这项工作是从埃利森医学基金会授予MK和曼谷的支持。 Mk为中老年埃利森医学基金会新学者。我们想感谢拍摄期间Soumya Kotireddy援助。

第1部分：准备株的复制寿命分析和板本节介绍的复制寿命试验和酵母细胞寿命分析的准备使用固体YEPD板的准备。 <ol><li>使用适当的消毒技术，，准备将用于培养酵母细胞和复制寿命分析的YEPD培养基琼脂平板（1％酵母提取物，细菌用2％的蛋白胨，2％琼脂，2％葡萄糖）。你应该准备至少2板寿命实验分析，每4-5株。板应准备至少2天前寿命试验，寿命试验开始前，允许干。如果实验内不会4-5天浇板发起的，他们应该被放置在冷藏孵化器，并在封口膜包裹或塑料，以防止dessication。 </li><li>删除从冻结的股票，到YEPD培养基琼脂平板上的单菌落连胜酵母菌株。截至6株可以很容易地在同一YEPD板挑染划分为同样大小的饼形楔板。 </li><li> 2天，在30 ° C培养的细胞。 </li><li>删除到一个新的YEPD板的细胞的孵化器和修补细胞从一个单一的殖民地。两个补丁程序，从两个不同的菌落应为每一株产生。在这个时候，要分析的菌株，应进行编码，以确保复制寿命试验是“盲点”，其中的dissectors不知道个别菌株的身份。 </li><li>孵育30的补丁，编码细胞° C，直到第二天晚上。 </li><li>删除每个编码株的细胞和新鲜YEPD板轻轻修补细胞，这将作为复制寿命​​分析所用的实验板。应修补细胞沿着垂直线左侧YEPD板（图1）。约3-5％的补丁盘是最优的，假设月20日从每一个补丁细胞复制寿命分析。这是没有必要也不可取，转移大量的细胞，新鲜的YEPD板，因为这将导致细胞生长在厚厚的孵育过夜，这可能会限制随后将其复制寿命。 </li><li>孵育新鲜修补板台式过夜。 </li></ol>第2部分：位置细胞复制寿命分析。 在本节中，我们描述如何定位板的酵母细胞和如何获得维尔京子细胞复制寿命分析。从此时起，所有应parafilmed板，除了正在进行清扫时，为了防止dessication。 <ol><li>使用酵母适合用显微切割设备配备一台显微镜，约50细胞从第一个修补应变转移到一个盘上远端修补细胞的位置。如果您的显微镜阶段等级，这些等级可以使用，以确保细胞将在随后的迭代中很容易找到。在蔡司Axioscope 40，从第一个修补应变细胞可以放置在坐标90 × 10和垂直排列从那里。 </li><li>清洁针反复接触琼脂表面的细胞，然后通过在琼脂孔针强行通过琼脂表面。这个洞将成为盘上在解剖和女儿细胞去除后续迭代作为东方您的标记。小心不要让酵母细胞中的孔，或他们成长，并能形成一个菌落。 </li><li>正上方的孔，垂直对齐个体酵母细胞为20个均匀分布的位置，与1-3针直径（〜100微米）之间的每个单元格的位置（图1）。每隔几个细胞，放置在相同的位置，而不是一个行的两个彼此相邻的细胞。这使得冗余在维尔京子细胞的选择，如果一个失败转移细胞划分。 </li><li> 10 日和11 日行位置，创建一个水平的7-8孔的琼脂系列。这将作为一个板块在随后的迭代的解剖和女儿细胞去除标记东方您。小心不要让酵母细胞中的孔，或他们成长，并能形成一个菌落。 </li><li>重复步骤为每个盘上的补丁2.1-2.4，继续沿同一轴线垂直阵列细胞。步骤2.2中，创建的孔数应与补丁号（一杆进洞的第一个补丁，第二两个洞，等）。 </li><li>一旦细胞被排列为每个补丁，封口膜板和孵育30℃，约2小时。 </li><li>在实验中重复步骤每盘2.1-2.6。 </li></ol>第3部分：获取维尔京子细胞的寿命分析在本节中，我们开始寿命分析，确保每一个细胞开始处女女儿细胞。 <ol><li>从孵化器中取出的钢板和验证，大部分的摆着细胞都发生了细胞分裂。如果需要更多的时间来完成，让细胞分裂，返回板的孵化器。 </li><li>与第一摆着单元开始，用针轻轻地贴壁细胞上的针分离的子细胞从母细胞。它有时是非常有用挖掘显微镜底座的一边针细胞休息。 </li><li>将分离的子细胞中的垂直线，更换母细胞和任何其他的子细胞，并移动它们暂时搁置。 </li><li>为阵列的每个细胞，重复步骤3.2和3.3。如果一个细胞不分化，从一个多余的细胞定位在步骤2.3一个子细胞。完成时，你应该有20个细胞寿命板垂直对齐每个补丁的女儿。 </li><li>收集所有的母细胞和成桩的额外子细胞，他们转移到附近的一个面积的修补程序（“墓地”）。重要的是要保持远离细胞进行寿命分析，以防止形成microcolonies和当地的养分枯竭不需要的细胞。 </li><li>封口膜板和30 ° C孵育约2小时。 </li><li>在实验中重复步骤每盘3.2-3.6。 </li></ol>第4部分：测量单个细胞的复制能力<ol><li>准备寿命数据表。一个寿命数据表可以是一个简单的网格，每一行对应于一个单独的母细胞，每一列对应一个年龄点（图2）。标签每个数据表，根据要分析的菌株数。 </li><li>从孵化器中取出的钢板和验证摆着细胞大部分已经历了至少一个细胞分裂。如果需要更多的时间来完成，让细胞分裂，返回板的孵化器。 </li><li>从第一盘上的第一个摆着细胞，直观地观察母亲和女儿细胞（S），并确定已发生的细胞分裂数量。它通常是很容易确定母亲有多少女儿细胞产生的，基于特征的细胞安排模式。记录在适当的方格上的寿命评分表由母细胞产生的子细胞的数目。 </li><li>使用针分离的子细胞从母细胞，在步骤3.2中所述。 </li><li>保留其在垂直线的位置在母细胞和子细胞（S）移动暂时搁置。 </li><li>重复步骤4.3-4.5为阵列的每个细胞。 </li><li>收集所有的被丢弃的女儿细胞，将它们移动到“墓地”，原来的补丁附近。 </li><li>封口膜板和孵化在30 ° C时2-3小时。 </li><li>在实验中重复步骤每盘4.3-4.8。 </li><li>重复步骤4.2-4.9，直到所有的母细胞停止分裂。由于母细胞年龄，细胞周期进程变慢，这将是可取的孵化盘为年龄点之间的时间较长。板可放置在4 ° C过夜。 </li></ol>第5部分：代表性的结果。 一个复制的寿命实验中产生的原始数据列表中的数字对应每个母细胞产生的子细胞，在每个年龄段的点（图2）。通过总结评分表的每一行，每个母细胞复制寿命。可以汇集来自同一个实验组的母细胞的数据，生成一个生存曲线（图3A），并进行统计计算，如平均寿命，寿命中位数，和标准差，。可以执行一个非参数检验，如Wilcoxon秩和检验，以确定是否在寿命的显着性差异实验组之间的检测。当实验工作正常，生存曲线应大致与姜氏Makeham显示早期死亡率低的一个S形十八动力学一致的，在生存的较快下降的曲线的扁平化了后世。大多数野生型菌株跨度在20至30代的范围内复制生命值，虽然有报道此范围之外的变化。 <img src="/files/ftp_upload/1209/1209fig1.jpg" alt="图1" /> <img src="/files/ftp_upload/1209/fig1.2.jpg" alt="图1.5" /> 图1。一个典型的复制寿命板图 。 3-5株，晚上开始酵母细胞显微切割前轻轻修补到一个YEPD板（红色框）在一条垂直线上的表面沿板的一侧。增长通宵之后，大约30个人从每一株细胞排列垂直插入一个包含20个职位。生命跨度analysiS将在V处女女儿从这些初始细胞获得的细胞。从显微切割（如不需要的子细胞）在实验过程中获得了额外的细胞被放置在“墓地”（灰色椭圆形），它位于远离细胞实验，以避免当地的养分枯竭殖民地形成。 <img src="/files/ftp_upload/1209/1209fig3.jpg" alt="图2" /> 图2。一个复制的寿命理货表从一个复制的寿命实验的原始数据 。所示。每一行对应一个单一的酵母母细胞和每一列对应一个年龄点。在每个方块中输入的数字是在这个年龄点，母细胞产生的子细胞的数目。每一株都带一个编码等个人剖析的酵母细胞有没有被分析菌株的身份的知识。 20细胞测定每一株。 <img src="/files/ftp_upload/1209/1209fig2.jpg" alt="图3" /> 图3。代表生存和生长曲线，从复制的寿命实验（一）生存曲线绘制母细胞的一小部分仍然活着的复制岁功能（细胞分裂的次数。获得（二）通过绘制生长曲线获得年龄点的功能作为一个给定的应变产生的子细胞的平均数目。，在这个例子中，＃2应变寿命更长，但增长较慢，所减少的增长曲线的初始斜率证明。

<ol>
	<li>Kaeberlein, M., Burtner, C. R., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+yeast+aging.">Recent developments in yeast aging.</a> PLoS Genet. 3, (2007).</li><li>Kaeberlein, M., Conn, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of models for human aging. , 109-120 (2006).</li><li>Smith, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evidence+for+conserved+longevity+pathways+between+divergent+eukaryotic+species.">Quantitative evidence for conserved longevity pathways between divergent eukaryotic species.</a> Genome Res. 18, 564-570 (2008).</li><li>Steinkraus, K. A., Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replicative+aging+in+yeast:+the+means+to+the+end.">Replicative aging in yeast: the means to the end.</a> Annu Rev Cell Dev Biol. 24, 29-54 (2008).</li><li>Murakami, C. J., Burtner, C. R., Kennedy, B. K., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+high-throughput+quantitative+analysis+of+yeast+chronological+life+span.">A method for high-throughput quantitative analysis of yeast chronological life span.</a> J Gerontol A Biol Sci Med Sci. 63, 113-121 (2008).</li><li>Jiang, J. C., Jaruga, E., Repnevskaya, M. V., Jazwinski, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+intervention+resembling+caloric+restriction+prolongs+life+span+and+retards+aging+in+yeast.">An intervention resembling caloric restriction prolongs life span and retards aging in yeast.</a> Faseb J. 14, 2135-2137 (2000).</li><li>Kaeberlein, M., Kirkland, K. T., Fields, S., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sir2-independent+life+span+extension+by+calorie+restriction+in+yeast.">Sir2-independent life span extension by calorie restriction in yeast.</a> PLoS Biol. 2, E296-E296 (2004).</li><li>Lin, S. J., Defossez, P. A., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+NAD+and+SIR2+for+life-span+extension+by+calorie+restriction+in+Saccharomyces+cerevisiae.">Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae.</a> Science. 289, 2126-2128 (2000).</li><li>Kaeberlein, M., Kennedy, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+in+yeast+of+conserved+ageing+genes.">Large-scale identification in yeast of conserved ageing genes.</a> Mech Ageing Dev. 126, 17-21 (2005).</li><li>Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+yeast+replicative+life+span+by+TOR+and+Sch9+in+response+to+nutrients.">Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients.</a> Science. 310, 1193-1196 (2005).</li></ol>

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measuring replicative life span in the budding yeast

老化是一种退化性的过程，特点是逐渐恶化的细胞成分和细胞器造成的死亡率。芽殖酵母<em&gt;酿酒酵母</em&gt;已被广泛用于研究生物学的老化，以及酵母长寿的几个因素已经证明，在多细胞真核生物中，包括蠕虫中保守，苍蝇和老鼠<sup&gt; 1</sup&gt;。由于缺乏容易量化的年龄相关的表型酵母中的老化，已经检测几乎完全通过测量细胞在不同语境中的寿命，具有两种不同的生活中常见的用法的跨度范式<sup&gt; 2</sup&gt;。按时间的寿命是指时间长度，母细胞可以生存在一个不分裂的，静止的状态，和建议，以作为在多细胞真核生物有丝分裂后细胞老化的典范。复制的寿命，相反，是指前，一位母亲细胞衰老所产生的子细胞的数目，被认为是提供了一个模式在分裂活跃的细胞老化。在这里，我们提出了一个测量的芽殖酵母中的母亲细胞中的复制寿命的广义协议。复制寿命试验的目的是确定每个母细胞芽多次。母亲和女儿的细胞可以很容易地使用一个标准的光镜下（总放大倍率160X），如蔡司Axioscope 40或其他类似的模式，由经验丰富的研究员有区别的。配备一个光纤针使用手册显微子细胞从母细胞的物理分离。典型的实验室酵母菌株的产生20-30％的母亲的女儿细胞和寿命实验需要2-3周。

Watch this Scientific Journal Video about Measuring Replicative Life Span in the Budding Yeast at JoVE.com

Measuring Replicative Life Span in the Budding Yeast

在这篇文章中，我们提出了一个用于测量酵母母细胞的复制寿命一般协议。

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 ...

measuring-replicative-life-span-in-the-budding-yeast

Research

JoVE Journal

Biology

20.5K 次观看.  University of Washington. 在这篇文章中，我们提出了一个用于测量酵母母细胞的复制寿命一般协议。

视频: Measuring Replicative Life Span in the Budding Yeast

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

测量线虫固体介质的寿命

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

科研

生物学

Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells

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

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

量化老化细胞生长的酵母年谱跨度

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

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

纯化 M。 magneticum应变AMB - 1磁小体相关蛋白MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

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

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

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 ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

测量线虫寿命在96孔板

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

Measuring the Kinetics of mRNA Transcription in Single Living Cells

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in vivo is of importance. Pol II kinetics have been measured using biochemical or molecular methods.1-3 In recent years, with the development of new visualization methods, it has become possible to follow transcription as it occurs in real time in single living cells.4 Herein we describe how to perform analysis of Pol II elongation kinetics on a specific gene in living cells.5, 6 Using a cell line in which a specific gene locus (DNA), its mRNA product, and the final protein product can be fluorescently labeled and visualized in vivo, it is possible to detect the actual transcription of mRNAs on the gene of interest.7, 8 The mRNA is fluorescently tagged using the MS2 system for tagging mRNAs in vivo, where the 3'UTR of the mRNA transcripts contain 24 MS2 stem-loop repeats, which provide highly specific binding sites for the YFP-MS2 coat protein that labels the mRNA as it is transcribed.9 To monitor the kinetics of transcription we use the Fluorescence Recovery After Photobleaching (FRAP) method. By photobleaching the YFP-MS2-tagged nascent transcripts at the site of transcription and then following the recovery of this signal over time, we obtain the synthesis rate of the newly made mRNAs.5 In other words, YFP-MS2 fluorescence recovery reflects the generation of new MS2 stem-loops in the nascent transcripts and their binding by fluorescent free YFP-MS2 molecules entering from the surrounding nucleoplasm. The FRAP recovery curves are then analyzed using mathematical mechanistic models formalized by a series of differential equations, in order to retrieve the kinetic time parameters of transcription.

RNA polymerase II transcriptional kinetics are measured on specific genes in living cells. mRNAs transcribed from the gene of interest are fluorescently tagged and using Fluorescence Recovery After Photobleaching (FRAP) the in vivo kinetics of transcriptional elongation are obtained.

测量单个活细胞中的mRNA转录的动力学

The transcriptional activity of RNA polymerase II (Pol II) is a dynamic process and therefore measuring the kinetics of the transcriptional process in ...

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 ...

Measuring the Osmotic Water Permeability Coefficient (Pf) of Spherical Cells: Isolated Plant Protoplasts as an Example

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is a simple and very efficient method for the determination of the osmotic water permeability coefficient (Pf) in plant protoplasts, applicable in principle also to other spherical cells such as frog oocytes. The first step of the assay is the isolation of protoplasts from the plant tissue of interest by enzymatic digestion into a chamber with an appropriate isotonic solution. The second step consists of an osmotic challenge assay: protoplasts immobilized on the bottom of the chamber are submitted to a constant perfusion starting with an isotonic solution and followed by a hypotonic solution. The cell swelling is video recorded. In the third step, the images are processed offline to yield volume changes, and the time course of the volume changes is correlated with the time course of the change in osmolarity of the chamber perfusion medium, using a curve fitting procedure written in Matlab (the &#8216;PfFit&#8217;), to yield Pf.

Measuring the Osmotic Water Permeability Coefficient Pf of Spherical Cells: Isolated Plant Protoplasts as an Example

Measuring the osmotic water permeability coefficient (Pf) of cells can help understand the regulatory mechanisms of aquaporins (AQPs). Pf determination in spherical plant cell protoplasts presented here involves protoplasts isolation and numerical analysis of their initial rate of volume change as a result of an osmotic challenge during constant bath perfusion.

测量渗透水渗透系数（P<sub&gt; F</sub&gt;）球形细胞：隔离植物原生质体为例。

Studying AQP regulation mechanisms is crucial for the understanding of water relations at both the cellular and the whole plant levels. Presented here is ...

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.