Trabajo en el laboratorio se Duennwald apoyado por becas de la Federación Americana para la Investigación del Envejecimiento (AFAR), la Fundación de Enfermedades Hereditarias (HDF) y la Fundación William Wood.

1. Expresión de Tóxicos de expansión polyQ-proteínas en la levadura Un análisis sistemático ha establecido la secuencia precisa de aminoácidos de una proteína polyQ-expansión que se requiere para producir toxicidad en la levadura 7. Este tóxico polyQ-expansión proteína contiene un amino-terminal-FLAG etiqueta seguido por 17 aminoácidos de la secuencia original de la proteína huntingtina, una región polyQ, y una fusión carboxi-terminal a una proteína fluorescente (GFP o bien PPC, véase la Figura 1 a). La expresión de las proteínas con las expansiones polyQ de 46 glutaminas o más (por ejemplo, 72 y 103 glutaminas) produce toxicidad en la levadura cuando se expresa bajo el control de la inducible y relativa fuerte promotor GAL1 7, 9. Como se describió anteriormente, la toxicidad polyQ en la levadura es sólo aparente en las células que llevan el Rnq1p proteína prión en su conformación, [RNQ +], por ejemplo, la cepa de levadura W303 9. El tóxico polyQ expansión de proteínas recapitulates aspectos centrales de la polyQ-biología en la levadura, tales como polyQ duración que dependen de la toxicidad (ver abajo) y la agregación (Figura 1 B). En particular, tóxicos expansión polyQ proteínas no matan a las células de levadura de inmediato, sino que más bien poner en peligro o detener el ciclo celular y devision celular, por lo tanto disminuir o inhibir el crecimiento de colonias de levaduras en las placas o cultivos líquidos (nuestros datos no publicados). 2. Posibles problemas con las células de levadura que expresan tóxicos de expansión polyQ proteínas Las células de levadura que expresan las proteínas polyQ otra tóxicos de expansión pueden no mostrar ningún defecto de crecimiento 9. La naturaleza genética de estos supresores de polyQ-toxicidad no parece estar basado en simples mutaciones mendelianas y por lo tanto puede ocurrir con una frecuencia relativamente alta (nuestros resultados no publicados). Los autores especulan que estos supresores espontáneos son causados ​​por el endurecimiento de los priones no identificadas que funcionan de manera similar a [RNQ +] en la determinación de toxicity.Thes polyQe supresores espontáneos polyQ de toxicidad puede poner en peligro el éxito de cualquier experimento que tiene como objetivo caracterizar la toxicidad polyQ o para identificar y caracterizar los modificadores de la toxicidad polyQ. Con el fin de evitar la ocurrencia frecuente de estos supresores espontáneas, seguir las medidas de precaución se describen a continuación, que han demostrado ser muy eficaz: <ol><li> Usar células frescas de levadura para los experimentos de toxicidad. No almacenar levadura durante períodos prolongados de tiempo. Frecuentemente recuperar las colonias de levadura fresca de material congelado. </li><li> Frecuentemente controlar la expresión y la agregación de expansión tóxicos polyQ-proteínas por microscopía de fluorescencia. </li><li> Mantener las células de levadura en los medios que reprimen la expresión de la toxicidad tóxico polyQ en todo momento (es decir, en medios selectivos que contienen glucosa como única fuente de carbono) antes de iniciar cualquier medición de toxicidad. </li><li> Use al menos tres transformantes independientes para cada experimento polyQ toxicidad. &lt;/ Li&gt; </ol> 3. Los ensayos de crecimiento <ol><li> Para cada condición experimental, inocular una colonia de cada tres transformantes independientes de células de levadura que albergan el tóxico polyQ-expansión de proteínas en 3 ml de medio de levadura selectivos con glucosa como única fuente de carbono. </li><li> Incubar estas culturas durante la noche a 30 ° C. Bajo estas condiciones, las células no se expresa la proteína polyQ-expansión debido a que la glucosa en el medio reprime su expresión. No deje que estos crecen en exceso las culturas; mantener el OD 600 (de absorción de luz de longitud de onda de 600 nm) de los cultivos de una noche por debajo de 1. </li><li> Habitualmente usamos tres ensayos diferentes para controlar los defectos de crecimiento de las células de levadura que expresan el tóxico polyQ expansión de proteínas: </li></ol> 3,1 Crecimiento en placas <ol><li> Diluir los cultivos de levaduras durante la noche (cultivados con agitación 220 rpm) hasta una DO 600 de 0.0005 (es decir, una dilución 1:1000 de un OD 600 = 0,5 la cultura) en selectiva mídia 7 de glucosa que contiene. </li><li> Repartir uniformemente 50 l (que resulta en ca. 700 colonias por placa) de cada cultivo diluido en una placa (10 cm de diámetro) con medio selectivo que contiene glucosa como única fuente de carbono y una placa con medio selectivo que contiene galactosa como única fuente de carbono. </li><li> Incubar las placas durante tres o cuatro días a 30 ° C. Después de la incubación, tomar fotografías de cada placa y contar el número de colonias en la glucosa y el número de colonias en las placas de galactosa. Bajo condiciones ideales, no debe haber ninguna o sólo muy pocas colonias en la placa de galactosa utilizando