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。

<table><tbody><tr><td>Nocodazole</td><td>Reagent</td><td>Sigma-Aldrich</td><td>M1404</td><td></td></tr><tr><td>Methylene Blue 1% (w/v)</td><td>Reagent</td><td>VWR international</td><td>VW6276-0</td><td></td></tr><tr><td>Calcofluor White</td><td>Reagent</td><td>Sigma-Aldrich</td><td>F3543</td><td></td></tr><tr><td>Petroleum Jelly</td><td>Reagent</td><td>VWR international</td><td>VW3339-2</td><td></td></tr></tbody></table>

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

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 &mu;m). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.

We describe here the operation of a microfluidic device that allows continuous and high-resolution microscopic imaging of single budding yeast cells during their complete replicative and/or chronological lifespan.

在芽殖酵母连续高分辨率显微观察复制性衰老

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The ...

科研

生物工程

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

萌芽酵母酿酒酵母中软骨蛋白定位的评估

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

生物化学

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

生物学

Analysis of Lipid Droplet Content in Fission and Budding Yeasts using Automated Image Processing

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are used as models in lipid metabolic research or for industrial lipid production. Lipid droplets are highly dynamic storage bodies and their cellular content represents a convenient readout of the lipid metabolic state. Fluorescence microscopy is a method of choice for quantitative analysis of cellular lipid droplets, as it relies on widely available equipment and allows analysis of individual lipid droplets. Furthermore, microscopic image analysis can be automated, greatly increasing overall analysis throughput. Here, we describe an experimental and analytical workflow for automated detection and quantitative description of individual lipid droplets in three different model yeast species: the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus, and the budding yeast Saccharomyces cerevisiae. Lipid droplets are visualized with BODIPY 493/503, and cell-impermeable fluorescent dextran is added to the culture media to help identify cell boundaries. Cells are subjected to 3D epifluorescence microscopy in green and blue channels and the resulting z-stack images are processed automatically by a MATLAB pipeline. The procedure outputs rich quantitative data on cellular lipid droplet content and individual lipid droplet characteristics in a tabular format suitable for downstream analyses in major spreadsheet or statistical packages. We provide example analyses of lipid droplet content under various conditions that affect cellular lipid metabolism.

Here, we present a MATLAB implementation of automated detection and quantitative description of lipid droplets in fluorescence microscopy images of fission and budding yeast cells.

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

Lipid metabolism and its regulation are of interest to both basic and applied life sciences and biotechnology. In this regard, various yeast species are ...

免疫与感染

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

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

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

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

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

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 ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

裂殖酵母性生命周期的显微镜

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...

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, ...

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

摘要

探索更多视频

此视频中的章节

在芽殖酵母连续高分辨率显微观察复制性衰老

萌芽酵母酿酒酵母中软骨蛋白定位的评估

酵母殖民地嵌入方法

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

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

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型

成长型测定和遗传需求生化确认的蛋白质降解

裂殖酵母性生命周期的显微镜

ODELAY:用于酵母生长多参数定量的大规模方法

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

摘要

探索更多视频

此视频中的章节

在芽殖酵母连续高分辨率显微观察复制性衰老

萌芽酵母酿酒酵母中软骨蛋白定位的评估

酵母殖民地嵌入方法

使用自动图像处理分析裂变和萌芽酵母中的脂滴含量

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

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

使用 体内 成像筛选哺乳动物宿主活动性感染期间 白色念珠 菌突变菌株的形态发生表型

成长型测定和遗传需求生化确认的蛋白质降解

裂殖酵母性生命周期的显微镜

ODELAY:用于酵母生长多参数定量的大规模方法

使用体内成像筛选哺乳动物宿主活动性感染期间白色念珠菌突变菌株的形态发生表型