constant dilution of multiple independent cultures. Here we set out to adapt these arrays for continuous culture of a morphologically and physiologically distinct yeast, the fission yeast Schizosaccharomyces pombe, with the goal of maintaining constant population density over time. First, we demonstrated that the original ministats are incompatible with growing fission yeast for more than a few generations, prompting us to modify different aspects of the system design. Next, we identified critical parameters for sustaining unbiased vegetative growth in these conditions. This requires deletion of the gsf2 flocculin-encoding gene, along with addition of galactose to the medium and lowering of the culture temperature. Importantly, we improved the flexibility of the ministats by developing a piezo-pump module for the independent regulation of the dilution rate of each culture. This made it possible to easily grow strains that have different generation times in the same assay. Our system therefore allows for maintaining multiple fission yeast cultures in exponential growth, adapting the dilution of each culture over time to keep constant population density for hundreds of generations. These multiplex culture systems open the door to a new range of long-term experiments using this model organism. © 2017 The Authors. Yeast published by John Wiley & Sons, Ltd.


Assuntos
Técnicas Microbiológicas/métodos , Schizosaccharomyces/crescimento & desenvolvimento , Meios de Cultura/química , Galactose/metabolismo , Deleção de Genes , Genética Microbiana/métodos , Proteínas de Membrana/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Temperatura
5.
Yeast ; 33(9): 483-92, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27145443

RESUMO

Synthetic biology is one of the most exciting strategies for the investigation of living organisms and lies at the intersection of biology and engineering. Originally developed in prokaryotes, the idea of deciphering biological phenomena through building artificial genetic circuits and studying their behaviours has rapidly demonstrated its potential in a broad range of fields in the life sciences. From the assembly of synthetic genomes to the generation of novel biological functions, yeast cells have imposed themselves as the most powerful eukaryotic model for this approach. However, we are only beginning to explore the possibilities of synthetic biology, and the perspectives it offers in a genetically amenable system such as yeasts are endless. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Células Artificiais , Leveduras/citologia , Leveduras/fisiologia , DNA Fúngico , Regulação Fúngica da Expressão Gênica , Engenharia Genética , Leveduras/genética
6.
PLoS Comput Biol ; 11(2): e1004056, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25658582

RESUMO

In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk) families, and the Anaphase Promoting Complex (APC). Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/fisiologia , Modelos Biológicos , Biologia Computacional , Quinases Ciclina-Dependentes/metabolismo , Fosforilação/fisiologia , Schizosaccharomyces/metabolismo , Schizosaccharomyces/fisiologia
7.
Nature ; 468(7327): 1074-9, 2010 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-21179163

RESUMO

Control of eukaryotic cell proliferation involves an extended regulatory network, the complexity of which has made it difficult to understand the basic principles of the cell cycle. To investigate the core engine of the mitotic cycle we have generated a minimal control network in fission yeast that efficiently sustains cellular reproduction. Here we demonstrate that orderly progression through the major events of the cell cycle can be driven by oscillation of an engineered monomolecular cyclin-dependent protein kinase (CDK) module lacking much of the canonical regulation. We show further that the CDK oscillator acts as the primary organizer of the cell cycle, imposing timing and directionality to a system of two CDK activity thresholds that define independent cell cycle phases. We propose that this simple core architecture forms the basic control of the eukaryotic cell cycle.


Assuntos
Relógios Biológicos , Ciclo Celular/fisiologia , Quinases Ciclina-Dependentes/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/enzimologia , Proliferação de Células , DNA/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Tempo
8.
J Cell Sci ; 125(Pt 20): 4703-11, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23223895

RESUMO

Fifteen years ago, it was proposed that the cell cycle in fission yeast can be driven by quantitative changes in the activity of a single protein kinase complex comprising a cyclin - namely cyclin B - and cyclin dependent kinase 1 (Cdk1). When its activity is low, Cdk1 triggers the onset of S phase; when its activity level exceeds a specific threshold, it promotes entry into mitosis. This model has redefined our understanding of the essential functional inputs that organize cell cycle progression, and its main principles now appear to be applicable to all eukaryotic cells. But how does a change in the activity of one kinase generate ordered progression through the cell cycle in order to separate DNA replication from mitosis? To answer this question, we must consider the biochemical processes that underlie the phosphorylation of Cdk1 substrates. In this Commentary, we discuss recent findings that have shed light on how the threshold levels of Cdk1 activity that are required for progression through each phase are determined, how an increase in Cdk activity generates directionality in the cell cycle, and why cell cycle transitions are abrupt rather than gradual. These considerations lead to a general quantitative model of cell cycle control, in which opposing kinase and phosphatase activities have an essential role in ensuring dynamic transitions.