las células de levadura que expresa un altamente tóxico polyQ-expansión proteína (103 octodecies, la Figura 2a). </li></ol> 3.2 Los ensayos Spotting Este ensayo es más cuantitativa que el ensayo de placas se ha descrito anteriormente y por lo tanto puede revelar incluso las diferencias sutiles en la toxicidad polyQ con el mismo experimento en la misma placa. <ol><li> Diluir el Overnculturas ight crecer en medio con glucosa a una DO 600 de 0,1. </li><li> Pipetear 200 l de estas culturas estériles diluidas en placas de 96 pocillos y preparar cinco diluciones en serie cinco veces en agua estéril utilizando una pipeta multicanal. </li><li> Usando un Frogger, (también llamado un observador, con 8 x 6 pines para la transferencia de las suspensiones celulares) transferir las suspensiones de células sobre placas que contienen medios selectivos con glucosa como única fuente de carbono y placas que contienen medios selectivos con galactosa como única fuente de carbono. </li><li> Dejar que las placas se seque antes de su incubación a 30 ° C durante tres a cuatro días. </li><li> Después de la incubación, tome fotografías de cada plato (Figura 2b). </li></ol> 3,3. Seguimiento de la toxicidad polyQ en la levadura por el crecimiento de cultivos líquidos Este protocolo es el más cuantitativo (OD 600 números) de los tres ensayos descritos aquí y puede incluso detectar diferencias muy sutiles en la toxicidad polyQ. Lo anterior ocCurrence de supresores de espontáneos, sin embargo, lo que potencialmente puede producir resultados engañosos. Por lo tanto, recomendamos que la combinación de este ensayo con al menos uno de los dos ensayos de galvanoplastia descritos anteriormente. Se propone utilizar el instrumento BioscreenC para estos experimentos. El BioscreenC es un instrumento que mide automáticamente la densidad óptica de cultivos de levadura en 100-placas mientras se incuba a temperaturas definidas con agitación definido. Otros métodos para el cultivo de células de levadura y la medición de su densidad óptica también se pueden aplicar. <ol><li> Lavar las células de levadura de medio mínimo que contienen glucosa como única fuente de carbono tres veces en 3 ml de agua estéril. </li><li> Diluir los cultivos de una noche que crecen en medio con galactosa a un OD 600 de 0,1. </li><li> Llenar cada pocillo de la placa 100-BioscreenC bien con 300 l de los cultivos de levaduras. </li><li> Abra el programa Easy Experimento Bioscreen. Determinar el número de muestras que desea monitorear (incluyendo espacios en blanco y único medio-controles), ajustar la temperatura a 30 ° C, fijar la duración de los experimentos de 3 días, establezca los intervalos de medición de 15 minutos, configurar el filtro para 600 nm / Brown, y establecer el modo de agitación a 15 segundos antes de cada medición en el de tipo medio. </li><li> El instrumento BioscreenC y el software adjunto, va a producir hojas de cálculo Excel de cada punto de datos tomados durante el experimento. </li><li> Preparar las curvas de crecimiento con Excel para cada muestra y comparar el crecimiento de diferentes muestras (Figura 2c). Una descripción detallada del análisis de los datos producidos por los experimentos BioscreenC se ha proporcionado antes 10. </li></ol> 4. Los resultados representativos <img alt="Figura 1" src="/files/ftp_upload/3461/3461fig1.jpg" /> Figura 1. La levadura polyQ modelo. a) Representación esquemática de la expansión tóxica polyQ proteínas. b) La microscopia de fluorescencia que muestra las células de levadura que expresan un corto, no tóxico polyQlas células de proteínas de expansión (25Q, panel izquierdo) y la levadura que expresa una larga, tóxico-polyQ expansión de las proteínas (panel de 103 octodecies, derecha). <img alt="Figura 2" src="/files/ftp_upload/3461/3461fig2.jpg" /> Figura 2. Los resultados representativos de los ensayos de crecimiento de células de levadura que expresan expansión polyQ-proteínas. a) Revestimiento de ensayo. Aproximadamente 700 células de levadura se extendió en placas y se incuba durante tres días a 30 ° C. El panel superior muestra una placa que contiene medio de glucosa, es decir, la expresión de la tóxico-polyQ expansión proteína (103 octodecies) no es inducida. El panel inferior muestra una placa de levadura que contiene el medio galactosa, es decir, la expresión de la tóxico-polyQ expansión proteína es inducida. Nótese que en el experimento mostrado aquí, no se produjo supresor espontánea. B) Manchado ensayo. Cinco serie de cinco diluciones de células de levadura albergar ya sea una proteína no tóxica polyQ (25Q) o un tóxico Protei polyQ-expansiónn (103 octodecies) fueron detectados en las placas de glucosa que reprimen la expresión de las proteínas (panel izquierdo) o en placas de galactosa que inducen su expresión. Las placas se incubaron entonces durante tres días a 30 ° C. c) experimento BioscreenC. El crecimiento de cultivos de células de levadura que expresan bien una proteína no tóxica polyQ (25Q) o un tóxico polyQ-expansión proteína (103 octodecies) fueron controlados por el instrumento BioscreenC. Las condiciones experimentales y el análisis de los experimentos BioscreenC se describen en el texto principal.

