Os autores agradecem Matthias Peter e seu grupo do Instituto de Bioquímica da ETH em Zurique, onde estes métodos têm sido desenvolvidos. Este trabalho foi financiado pela Fundação de Ciência Nacional Suíço.

1. Medidas Microscopia <ol><li> Inocular 5 ml de meio sintético com levedura. </li><li> Crescer as células a 30 ° C durante a noite. </li><li> Medir a OD 600 da cultura durante a noite a na manhã seguinte. </li><li> Dilui-se a cultura durante a noite em 5 ml de meio sintético de OD 600 de 0,05. </li><li> Crescer a cultura diluída a 30 ° C durante pelo menos 4 horas. </li><li> Preparar 3 vezes concentrado (0,6 M) de NaCl em meio sintético. </li><li> Prepara-se uma solução de 1 mg / ml de Concanavalina A (ConA) em PBS. </li><li> Filtrado ~ 200 mL de solução para o poço de ConA corrediça. </li><li> Deixe descansar por 30 min. </li><li> Remover solução ConA. </li><li> Adicionar 150 mL de H 2 O para o poço. </li><li> Retirar H 2 O. </li><li> Medida OD600 da cultura de células. </li><li> Prepare 300 mL de cultura de células em OD 600 = 0,02 em um microtubo de 1,5 ml. </li><li> Sonicar as células num banho de água durante 45 segundos. </li><li> Vortex brevemente a microtube. </li><li> Sonicar as células num banho de água durante 45 segundos. </li><li> Vortex brevemente o microtubo. </li><li> Adicionar 200 mL de cultura de células para o bem. </li><li> Esperar 30 minutos para permitir que as células se depositam no fundo do poço. </li><li> Colocar o bem corrediça sobre o microscópio. </li><li> Selecione as configurações de iluminação para o canal fluorescente para ser gravado. </li><li> Seleccionar as configurações de iluminação por duas imagens de campo claro (um pouco fora do foco: 3 um abaixo do plano focal para permitir uma adequada segmentação das células). </li><li> Selecione os intervalos de tempo para a medição de lapso de tempo </li><li> Selecione os campos de visão a imagem. </li><li> Iniciar a aquisição de alguns fotogramas. </li><li> Pausa a aquisição para adicionar os 100 ml de 0,6 M NaCl médio. </li><li> Retomar imagem. </li></ol> 2. Medidas Citometria de Fluxo <ol><li> Inocular 5 ml de meio sintético com levedura. </li><li> Crescer a 30 ° C durante a noite. </li><li> Medidaa DO600 da cultura durante a noite a na manhã seguinte. </li><li> Dilui-se a cultura durante a noite em 5 ml de meio sintético de OD 600 de 0,1. </li><li> Crescer a 30 ° C durante pelo menos 4 horas, para atingir uma DO 600 de 0,2-0,4. </li><li> Preparar a solução de cicloheximida 1 mg / ml em H 2 O. </li><li> Preparar 3 vezes concentrado (0,6 M) de NaCl em meio sintético. </li><li> Preparar um microtubo de 1,5 mL com 100 uL de 0,6 M de NaCl média para cada ponto de tempo a ser medidos. </li><li> No tempo 0, adicionar 200 mL de cultura de células a cada microtubo. </li><li> Incubar, sob agitação, a 30 ° C. </li><li> No tempo t, adicionar 30 mL cicloheximida para um microtubo. </li><li> Repita o passo 2.11 para todos os momentos desejados. </li><li> Incubar todas microtubos durante pelo menos 45 minutos e até 3 horas a 30 ° C com agitação. </li><li> Prepare os tubos de FACS com 400 mL PBS. </li><li> Sonicar das células em banho de água durante 1 min. </li><li> Vortex brevemente os microtubos. </li><li> Filhoicate das células em banho de água durante 1 min. </li><li> Vortex brevemente os microtubos. </li><li> Adicionar 100 ul de cultura de células a partir de cada microtubo no seu correspondente tubo de FACS. </li><li> Selecione o laser de excitação 488 nm e 530/30 nm filtro passa-banda para a detecção. </li><li> Teste não expressar e expressar totalmente amostra para verificar se eles estão na faixa de sensibilidade do detector. </li><li> Medir 10.000 células por cada amostra adquirida. </li></ol>