Assuntos
Proteína Quinase CDC2 , Pontos de Checagem do Ciclo Celular/genética , Mitose/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Replicação do DNA , Humanos , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Pontos de Checagem da Fase S do Ciclo Celular/genética , Saccharomyces cerevisiae/genética
9.
Methods Mol Biol ; 2740: 89-105, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38393470

RESUMO

Cell cycle control is a central aspect of the biology of proliferating eukaryotic cells. However, progression through the cell cycle relies on a highly complex network, making it difficult to unravel the core design principles underlying the mechanisms that sustain cell proliferation and the ways in which they interact with other cellular pathways. In this context, the use of a synthetic approach to simplify the cell cycle network in unicellular genetic models such as fission yeast has opened the door to studying the biology of proliferating cells from unique perspectives. Here, we provide a series of methods based on a minimal cell cycle module in the fission yeast Schizosaccharomyces pombe that allows for an unprecedented artificial control of cell cycle events, enabling the rewiring and remodeling of cell cycle progression.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Ciclo Celular , Divisão Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
10.
Dev Cell ; 59(4): 545-557.e4, 2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38228139

RESUMO

Cyclin-dependent kinase (CDK) determines the temporal ordering of the cell cycle phases. However, despite significant progress in studying regulators of CDK and phosphorylation patterns of CDK substrates at the population level, it remains elusive how CDK regulators coordinately affect CDK activity at the single-cell level and how CDK controls the temporal order of cell cycle events. Here, we elucidate the dynamics of CDK activity in fission yeast and mammalian cells by developing a CDK activity biosensor, Eevee-spCDK. We find that although CDK activity does not necessarily correlate with cyclin levels, it converges to the same level around mitotic onset in several mutant backgrounds, including pom1Δ cells and wee1 or cdc25 overexpressing cells. These data provide direct evidence that cells enter the M phase when CDK activity reaches a high threshold, consistent with the quantitative model of cell cycle progression in fission yeast.


Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Animais , Fosforilação , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Mitose , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Mamíferos/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
11.
Dev Cell ; 14(1): 140-7, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18160347

RESUMO

Wnt proteins are secreted signaling molecules that play a central role in development and adult tissue homeostasis. We have previously shown that Wnt signaling requires retromer function in Wnt-producing cells. The retromer is a multiprotein complex that mediates endosome-to-Golgi transport of specific sorting receptors. MIG-14/Wls is a conserved transmembrane protein that binds Wnt and is required in Wnt-producing cells for Wnt secretion. Here, we demonstrate that in the absence of retromer function, MIG-14/Wls is degraded in lysosomes and becomes limiting for Wnt signaling. We show that retromer-dependent recycling of MIG-14/Wls is part of a trafficking pathway that retrieves MIG-14/Wls from the plasma membrane. We propose that MIG-14/Wls cycles between the Golgi and the plasma membrane to mediate Wnt secretion. Regulation of this transport pathway may enable Wnt-producing cells to control the range of Wnt signaling in the tissue.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/fisiologia , Proteínas de Transporte/fisiologia , Proteínas Wnt/fisiologia , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , Linhagem Celular , Membrana Celular/fisiologia , Endossomos/fisiologia , Complexo de Golgi/fisiologia , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Rim , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Wnt/genética
12.
bioRxiv ; 2023 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-37034650

RESUMO

Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently-developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new standalone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled setup for long-term experimental evolution.

13.
Open Biol ; 13(7): 230118, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37491941

RESUMO

Experimental evolution using fast-growing unicellular organisms is a unique strategy for deciphering the principles and mechanisms underlying evolutionary processes as well as the architecture and wiring of basic biological functions. Over the past decade, this approach has benefited from the development of powerful systems for the continuous control of the growth of independently evolving cultures. While the first devices compatible with multiplexed experimental evolution remained challenging to implement and required constant user intervention, the recently developed eVOLVER framework represents a fully automated closed-loop system for laboratory evolution assays. However, it remained difficult to maintain and compare parallel evolving cultures in tightly controlled environments over long periods of time using eVOLVER. Furthermore, a number of tools were lacking to cope with the various issues that inevitably occur when conducting such long-term assays. Here we present a significant upgrade of the eVOLVER framework, providing major modifications of the experimental methodology, hardware and software as well as a new stand-alone protocol. Altogether, these adaptations and improvements make the eVOLVER a versatile and unparalleled set-up for long-term experimental evolution.