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No hay conflictos de interés declarado.

Este manuscrito describe tres enfoques complementarios experimentales destinados a medir polyQ de toxicidad en el organismo modelo la levadura sobre la base de la reducción del crecimiento de células de levadura que expresan tóxicos de expansión polyQ proteínas. Trabajo en la levadura se ofreció reflexiones profundas sobre los mecanismos celulares y moleculares básicos de mal plegamiento de proteínas y su toxicidad resultante, incluyendo el mal plegamiento y la toxicidad de las proteínas de expansión poly...

<table border="1"><tbody><tr><td> Nombre del instrumento </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Frogger (6x8 pines) </td><td> V &amp; P Científica, en San Diego </td><td> VP 407 AH </td></tr><tr><td> BioscreenC </td><td> Growthcurves EE.UU. </td><td> 5101370 </td></tr><tr><td> 100 y placas alveolares </td><td> Growthcurves EE.UU. </td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

Mal plegamiento de proteínas se asocia con muchas enfermedades humanas, especialmente enfermedades neurodegenerativas, como la enfermedad de Alzheimer, la enfermedad de Parkinson y la enfermedad de Huntington 1. La enfermedad de Huntington (HD) es causada por la expansión anormal de una poliglutaminas (polyQ) región dentro de la huntingtina proteína. La proteína huntingtina polyQ expandido alcanza una conformación aberrante (es decir, misfolds) y causa la toxicidad celular 2. Al menos ocho enfermedades neurodegenerativas adicionales son causados ​​por las expansiones polyQ, incluyendo la espinocerebelosa Ataxias y la enfermedad de Kennedy 3. El organismo modelo la levadura ha facilitado una mejor aproximación a las bases celulares y moleculares de polyQ toxicidad, incluyendo el impacto de los factores intra-e inter-molecular de polyQ toxicidad, y la identificación de las vías celulares que se dañan en las células que expresan polyQ expansión proteínas 3-8. Importantemente, muchos aspectos de la polyQ de toxicidad que se encuentran en la levadura se reproduce en otros sistemas experimentales y en cierta medida en las muestras de los pacientes en HD, lo que demuestra la importancia del modelo de levadura para el descubrimiento de los mecanismos básicos que sustentan polyQ toxicidad. Una forma directa y relativamente simple para determinar polyQ de toxicidad en la levadura es la medición de los defectos de crecimiento de las células de levadura que expresan las proteínas polyQ expansión. Este manuscrito describe tres enfoques complementarios experimentales para determinar polyQ-toxicidad en la levadura mediante la medición del crecimiento de células de levadura que expresan expansión polyQ-proteínas. Los dos primeros métodos experimentales monitorear el crecimiento de levaduras en las placas, el tercer enfoque monitorea el crecimiento de cultivos de levaduras líquidas utilizando el instrumento BioscreenC. Por otra parte, este manuscrito se describen las dificultades experimentales que pueden ocurrir cuando el manejo de los modelos de levadura polyQ y describe las estrategias que ayuden a evitar ominimizar estas dificultades. Los protocolos descritos aquí se pueden utilizar para identificar y caracterizar vías genéticas y pequeñas moléculas que modulan polyQ-toxicidad. Por otra parte, los ensayos descritos pueden servir de modelo para análisis precisos de la toxicidad causada por otras enfermedades asociadas a proteínas mal plegadas en los modelos de la levadura.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

Este trabajo describe tres protocolos complementarios para evaluar la toxicidad de poliglutaminas (polyQ)-expansión proteínas en la levadura<em&gt; Saccharomyces cerevisiae</em&gt;. Estos protocolos pueden ser fácilmente modificado para controlar la toxicidad de otras proteínas plegadas incorrectamente en la levadura.