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+protein+expression+in+yeast.">Global analysis of protein expression in yeast.</a> Nature. 425 (6959), 737-741 (2003).</li><li>Wu, J. -. Q., Pollard, T. 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B., Levine, A. J., Siggia, E. D., Swain, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+gene+expression+in+a+single+cell.">Stochastic gene expression in a single cell.</a> Science. 297 (5584), 1183-1186 (2002).</li><li>Pelet, S., Dechant, R., Lee, S. S., van Drogen, F., Peter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+image+analysis+platform+to+quantify+signal+transduction+in+single+cells.">An integrated image analysis platform to quantify signal transduction in single cells.</a> Integrative biology : quantitative biosciences from nano to macro. 4 (10), 1274-1282 (2012).</li><li>Carpenter, A. E., Jones, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome biology. 7 (10), R100 (2006).</li><li>Chernomoretz, A., Bush, A., Yu, R., Gordon, A., Colman-Lerner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+Cell-ID+1.4+with+R+for+microscope-based+cytometry.">Using Cell-ID 1.4 with R for microscope-based cytometry.</a> Current protocols in molecular biology. 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N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (20), 7165-7170 (2008).</li><li>Pepperkok, R., Ellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+fluorescence+microscopy+for+systems+biology.">High-throughput fluorescence microscopy for systems biology.</a> Nature reviews Molecular cell biology. 7 (9), 690-696 (2006).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Microscopia O tratamento do poço com ConA é um passo essencial para assegurar uma imagem adequada das células. Porque ConA tem uma baixa solubilidade em PBS (5 mg / ml), o processo de filtração permite a remoção de grandes agregados que se encontram presentes na solução não filtrada e interferem com a imagem latente. Células prender de forma relativamente forte à superfície tratada e a adição da solução de indução não deve perturbar a localização das células, o que permit...