Assuntos
Evolução Biológica , Software
14.
Nat Commun ; 14(1): 5104, 2023 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-37607906

RESUMO

Histone post-translational modifications promote a chromatin environment that controls transcription, DNA replication and repair, but surprisingly few phosphorylations have been documented. We report the discovery of histone H3 serine-57 phosphorylation (H3S57ph) and show that it is implicated in different DNA repair pathways from fungi to vertebrates. We identified CHK1 as a major human H3S57 kinase, and disrupting or constitutively mimicking H3S57ph had opposing effects on rate of recovery from replication stress, 53BP1 chromatin binding, and dependency on RAD52. In fission yeast, mutation of all H3 alleles to S57A abrogated DNA repair by both non-homologous end-joining and homologous recombination, while cells with phospho-mimicking S57D alleles were partly compromised for both repair pathways, presented aberrant Rad52 foci and were strongly sensitised to replication stress. Mechanistically, H3S57ph loosens DNA-histone contacts, increasing nucleosome mobility, and interacts with H3K56. Our results suggest that dynamic phosphorylation of H3S57 is required for DNA repair and recovery from replication stress, opening avenues for investigating the role of this modification in other DNA-related processes.


Assuntos
Histonas , Vírus da Influenza A , Humanos , Animais , Fosforilação , Processamento de Proteína Pós-Traducional , Reparo do DNA , Cromatina
15.
Traffic ; 10(3): 334-43, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19207483

RESUMO

Little is known about the release and intercellular transport of Wnt proteins from mammalian cells. Lipoproteins may act as carriers for the intercellular movement and gradient formation of the lipid-linked morphogens Wingless and Hedgehog in Drosophila. To investigate whether such a mechanism can occur in mammals, we have studied Wnt release in cultured mammalian cells. Wnt3a associated with lipoproteins in the culture medium and not with extracellular vesicles or exosomes. Although Wnt3a was associated with both high-density lipoproteins (HDL) and low-density lipoproteins, only HDL allowed Wnt3a release from mouse fibroblasts. Remarkably, Wnt3a lacking its palmitate moiety was released in a lipoprotein-independent manner, demonstrating the dual role of palmitoylation in membrane and lipoprotein binding. We additionally found that Wnt3a can be released from enterocyte cell lines on endogenously expressed lipoproteins. We further discuss the physiological implications of our findings.


Assuntos
Lipoproteínas/metabolismo , Proteínas Wnt/metabolismo , Animais , Linhagem Celular , Cricetinae , Proteínas de Transporte de Ácido Graxo/metabolismo , Proteínas Wnt/genética , Proteína Wnt3 , Proteína Wnt3A
16.
Methods Cell Biol ; 147: 3-28, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30165961

RESUMO

Microfluidic technologies have become a standard tool in cell biological studies, offering unprecedented control of the chemical and physical environment of cells grown in microdevices, the possibility of multiplexing assays, as well as the capacity to monitor the behavior of single cells in real time while dynamically manipulating their growth medium. However, the properties of the materials employed for the fabrication of microchips that are compatible with live-cell imaging has limited the use of these techniques for a broad range of experiments. In particular, the strong absorption of a large panel of small molecules by these materials prevents the accurate delivery of compounds of interest. Here we describe a novel microsystem dedicated to live-cell imaging that (1) uses alternative materials devoid of absorptive properties, and (2) allows for dynamic in-chip control of sample temperature. Based on a proof-of-concept design that we have routinely used with non-adherent fission yeast cells, this chapter details all the steps for the fabrication and utilization of these microdevices.