Los ensayos de crecimiento para evaluar la toxicidad de poliglutaminas en la levadura

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

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Research

JoVE Journal

Biology

13.5K vistas.  Boston Biomedical Research Institute. Este trabajo describe tres protocolos complementarios para evaluar la toxicidad de poliglutaminas (polyQ)-expansión proteínas en la levadura Saccharomyces cerevisiae. Estos protocolos pueden ser fácilmente modificado para controlar la toxicidad de otras proteínas plegadas incorrectamente en la levadura.

Video: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identificación de los fenotipos de inhibición del crecimiento inducida por la expresión de tipo bacteriana efectores III en la levadura

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Investigación

Biología

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Modificado levadura sistema híbrido de dos para identificar proteínas que interactúan con el factor de crecimiento progranulina

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Métodos para evaluar subcelulares compartimentos de músculo en<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...

Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 and minimum levels of 0.03 ng ml&#8722;1) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.

The proposed work assesses the diagnostic potential of direct and amplified surface plasmon resonance imaging (SPRi) assays, particularly for the detection of recombinant human growth hormone in spiked human serum, by comparing SPRi results directly with commercially available enzyme-linked immunosorbent assay (ELISA) kit.

Análisis comparativo de la hormona de crecimiento humano en suero utilizando SPRI, Nano-SPRI y ELISA Ensayos

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, log-phase, and stationary-phase. Each growth phase can reveal different aspects of fitness that are related to various environmental and genetic conditions. High-resolution and quantitative measurements of all 3 phases of growth are generally difficult to obtain. Here we present a detailed method to characterize all 3 growth phases on solid media using an assay called One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). ODELAY quantifies growth phenotypes of individual cells growing into colonies on solid media using time-lapse microscopy. This method can directly observe population heterogeneity with each growth parameter in genetically identical cells growing into colonies. This population heterogeneity offers a unique perspective for understanding genetic and epigenetic regulation, and responses to genetic and environmental perturbations. While the ODELAY method is demonstrated using yeast, it can be utilized on any colony forming microorganism that is visible by bright field microscopy.

We present a method for quantifying growth phenotypes of individual yeast cells as they grow into colonies on solid media using time-lapse microscopy termed, One-cell Doubling Evaluation of Living Arrays of Yeast (ODELAY). Population heterogeneity of genetically identical cells growing into colonies can be directly observed and quantified.

ODELAY: Un método a gran escala para la cuantificación multiparamétrica del crecimiento de la levadura

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Efecto de proteínas fluorescentes de fusión socios en ensayos de toxicidad de poliglutaminas de levadura

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm cell to penetrate the zona pellucida and fertilize the egg. It is therefore of great interest to test compounds (e.g., environmental chemicals or drug candidates) for their effect on Ca2+-signaling and acrosome reaction in human sperm either to examine the potential adverse effects on human sperm cell function or to investigate a possible role as a contraceptive. Here, two medium-throughput assays are described: 1) a fluorescence-based assay for assessment of effects on Ca2+-signaling in human sperm, and 2) an image cytometric assay for assessment of acrosome reaction in human sperm. These assays can be used to screen a large number of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm. Furthermore, the assays can be used to generate highly specific dose-response curves of individual compounds, determine potential additivity/synergism for two or more compounds, and to study the pharmacological mode of action through competitive inhibition experiments with CatSper inhibitors.

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Here, two medium-throughput assays for assessment of effects on Ca2+-signaling and acrosome reaction in human sperm are described. These assays can be used to quickly and easily screen large amounts of compounds for effects on Ca2+-signaling and acrosome reaction in human sperm.

Rendimiento medio de selección de ensayos para la evaluación de efectos sobre la Ca2 +-señalización y reacción acrosómica en espermatozoides humanos

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

Examen de la dinámica nuclear de levadura de ficción mitotica y meiótica por microscopía de células vivas de fluorescencia

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

Medición de la toxicidad del péptido RAN en C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...