A sinalização por meio de cascatas de transdução de muitas vezes termina com a expressão de proteínas. A caracterização desse perfil de expressão é um elemento chave para a compreensão da função das vias biológicas. A identificação do espectro de sobre-regulada de proteínas e sua dinâmica de activação pode ser alcançada por diversas técnicas, tais como as micro-matrizes, transferências de Northern ou Western blot 1-3. No entanto, essas técnicas de média a resposta de uma população inteira de células. Para compreender a regulação fina da expressão de proteínas, é desejável reunir as medições ao nível da célula individual. O ideal é que estas medidas também deve fornecer dados quantitativos passíveis de desenvolver modelos matemáticos da via subjacente. Microscopia e citometria de fluxo são duas técnicas, as quais são idealmente adequados para entregar tais medições quantitativas de uma única célula. A marcação de proteínas endógeno com uma proteína fluorescente pode be utilizada para quantificar o nível de expressão 4. No entanto, uma vez que a adição de uma grande porção fluorescente no terminal da proteína pode torná-la não-funcional, isto é, muitas vezes mais desejável gerar repórteres de expressão específicas com base em um promotor dirigindo a expressão de uma construção fluorescente. Esta proteína repórter é exógeno ao sistema celular e, portanto, não influencia os eventos de sinalização que estão ocorrendo na célula. Ambos microscopia e citometria de fluxo têm sido amplamente utilizados no campo de sinalização de levedura. Como exemplos; Colman-Lerner e colaboradores correlacionados, em células individuais, a expressão de acasalamento repórteres específicos e constitutivamente expressos por microscopia para quantificar o ruído em levedura acasalamento cascata de transdução de sinal 5, Acar e colegas utilizados citometria de fluxo para estudar a rede reguladora controlando a expressão dos genes GAL 6. Em um estudo anterior 7, nós usamos uma associaçem uma dessas duas técnicas para estudar a saída expressão do alto glicerol osmolaridade (HOG) via em brotamento da levedura. Este mitógeno proteína quinase ativada (MAPK) é desencadeada por estresse hiper-osmótico. Isso resulta na activação dos Hog1 MAPK, que transloca-se para o núcleo da célula para induzir um programa transcricional resultando na expressão de cerca de 300 genes. Para estudar este processo, que tinha engenharia um repórter com base na expressão do promotor STL1 (um gene induzido especificamente em resposta à actividade Hog1 8) dirigindo a expressão de uma proteína fluorescente Vénus quádruplo (p STL1-qV). A citometria de fluxo medições revelaram a presença de duas populações de células no nível de tensão intermédia (NaCl 0,1 M), com apenas uma fracção da população que expressa o repórter fluorescente. Nós usamos a microscopia para investigar mais este comportamento e descobriu que este ruído na expressão da proteína foi governada por fatores intrínsecos 9. Nós could observar, ainda, que as células com um nível semelhante de atividade Hog1 poderia exibir resultados muito diferentes de expressão. A combinação destas duas técnicas permitiram-nos demonstrar como a remodelação lenta dos genes de resposta ao stress influenciou a expressão resultados ao nível da célula individual 7. Neste trabalho, usamos a expressão da p STL1-qV repórter induzida pelo choque hiper-osmótico como um exemplo da quantificação da expressão da proteína por microscopia e citometria de fluxo. A mesma cepa submetidos a 0,2 M NaCl estresse foi estudado com ambas as técnicas. Isto irá permitir-nos para destacar algumas das principais diferenças nestas duas técnicas altamente complementares.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Reagent&lt;/strong&gt;</td></tr><tr><td>Yeast Nitrogen Base</td><td>ForMedium</td><td>CYN6202</td><td></td></tr><tr><td>CSM amino-acid mix</td><td>ForMedium</td><td>DCS0011</td><td></td></tr><tr><td>Concanavalin A</td><td>GE Healthcare</td><td>17-0450-01</td><td></td></tr><tr><td>Cycloheximide</td><td>Fluka Aldrich Sigma</td><td>C7698</td><td>Toxic</td></tr><tr><td>ySP9</td><td></td><td></td><td>W303Mata leu2::LEU2-pSTL1-quadrupleV enus</td></tr><tr><td>pSP34</td><td></td><td></td><td>pRS305 pSTL1 (-800 -0) - quadruple V enus</td></tr><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Ti-Eclipse</td><td></td></tr><tr><td>Microscope control software</td><td>Micro-manager</td><td>Ver 1.4.11</td><td></td></tr><tr><td>Incubation chamber</td><td>LIS</td><td>The Box</td><td></td></tr><tr><td>Fluorescence light source</td><td>Lumencor</td><td>SpectraX</td><td></td></tr><tr><td>Camera</td><td>Hamamatsu</td><td>Flash 4.0</td><td></td></tr><tr><td>Flow cytometer</td><td>BD</td><td>FACS calibur</td><td></td></tr><tr><td>Sonicator bath</td><td>Telesonic</td><td>TUC-150</td><td></td></tr><tr><td>8-well-slide </td><td>Thermo Lab-tek</td><td>155409</td><td></td></tr><tr><td>96-well-plate</td><td>Matrical bioscience</td><td>MGB096-1-2-LG</td><td></td></tr><tr><td>FACS tube</td><td>BD Falcon</td><td>352054</td><td></td></tr></tbody></table>

temporal quantification of mapk induced expression in single yeast cells

A quantificação da expressão gênica no nível da célula única descobre novos mecanismos de regulação obscurecidos em medições realizadas a nível da população. Dois métodos baseados em citometria de fluxo e microscopia são apresentados para demonstrar a forma como esses dados podem ser adquiridos. A expressão de um repórter fluorescente induzida após a activação da MAPK alta osmolaridade glicerol via em levedura é usada como um exemplo. As vantagens específicas de cada método são realçados. A citometria de fluxo mede um grande número de células (10.000) e proporciona uma medida direta da dinâmica da expressão proteica independente da cinética de maturação lenta da proteína fluorescente. Imagiologia de células vivas por microscopia por contraste é limitada para a medição da forma amadurecida do repórter no menor número de células. No entanto, os conjuntos de dados gerados por esta técnica pode ser extremamente ricos graças às combinações de vários jornalistas e à informação espacial e temporalobtido a partir de células individuais. A combinação destes dois métodos de medição pode oferecer novos insights sobre a regulação da expressão de proteínas por vias de sinalização.

Microscopia As células de levedura que ostentam a expressão repórter p-STL1 qV (ySP9 7) foram ligados ao fundo do poço de deslizamento e colocado sob o microscópio. As células foram estimuladas pela adição de meio de pressão directamente para dentro do poço, durante o decurso da sessão de imagem. Isso nos permite adquirir algumas imagens das células antes da indução da via e seguir o seu destino após a estimulação. No presente caso, as células foram seguido...

Watch this Scientific Journal Video about Temporal Quantification of MAPK Induced Expression in Single Yeast Cells at JoVE.com

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Dois métodos complementares baseados em citometria de fluxo e microscopia são apresentadas, que permitem a quantificação, no nível de uma única célula, da dinâmica da expressão de genes induzidos pela activação de uma via de MAPK em levedura.

A quantificação temporal da expressão MAPK induzida em células de levedura Solteiro

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

temporal-quantification-mapk-induced-expression-single-yeast

Research

JoVE Journal

Biology

8.5K visualizações.  University of Lausanne.  Dois métodos complementares baseados em citometria de fluxo e microscopia são apresentadas, que permitem a quantificação, no nível de uma única célula, da dinâmica da expressão de genes induzidos pela activação de uma via de MAPK em levedura. 

Vídeo: Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Ensaio para a Adesão e Invasão Agar em S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Biologia

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Medição de níveis de mRNA ao longo do tempo durante a levedura s. cerevisiae hipóxica resposta

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

Genética

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in mRNA synthesis has been shown to be compensated by a simultaneous decrease in mRNA degradation to restore normal steady-state levels. Hence, the genome-wide quantification of mRNA synthesis, independently from mRNA decay, is the best direct reflection of RNA polymerase II transcriptional activity. Here, we discuss a method using non-perturbing metabolic labeling of nascent RNAs in Saccharomyces cerevisiae (S. cerevisiae). Specifically, the cells are cultured for 6 min with a uracil analog, 4-thiouracil, and the labeled newly transcribed RNAs are purified and quantified to determine the synthesis rates of all individual mRNA. Moreover, using labeled Schizosaccharomyces pombe cells as internal standard allows comparing mRNA synthesis in different S. cerevisiae strains. Using this protocol and fitting the data with a dynamic kinetic model, the corresponding mRNA decay rates can be determined.

Saccharomyces cerevisiae Metabolic Labeling with 4-thiouracil and the Quantification of Newly Synthesized mRNA As a Proxy for RNA Polymerase II Activity

The protocol described here is based on the genome-wide quantification of newly synthesized mRNA purified from yeast cells labeled with 4-thiouracil. This method allows to measure mRNA synthesis uncoupled from mRNA decay and, thus, provides an accurate measurement of RNA polymerase II transcription.

Saccharomyces cerevisiae Metabólica de rotulagem com 4-thiouracil e a quantificação dos recém sintetizado mRNA como um Proxy para a RNA polimerase II atividade

Global defects in RNA polymerase II transcription might be overlooked by transcriptomic studies analyzing steady-state RNA. Indeed, the global decrease in ...

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and chronological life spans are two established paradigms used to study aging in yeast. Replicative aging is defined as the number of daughter cells a single yeast mother cell produces before senescence; chronological aging is defined by the length of time cells can survive in a non-dividing, quiescence-like state 2. We have developed a high-throughput method for quantitative measurement of chronological life span. This method involves aging the cells in a defined medium under agitation and at constant temperature. At each age-point, a sub-population of cells is removed from the aging culture and inoculated into rich growth medium. A high-resolution growth curve is then obtained for this sub-population of aged cells using a Bioscreen C MBR machine. An algorithm is then applied to determine the relative proportion of viable cells in each sub-population based on the growth kinetics at each age-point. This method requires substantially less time and resources compared to other chronological lifespan assays while maintaining reproducibility and precision. The high-throughput nature of this assay should allow for large-scale genetic and chemical screens to identify novel longevity modifiers for further testing in more complex organisms.

Chronological aging in yeast refers to the loss of cell viability associated with time in stationary phase. Here we describe a high-throughput method for quantitatively determining yeast chronological life span.

Quantificar Yeast Life Span cronológica por conseqüência de células envelhecidas

The budding yeast Saccharomyces cerevisiae has proven to be an important model organism in the field of aging research 1. The replicative and ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

Rotulagem e Visualização de Produtos de Expressão do Genoma Mitocondrial no Saccharomyces cerevisiae de Levedura de Baker

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Bioquímica

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Quantitativas de células vivas de Análise de microscopia de fluorescência de levedura

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

Aquisição de fluorescência Filmes Time-lapse da levedura de brotamento e análise da dinâmica de células simples utilização de enxertos

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Imagiologia reorganização espacial de uma via de sinalização MAPK Utilizar o sistema de expressão transitória do Tabaco

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

Análise espacial e temporal da Cytokinetic Eventos na levedura de fissão

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...