Assuntos
Sistemas de Liberação de Medicamentos , Técnicas Analíticas Microfluídicas/métodos , Temperatura , Alcenos/química , Animais , Calibragem , Sobrevivência Celular , Imageamento Tridimensional , Polímeros/química
17.
Lab Chip ; 17(15): 2581-2594, 2017 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-28656191

RESUMO

One of the most important areas of research on microfluidic technologies focuses on the identification and characterisation of novel materials with enhanced properties and versatility. Here we present a fast, easy and inexpensive microstructuration method for the fabrication of novel, flexible, transparent and biocompatible microfluidic devices. Using a simple hot press, we demonstrate the rapid (30 s) production of various microfluidic prototypes embossed in a commercially available soft thermoplastic elastomer (sTPE). This styrenic block copolymer (BCP) material is as flexible as PDMS and as thermoformable as classical thermoplastics. It exhibits high fidelity of replication using SU-8 and epoxy master molds in a highly convenient low-isobar (0.4 bar) and iso-thermal process. Microfluidic devices can then be easily sealed using either a simple hot plate or even a room-temperature assembly, allowing them to sustain liquid pressures of 2 and 0.6 bar, respectively. The excellent sorption and biocompatibility properties of the microchips were validated via a standard rhodamine dye assay as well as a sensitive yeast cell-based assay. The morphology and composition of the surface area after plasma treatment for hydrophilization purposes are stable and show constant and homogenous distribution of block nanodomains (∼22° after 4 days). These domains, which are evenly distributed on the nanoscale, therefore account for the uniform and convenient surface of a "microfluidic scale device". To our knowledge, this is the first thermoplastic elastomer material that can be used for fast and reliable fabrication and assembly of microdevices while maintaining a high and stable hydrophilicity.

18.
Nat Commun ; 7: 11161, 2016 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-27045731

RESUMO

Cell proliferation is regulated by cyclin-dependent kinases (Cdks) and requires the periodic expression of particular gene clusters in different cell cycle phases. However, the interplay between the networks that generate these transcriptional oscillations and the core cell cycle machinery remains largely unexplored. In this work, we use a synthetic regulable Cdk1 module to demonstrate that periodic expression is governed by quantitative changes in Cdk1 activity, with different clusters directly responding to specific activity levels. We further establish that cell cycle events neither participate in nor interfere with the Cdk1-driven transcriptional program, provided that cells are exposed to the appropriate Cdk1 activities. These findings contrast with current models that propose self-sustained and Cdk1-independent transcriptional oscillations. Our work therefore supports a model in which Cdk1 activity serves as a quantitative platform for coordinating cell cycle transitions with the expression of critical genes to bring about proper cell cycle progression.


Assuntos
Proteína Quinase CDC2/genética , Ciclo Celular/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Transcrição Gênica , Proteína Quinase CDC2/metabolismo , Ciclo Celular/efeitos dos fármacos , Perfilação da Expressão Gênica , Família Multigênica , Periodicidade , Purinas/farmacologia , Schizosaccharomyces/efeitos dos fármacos , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais
19.
Open Biol ; 6(8)2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27512142

RESUMO

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.


Assuntos
Desenho de Equipamento/métodos , Microfluídica/instrumentação , Células HeLa/ultraestrutura , Humanos , Reprodutibilidade dos Testes , Temperatura , Leveduras/ultraestrutura
20.
Curr Biol ; 20(12): 1053-64, 2010 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-20605454

RESUMO

BACKGROUND: The switch from cellular proliferation to differentiation occurs to a large extent through specific programs of gene expression. In fission yeast, the master regulator of sexual differentiation, ste11, is induced by environmental conditions leading to mating and meiosis. RESULTS: We show that phosphorylation of serine 2 (S2P) in the C-terminal domain of the largest subunit of the RNA polymerase II (PolII) enzyme by the Lsk1 cyclin-dependent kinase has only a minor impact on global gene expression during vegetative growth but is critical for the induction of ste11 transcription during sexual differentiation. The recruitment of the Lsk1 kinase initiates in the vicinity of the transcription start site of ste11, resulting in a marked increase of S2P on the ste11 unit, including an extended 5' untranslated region (5'UTR). This pattern contrasts with the classical gradient of S2P toward the 3' region. In the absence of S2P, both PolII occupancy at the ste11 locus and ste11 expression are impaired. This results in sterility, which is rescued by expression of the ste11 coding sequence from the adh1 promoter. CONCLUSION: Thus, the S2P polymerase plays a specific, regulatory role in cell differentiation through the induction of ste11.


Assuntos
RNA Polimerase II/metabolismo , Schizosaccharomyces/enzimologia , Eletroforese em Gel de Poliacrilamida , Regulação Fúngica da Expressão Gênica , Fosforilação , Schizosaccharomyces/citologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA