衰老研究（远方），遗传性疾病基金会（高密度纤维板）和威廉·伍德基金会从美国联邦赠款支持在Duennwald实验室工作。

1。 polyQ蛋白膨胀有毒蛋白在酵母中的表达系统分析建立了一个polyQ蛋白膨胀蛋白所必需的生产在酵母中的毒性7精确的氨基酸序列。这种有毒的polyQ蛋白膨胀蛋白包含一个氨基末端的标志，标签从原始序列的亨廷顿蛋白质，polyQ蛋白地区和羧基端融合荧光蛋白（GFP或CFP资格由17个氨基酸，见图1 A）。表达的蛋白质与polyQ蛋白46 glutamines或更多扩展（例如72和103 glutamines的）生产酵母中的毒性控制下的诱导和相对强GAL1启动7，9时表示。 如前所述，polyQ蛋白在酵母中的毒性是唯一明显的细胞携带的朊蛋白构象的蛋白质Rnq1p [RNQ +]，如酵母菌株W303 9。有毒polyQ蛋白膨胀蛋白ŕecapitulates中央polyQ蛋白在酵母细胞生物学方面的，如polyQ蛋白长度依赖毒性（见下文）和聚集（图1）。值得注意的是，有毒polyQ蛋白膨胀蛋白不杀死酵母细胞立即而不是削弱或逮捕的细胞周期和细胞及试产，从而减缓或抑制酵母片或液体培养（未发表资料）殖民地的增长。 2。对有毒polyQ蛋白膨胀蛋白与酵母细胞的潜在问题 polyQ蛋白扩张，否则有毒蛋白质的酵母细胞中表达，可能不会显示任何增长的缺陷9。这些抑制器polyQ蛋白毒性的遗传性质，不会出现简单的孟德尔遗传突变的基础上，从而可以发生的频率比较高（未公布结果）。我们推测，这些自发的抑制是造成类似[RNQ]在确定polyQ蛋白toxicity.Thes功能不明的朊病毒固化polyQ蛋白毒性的é自发抑制器可危及任何实验，旨在刻画polyQ蛋白毒性或识别和描述polyQ蛋白的毒性修饰符取得圆满成功。 为了避免这些自发抑制频繁发生，请按照下述的预防措施，这已被证明是非常有效的： <ol><li>使用鲜酵母细胞毒性实验。不要长时间储存​​的酵母。经常从冷冻股票检索鲜酵母殖民地。 </li><li>经常监测荧光显微镜有毒polyQ蛋白膨胀蛋白的表达和聚集。 </li><li>保持在镇压开始前任何毒性测量polyQ蛋白的毒性在所有时代的毒性（即含葡萄糖为唯一碳源的选择性培养基）的表达媒体的酵母细胞。 </li><li>每个polyQ蛋白毒性实验中使用的至少有三个独立的转化。&lt;/ LI&gt; </ol> 3。生长实验<ol><li>对于每个实验条件下，接种一个殖民地polyQ蛋白膨胀在3毫升选择性酵母媒体有毒蛋白细胞窝藏葡萄糖作为唯一碳源的酵母从三个独立的转化。 </li><li>这些文化过夜孵育30°C。在这些条件下，细胞不表达polyQ蛋白膨胀蛋白，因为葡萄糖在中期抑制其表达。不要让这些文化长满;保持外径600（600 nm波长的光吸收）低于1隔夜文化。 </li><li>我们经常使用三种不同的化验，以监察表达有毒polyQ蛋白膨胀蛋白的酵母细胞的生长缺陷： </li></ol> 3.1平板上的生长<ol><li>选择性稀释隔夜酵母培养（220转震动成长）600 0.0005 OD（即一个OD600值= 0.5文化1:1000稀释）直径7含葡萄糖。 </li><li>均匀地涂抹上含有葡萄糖为唯一碳源的选择性介质和含半乳糖为唯一碳源的选择性培养基板一板（直径10厘米）50μl各摊薄文化的（CA 700菌落每盘）。 </li><li>在30°C孵育三到四天的板孵化后，每块板的照片和葡萄糖和半乳糖板菌落数菌落计数的数量。在理想的条件下，应该没有或只有很少的殖民地上的半乳糖板时，利用酵母细胞表达一种剧毒polyQ蛋白膨胀蛋白（103Q，图2a）。 </li></ol> 3.2合模分析此法比电镀法上面介绍更多的定量，从而可以揭示polyQ蛋白毒性哪怕是细微的差异与同一板块上的相同的实验。 <ol><li>稀释的overn生长在培养基葡萄糖0.1的外径600的飞行文化。 </li><li>吸取200μL无菌96孔板和这些摊薄文化的准备五个五倍串行无菌水稀释，使用多通道移液器。 </li><li>用青蛙过河（也称为去污剂与8×6针细胞悬液转移）转移到选择性培养基含葡萄糖作为唯一碳源和含半乳糖为唯一碳源的选择性培养基平板板的细胞悬液。 </li><li>允许板晾干后方可孵化30°Ç三到四天。 </li><li>孵化后，每块板的照片（图2b）。 </li></ol> 3.3。监测polyQ蛋白在酵母中的毒性液体培养的生长此协议是最定量这里描述的三个实验（OD600值的数字），甚至可以检测polyQ蛋白的毒性非常细微的差别。上述业主立案法团然而，自发抑制currence，可能产生误导的结果。因此，我建议至少有一个以上所述的两个电镀检测相结合的检测。我们建议使用这些实验BioscreenC仪器。 BioscreenC是仪器自动测量光密度在100孔板酵母培养，而培养与定义搅拌定义的温度。日益增长的酵母细胞，并测量其光密度等方法也可申请。 <ol><li>从最小的媒体含有葡萄糖作为唯一碳源，在3毫升无菌水三次洗的酵母细胞。 </li><li>稀释与半乳糖的外径600 0.1在培养基上生长的过夜培养。 </li><li>填补300μl酵母菌文化的每个井，100井BioscreenC板。 </li><li>打开简易BIOSCREEN实验方案。确定要监视（包括空白和中唯一的样本数量控制），设定温度至30°C，设置实验的长度为3天，设置了测量时间间隔为15分钟，设置过滤器以600海里/布朗，并设置每次测量前15秒震动模式中等强度。 </li><li>的BioscreenC文书和所附的软件会产生在实验过程中所采取的每个数据点的Excel电子表格。 </li><li>准备与Excel的生长曲线，每个样品和比较不同样本（图2c）的增长。 10日前已提供BioscreenC实验产生的数据进行分析的详细描述。 </li></ol> 4。代表结果<img alt="图1" src="/files/ftp_upload/3461/3461fig1.jpg" /> 图1。 polyQ蛋白酵母模型。荧光显微镜一）有毒polyQ蛋白膨胀的蛋白质，B的示意图），显示酵母细胞中表达了一个简短的，无毒的polyQ蛋白扩张蛋白（25Q，左侧面板）和酵母细胞长，有毒polyQ蛋白膨胀蛋白（103Q，右图）。 <img alt="图2" src="/files/ftp_upload/3461/3461fig2.jpg" /> 图2。代表表达polyQ蛋白膨胀蛋白质的酵母细胞的生长实验结果。一）电镀法。约700个酵母细胞伸长平板上，三天在30℃孵育上游面板显示板含葡萄糖培养基，即有毒polyQ蛋白膨胀蛋白（103Q）的表达， 不诱导。较低的面板显示含有半乳糖培养基酵母片，即有毒polyQ蛋白膨胀蛋白表达诱导 。请注意，这里显示实验中，没有发生自发抑制B）合模试验。五串行5倍稀释的酵母细胞，窝藏一个无毒polyQ蛋白蛋白（25Q）或有毒polyQ蛋白膨胀proteiN（103Q）被发现葡萄糖板压制表达蛋白（左侧面板）或半乳糖诱导其表达的板块。板，然后孵育3天30°C。C）BioscreenC实验。酵母细胞中表达一个无毒polyQ蛋白蛋白（25Q）或有毒polyQ蛋白膨胀蛋白（103Q）文化的增长，由监察BioscreenC仪器。在主文本描述的实验条件和分析的BioscreenC实验。

<ol>
	<li>Soto, C., Estrada, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+and+neurodegeneration.">Protein misfolding and neurodegeneration.</a> Arch. Neurol. 65, 184-189 (2008).</li><li>Ross, C. A., Tabrizi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+from+molecular+pathogenesis+to+clinical+treatment.">Huntington's disease: from molecular pathogenesis to clinical treatment.</a> Lancet Neurol. 10, 83-98 (2011).</li><li>Zoghbi, H. Y., Orr, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+repeats+and+neurodegeneration.">Glutamine repeats and neurodegeneration.</a> Annu. Rev. Neurosci. 23, 217-247 (2000).</li><li>Meriin, A. 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没有利益冲突的声明。

这手稿概述了三种互补的实验方法来衡量polyQ蛋白表达有毒polyQ蛋白膨胀蛋白的酵母细胞减少增长的基础上在模式生物酵母毒性。在酵母中的工作提供了深刻的见解成蛋白质错误折叠的基本细胞和分子机制及其随后的毒性，包括错误折叠和毒性polyQ蛋白膨胀蛋白9,11,12。在这里提出的协议为基础的实验已经帮助识别和描述polyQ蛋白毒性增强或抑制基因，polyQ蛋白毒性的小分子调节剂和细胞机制...

<table border="1"><tbody><tr><td> 仪器名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>青蛙过河（6X8针） </td><td> V＆P科学，圣地亚哥</td><td>副总裁407的AH </td></tr><tr><td> BioscreenC </td><td> growthcurves美国</td><td> 5101370 </td></tr><tr><td> 100蜂窝板</td><td> growthcurves美国</td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

蛋白质错误折叠与人类的许多疾病，特别是神经退行性疾病，如阿尔茨海默氏症，帕金森氏病，亨廷顿氏病1。亨廷顿氏病（HD），是由异常扩张的多聚谷氨酰胺（polyQ蛋白）区域内的蛋白质亨廷顿。亨廷顿蛋白质的polyQ蛋白膨胀达到异常的构象（即它错误折叠），并导致细胞毒性2。至少有八个进一步的神经退行性疾病造成polyQ蛋白的扩展，包括脊髓小脑性共济失调和肯尼迪的疾病3。 模式生物酵母促进polyQ蛋白毒性的细胞和分子基础的重要见解，包括polyQ蛋白毒性内和分子间的因素的影响，并在细胞受损的细胞通路的识别表达polyQ蛋白膨胀蛋白质3-8。重要polyQ蛋白毒性在酵母中发现的许多方面，LY，复制在其他的实验系统，并一定程度上在血液透析病人的样本，从而表明polyQ蛋白毒性支撑的基本机制的发现酵母模型的意义。 直接和相对简单的方法来确定polyQ蛋白在酵母毒性是衡量表达polyQ蛋白膨胀蛋白质的酵母细胞的生长缺陷。这手稿描述了三种互补的实验方法，确定酵母测量表达polyQ蛋白膨胀蛋白质的酵母细胞的生长，polyQ蛋白毒性。前两个实验方法监测酵母的生长，对板的第三种方法监测液体酵母文化使用BioscreenC文书的增长。 此外，这个手稿描述实验处理酵母polyQ蛋白模型时可能出现的困难，并概述了战略，这将有助于避免或最大限度地减少这些困难。这里所描述的协议，可以用来识别和特征的遗传途径和调节polyQ蛋白毒性的小分子。此外，所述的检测可以作为模板在酵母模型由其他疾病相关的错误折叠的蛋白质引起的毒性的准确分析。

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

这手稿描述了三个评估毒性的多聚谷氨酰胺（polyQ蛋白）扩张蛋白在酵母的补充协议<em&gt;酿酒酵母</em&gt;。这些协议可以很容易地进行修改，以监察在酵母中错误折叠的蛋白质的毒性。

生长实验，以评估在酵母中的多聚谷氨酰胺毒性

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

growth-assays-to-assess-polyglutamine-toxicity-in-yeast

Research

JoVE Journal

Biology

13.5K 次观看.  Boston Biomedical Research Institute. 这手稿描述了三个评估毒性的多聚谷氨酰胺（polyQ蛋白）扩张蛋白在酵母的补充协议酿酒酵母。这些协议可以很容易地进行修改，以监察在酵母中错误折叠的蛋白质的毒性。

视频: 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.

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

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 ...

科研

生物学

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.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

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.

致命的甲壳类动物长期毒性试验卤虫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.

方法来评估肌肉的亚细胞中<em&gt;温度。线虫</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.

人生长激素测定血清SPRI，纳米单层屋面和酶联免疫吸附测定比较分析

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：用于酵母生长多参数定量的大规模方法

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.

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

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.

中通量筛选法评估对精子中 ca2 +信号和反应的影响

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 ...

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.

测量在C.elegan的RAN肽毒性

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

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

使用原子力显微镜表征活植物器官中原代细胞壁的机械性能

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...