我们感谢布莱恩G.库恩和克劳迪亚B。汉纳有益的讨论和支持。这个项目是从DEP的启动资金支持。生物科学，普渡大学的R.克劳己立和美国癌症协会的机构研究资助通过普渡大学癌症中心的R.克劳阿吉拉尔。

A.细胞培养和蛋白质感应本节介绍了细胞培养条件下，从regulatable启动子在酵母细胞中蛋白表达诱导细胞周期同步，与噻氨酯哒唑。 <ol><li>在野生型酵母株W303（LEU2 - 3，112 TRP1 - 1 CAN1 - 100 URA3 - 1 ADE2 - 1 HIS3 - 11，15）是转变高复制（2μ）的质粒DNA，包含的控制下我们感兴趣的基因（ENTH2）蛋氨酸repressible子（MET25）。 2MM蛋氨酸足以抑制启动子活性，而缺乏蛋氨酸的媒体允许的最大表达。因此，蛋氨酸浓度范围内可用于表达蛋白质的理想水平。 1 W303细胞的质粒DNA转化LiAc - TE的方法是使用按标准程序。 注意:此试验应执行中存在2mm的蛋氨酸，以确保只在背景水平蛋白表达的细胞生长隔夜。第二天早晨，蛋白表达应引起resuspending细胞缺乏蛋氨酸紧随细胞周期同步（约4小时）的媒体 。 然而，我们发现，ENTH2依赖细胞分裂表型诱导后，才在相当集中的ENTH2已建成的细胞（通常在4 - 6H蛋白诱导3），使所有表现形式的分析日久非常冗长（至少16小时）的表型。我们发现，越来越多的细胞在0.2MM蛋氨酸的存在一夜之间压抑形态的表型，但允许亚临界蛋白浓度，可以迅速提高到表型诱导水平转移到媒体缺乏蛋氨酸。 这允许在相对 ​​较短的时间内细胞分裂缺陷严重的表现形式进行详细的分析 。 因此，我们建议实验者确定蛋氨酸的浓度是在逐案基础上的表型诱导的亚阈值。 </li><li>挑选一个含有50毫升蛋氨酸在灭菌的锥形烧瓶中使用标准的无菌技术与0.2MM的选择性培养基板最近转化的细胞和接种的6个殖民地。孵育过夜30 ° C的晃动在200转（标准的酵母培养条件的详细信息，参见文献4） </li><li>第二天早上，估计在600nm处测量光密度的文化细胞密度。转移到无菌的离心管和收获相当于20 外径600nm处的细胞在室温下为5分钟旋转2200转。 </li><li>在悬浮细胞YPD在0.4 OD 600nm处 /毫升的浓度，并添加到终浓度为15μg/ml的股票从DMSO为1mg/ml噻氨酯哒唑。在30℃孵育4h后晃动在200rpm。 注:可用于替代逮捕的方法，包括在使用α-因子的G0期或G1 - S期使用羟基脲逮捕。我们已经发现，在有丝分裂逮捕，用噻氨酯哒唑是简单而有效的，对于我们的目的的。 </li><li> 4h后，检查被捕的有丝分裂细胞计数与相同大小的芽细胞的数量，在显微镜下观看的百分比。如果逮捕的是大于90％，继续以下步骤。否则，维持细胞在噻氨酯哒唑，直到实现所需的百分比有丝分裂逮捕。 </li><li>在2200转5min后，用冰水洗脸三次，离心收集细胞。最后一次洗涤后，重悬沉淀〜0.5 OD 600nm处 / ml的细胞密度在缺乏蛋氨酸选择性媒体。转移到一个新的无菌管细胞体积的一半，最后的0.2mm的浓度和添加蛋氨酸。这将作为一个控制蛋白表达的基础水平的影响。这标志着实验（时间= 0H）第一次点。分析细胞每隔一小时为6H。 </li></ol> B.分析表型发展 注意:下面描述的所有的分析方法并不需要同时进行 。 Western印迹法测定蛋白质表达水平<ol><li>在每个时间点，在2200转离心5分钟测量的文化和收获2外径600nm处等值细胞的OD 600nm处。 </li><li>在1毫升无菌水重悬沉淀和暂停转移到1.5ml的Eppendorf管中。离心重复获得沉淀。吸出上清液。 </li><li> 50μL的SDS - PAGE Laemmli样品缓冲液（50MM三pH值6.8，2％SDS，10％甘油，2％β-巯基乙醇和0.006％溴酚蓝）重悬沉淀。加入50μL酸洗玻璃珠（直径0.2-0.4微米）和1分钟的旋涡。 </li><li>在95℃加热BLO煮沸管CK为2分钟，旋涡，再次为30秒，恢复煮沸5分钟。冻结在-20 ° C的蛋白质样品</li><li>运行在12％SDS - PAGE凝胶转移到硝酸纤维素膜和蛋白质样品进行西医结合相应的抗体杂交检测感兴趣的蛋白质。在这个例子中，我们探测血凝素标记的鼠抗HA抗体（Covance公司，普林斯顿，新泽西州）的孵化1:2000稀释烂醉的吐温（1XPBS，5％的非脱脂奶粉ENTH域（"房委会"）的存在，0.1％吐温）。在1:2500稀释辣根过氧化物酶标记的羊抗鼠二抗（皮尔斯），也是在烂醉的吐温，允许绑定上半年的膜在室温下的主要抗体。的Supersignal西Pico化学发光底物是用来检测辣根过氧化物酶标记的抗鼠抗体。 </li><li>光密度量化蛋白表达水平。 </li></ol>本节介绍两种方法跟踪蛋白浓度增加的功能表型的发展。第一种方法是细胞人口为基础的，而第二个研究它在单个细胞的水平。 量化表型的细胞分裂和细胞死亡<ol><li>在每一个时间点，转移1毫升细胞悬液以无菌Eppendorf管中，在2200转离心5分钟使用无菌技术和收获细胞。 </li><li>吸上清颗粒，悬浮在70μl媒体30μl甲基蓝从无菌水的股票为1mg/ml亚甲蓝是一个至关重要的蓝色染料，因此死亡的细胞可以通过他们的蓝色确定细胞质减少环境变得无色。 </li><li>移液器〜6μl这种悬挂在洁净的载玻片和盖玻片放置在下降。轻轻按下盖玻片，形成一层很薄的一层均匀，细胞之间的玻璃接口。 </li><li>分成9象限盖玻片，并采取3个象限的图像，如图所示。 2。细胞/场的数量应不超过30个细胞，以便准确定量。约15分钟可成像每个盖玻片，气泡破坏前的准备。 </li><li>统计显示所研究的表型的细胞数量。后ENTH2过度表达引起的细胞分裂的缺陷，导致细胞死亡，因此，我们也算他们的蓝色标识的死细胞的数量。 </li><li>计算表型细胞的百分比。 </li><li>绘制如图所示的数据。 3。 </li></ol>用荧光显微镜的特定表型缺陷的可视化有缺陷的细胞，细胞分裂ENTH2过度表达时，往往细胞壁缺陷和septin mislocalization3（GFP融合蛋白的可视化）的特点。芽殖酵母的细胞壁可以很容易地Calcofluor白（CFW）不可逆转地结合甲壳素在酵母细胞壁染色观察。 <ol><li>在每一个时间点，文化等分在无菌Eppendorf管中，并在室温下5分钟2200转离心收获细胞1毫升。 </li><li> 1mg/ml的无菌水，并培育CFW溶液100μL重悬细胞沉淀在室温下5分钟，在黑暗中。 </li><li>离心收集细胞，用1X PBS洗3次。 </li><li>在100X的放大倍率，重悬于100μL1X PBS颗粒和使用紫外线光学显微镜下的图像（细胞壁）和FITC过滤器（GFP - septin）。 </li><li>通过以下步骤4-6在第B.2.1节细胞的百分比量化细胞壁缺陷和septin mislocalization </li></ol> B.3。在单细胞水平测定表型的发展:定时视频显微镜。 <ol><li>用70％酒精清洁玻片上，一个凹不景气使空气干燥。 </li><li> 5毫升选择性培养基，缺乏蛋氨酸在微波无菌玻璃试管，直到琼脂糖完全溶解，煮沸0.06克琼脂糖。 </li><li>快速吸管琼脂糖200μL包含在幻灯片中抑郁症的媒体和反转另一块干净的载玻片，确保被困于车底，无气泡。 </li><li>凝胶固化时，在上面移动幻灯片顺利，远离抑郁症的幻灯片，在任何时候都保持其表面平行。这将确保琼脂糖床表面与表面抑郁幻灯片平齐。清洁幻灯片周边地区琼脂糖床用细腻的组织。幻灯片是现在可以使用了。 </li><li>在时间为4小时，从文化细胞转移到无菌Eppendorf管中，并在室温下5分钟2200转离心收获1毫升。 注:建议以后的时间点选择，以保证蛋白的表达水平高，使表型的发展是保证 。 试点实验，以确定E对诱导时间的案件逐案基础上。 </li><li>吸新鲜的选择性培养基，缺乏蛋氨酸100μL上清，悬浮细胞。 </li><li>凡士林盖玻片边缘。然后下降琼脂糖床中央放置一个细胞悬液和扩散均匀，轻轻按压盖玻片。 </li><li>用凡士林密封盖玻片边缘和修复衬里用指甲油的边缘地位。等待，直到指甲油变干。 </li><li>放置在显微镜加热阶段的幻灯片，然后选择一个字段，它包含足够的间隔不超过10个细胞。随着细胞分裂时，该领域将随着时间的推移显得很拥挤。 </li><li>以一个领域的形象〜5小时，每5分钟。表型的进展，可组装成的图像，使用ImageJ软件（ <a name="cite">电影Rasband，WS，ImageJ，美国，马里兰州贝塞斯达，美国，http://rsb.info.nih.gov/ij/，1997年国家</a>卫生研究院的研究<a name="cite">-2005</a> ） </li></ol> 请注意:我们使用ZEISS AXIOVERT 200M显微镜，蔡司Axiocam MRM单色数码相机和卡尔蔡司Axiovision图像采集软件（4.4版）为活细胞成像。温度控制是通过将保持在30 ° C的PeCon Tempcontrol 37设备由一个加热阶段的幻灯片 。 为了避免再反复调整的重点，这是方便编程的图像采集软件，在每一个时间点自动获取数Z - Stack的图像和装配动画时，请选择最佳的聚焦图像。 一些图像采集软件的自动对焦时间课程实验的选项 。 这是方便使用该选项时可用。 野生型酵母细胞生长最好在30 °彗星，因此温度控制是至关重要的。为了保证足够的热量，这是有助于提供一个外部的热量来源，在加热阶段 。 例如，我们离开对整个工期的成像显微镜传播的白光 。 这也很关键，以避免气泡时准备琼脂糖床和细胞夹心。随着时间的推移加热时，往往以推动细胞成像领域内的气泡夹带的空气膨胀。 同样重要的是，以确保该领域内的单个细胞职位没有变化明显，在成像过程的持续时间，尤其是在试图重新聚焦。 影像稳定器插件是可用的，ImageJ到整顿后的影片已被组装在单元格的位置小变化。 （ <a href="http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html">http://www.cs.cmu.edu/〜康丽/代码/ Image_Stabilizer.html</a> ）。

<ol>
	<li>R&#246;nicke, V., Graulich, W., Mumberg, D., M&#252;ller, R., Funk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+conditional+promoters+for+expression+of+heterologous+proteins+in+Saccharomyces+cerevisiae.">Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae.</a> Methods Enzymol. 283, 313-322 (1997).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> High efficiency transformation with lithium acetate in Molecular Genetics of Yeast: A Practical Approach. , 121-134 (1994).</li><li>Mukherjee, D., Coon, B. G., Edwards, D. F., Hanna, C. B., Longhi, S. A., McCaffery, J. M., Wendland, B., Retegui, L. A., Bi, E., Aguilar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+endocytic+protein+Epsin+2+functions+in+a+cell-division+signaling+pathway.">The yeast endocytic protein Epsin 2 functions in a cell-division signaling pathway.</a> J Cell Sci. 122, 2453-2463 (2009).</li><li>Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Getting started with yeast in Guide to Yeats genetics and Molecular Biology. , 3-21 (1991).</li></ol>

本协议描述了如何监督的形态学表型蛋白表达（酵母细胞无法进行正常的细胞分裂）后的发展。执行此过程时，重要的是要记住造粒建议离心速度更快的速度可能会损伤细胞和晦涩的结果收获的酵母细胞。应增加活细胞成像前，因为它们是有毒的亚甲基蓝和Calcofluor白色。此过程也可以很容易地适应蛋白质镇压条件下观察到表型的目标是从一个可控的发起人表示。

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analysis of the development of a morphological phenotype as a function of protein concentration in budding yeast

基因缺失和蛋白表达研究蛋白质功能的常用方法。在这篇文章中，我们描述了作为一个人口功能的蛋白浓度和单细胞水平分析表型的发展协议<em&gt;酿酒酵母</em&gt;。虽然此协议基础上的一种蛋白质的过度表达，它可以很容易地适应依赖于抑制蛋白表达的形态学表型。我们的实验室是在兴趣学习吞适配器蛋白epsin的信号。为了这个目的，我们使用了一个占主导地位的消极做法，在我们过度表达的保守<u&gt;电子</u&gt; psin<u&gt; N</u&gt; -<u&gt; T</u&gt; erminal<u&gt; H</u&gt; omology（ENTH）域，以干扰内源性epsin - 2（Ent2或YLR206W）的功能。我们观察到，在野生型细胞，一个细胞分裂的缺陷，是依赖于一个家庭的骨架蛋白，septins mislocalization Ent2（ENTH2）ENTH域的过度表达。

Watch this Scientific Journal Video about Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast at JoVE.com

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

基因缺失和蛋白表达研究蛋白质功能的常用方法。在这篇文章中，我们描述了作为一个人口功能的蛋白浓度和单细胞水平分析表型的发展协议<em&gt;酿酒酵母</em&gt;。

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

analysis-development-morphological-phenotype-as-function-protein

Research

JoVE Journal

Biology

10.7K 次观看.  Purdue University. 基因缺失和蛋白表达研究蛋白质功能的常用方法。在这篇文章中，我们描述了作为一个人口功能的蛋白浓度和单细胞水平分析表型的发展协议酿酒酵母。

视频: Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Use of Rotorod as a Method for the Qualitative Analysis of Walking in Rat

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a rotorod, animals remain in approximately the same place making repetitive movements of stepping. Thus the task provides a rich source of information on the details of foot stepping movements. Subjects were hemi-Parkinson analogue rats, produced by injection of 6-hydroxydopamine (6-OHDA) into the right nigrostriatal bundle to deplete nigrostriatal dopamine (DA). The present report provides a video analysis illustration of animals previously were filmed from frontal, lateral, and posterior views as they walked (15). Rating scales and frame-by-frame replay of the video records of stepping behavior indicated that the hemi-Parkinson rats were chronically impaired in posture and limb use contralateral to the DA-depletion. The contralateral limbs participated less in initiating and sustaining propulsion than the ipsilateral limbs. These deficits secondary to unilateral DA-depletion show that the rotorod provides a use task for the analysis of stepping movements.

The rotorod test is used to assess motor status in the walking movement of hemi-Parkinson analogue rats.

走在大鼠作为定性分析方法使用Rotorod

High speed videoanalysis of the details of movement can provide a source of information about qualitative aspects of walking movements. When walking on a ...

科研

生物学

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

正确的蛋白浓度的动力学结构与功能分析和亲和力测定的重要性

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

肾单位的组成与功能的成年斑马鱼肾脏分析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

高分辨率成像和芽殖酵母个人星体微管动力学分析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...