Name: high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast
Uploaded: 2017-04-20T13:00:00
Description: Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

我们感谢克里·布卢姆（北卡罗莱纳大学），凯·李（NCI），史蒂文·马库斯（科罗拉多州立大学）和埃尔马·希贝尔（海德堡大学）分享各种FP-TUB1质粒。我们是在滑动室制备方法训练我们感谢梅利莎·加德纳（明尼苏达大学）。这项工作是由美国国立卫生研究院（NIH）授予R01GM112893-01A1（以JKM）和T32GM008730（到CE）的支持。

1.准备-Leu降板<ol><li>添加800毫升的无菌去离子水（DIH 2 O）到2L烧瓶中。添加26.71克漏失碱（DOB）粉末，0.69克CSM-的Leu，和20g琼脂。用磁力搅拌棒混合。带来至1L DIH 2 O. </li><li>高压釜在121℃和19磅30分钟。 </li><li>除去从高压釜的烧瓶中，并使其冷却至〜65℃，温和搅拌。 </li><li>倾30毫升/板放入100mm直径板。让这些坐在室温下36-48小时，在4℃下储存之前。 </li></ol> 2.整?...

<ol>
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L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Tubulin+C354+mutations+that+severely+decrease+microtubule+dynamics+do+not+prevent+nuclear+migration+in+yeast.">beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast.</a> Mol Biol Cell. 13 (8), 2919-2932 (2002).</li><li>Moore, J. K., Cooper, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+mitosis+with+cell+polarity:+Molecular+motors+at+the+cell+cortex.">Coordinating mitosis with cell polarity: Molecular motors at the cell cortex.</a> Semin Cell Dev Biol. 21 (3), 283-289 (2010).</li><li>Gartz Hanson, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+alpha-tubulin+mutation+disrupts+neural+development+and+tubulin+proteostasis.">Novel alpha-tubulin mutation disrupts neural development and tubulin proteostasis.</a> Dev Biol. 409 (2), 406-419 (2016).</li><li>Fees, C. P., Aiken, J., O'Toole, E. T., Giddings, T. H. J., Moore, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+negatively+charged+carboxy-terminal+tail+of+beta-tubulin+promotes+proper+chromosome+segregation.">The negatively charged carboxy-terminal tail of beta-tubulin promotes proper chromosome segregation.</a> Mol Biol Cell. 27 (11), 1786-1796 (2016).</li><li>Song, S., Lee, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+function+of+Saccharomyces+cerevisiae+CDC5+in+cytokinesis.">A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.</a> J Cell Biol. 152 (3), 451-469 (2001).</li><li>Knop, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+tagging+of+yeast+genes+using+a+PCR-based+strategy:+more+tags+and+improved+practical+routines.">Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines.</a> Yeast. 15 (10B), 963-972 (1999).</li><li>Kosco, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+microtubule+dynamics+by+Stu2p+is+essential+for+spindle+orientation+and+metaphase+chromosome+alignment+in+yeast.">Control of microtubule dynamics by Stu2p is essential for spindle orientation and metaphase chromosome alignment in yeast.</a> Mol Biol Cell. 12 (9), 2870-2880 (2001).</li><li>Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B., Wilson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+stabilization+of+microtubule+dynamic+instability+in+vitro+by+vinblastine.">Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine.</a> Biochemistry. 32 (5), 1285-1293 (1993).</li><li>Aiken, J., Sept, D., Costanzo, M., Boone, C., Cooper, J. A., Moore, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+reveals+novel+and+discrete+functions+for+tubulin+carboxy-terminal+tails.">Genome-wide analysis reveals novel and discrete functions for tubulin carboxy-terminal tails.</a> Curr Biol. 24 (12), 1295-1303 (2014).</li><li>Straight, A. F., Marshall, W. F., Sedat, J. W., Murray, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitosis+in+living+budding+yeast:+anaphase+A+but+no+metaphase+plate.">Mitosis in living budding yeast: anaphase A but no metaphase plate.</a> Science. 277 (5325), 574-578 (1997).</li><li>Khmelinskii, A., Lawrence, C., Roostalu, J., Schiebel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdc14-regulated+midzone+assembly+controls+anaphase+B.">Cdc14-regulated midzone assembly controls anaphase B.</a> J Cell Biol. 177 (6), 981-993 (2007).</li><li>Markus, S. M., Omer, S., Baranowski, K., Lee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Plasmids+for+Fluorescent+Protein+Tagging+of+Microtubules+in+Saccharomyces+cerevisiae.">Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae.</a> Traffic. 16 (7), 773-786 (2015).</li></ol>

芽殖酵母模型聚集在体内环境的微管动力学的高分辨率测量，包括对时间的能力，形象单一微管和操纵使用酵母遗传学的工具，微管蛋白和微管监管机构的能力提供了重要的优势。 所述涂覆的伴刀豆球蛋白室提供许多优于先前所描述的设备中，包含熔化的琼脂垫。与腔室玻片可以被预先制成并存储长期或可以立即使用。此外，该涂覆的伴刀豆球蛋白室能够培养的酵母?...

微管是由αβ微管蛋白的蛋白质亚基的细胞骨架的聚合物和在多种细胞环境中，包括胞内转运，细胞分裂，形态发生和运动性的被使用。要建立微管网络对于这些不同的角色，细胞必须仔细调节何时何微管形式。这种调节是通过控制反应完成，要么组装αβ微管蛋白亚基插入微管聚合物或拆卸的微管转化为游离亚基;这被称为微管动力学。 微管字段的一个主要目标是阐明调节微管动力学，包括结合于微管蛋白和/或微管的αβ微管蛋白亚单元和外在调节研究的分子机制。行之有效的实验方法是在体外用纯化的αβ微管蛋白的蛋白质在COM重建该系统中，常常bination用纯化的外在调节器。虽然这是一个有用的方法，很显然，在重构的系统微管动力学从活细胞中观察到的强烈不同。例如，微管生长更快和收缩体内比体外慢。这些差异可以归因于已知的外在调节器1的可用性，以及在细胞中尚未未定义的因素。因此，关键是要确定微管调节剂和破坏在天然细胞环境动力学突变体的活动。 虽然后生动物模型已经被证明是研究微管功能和高阶组织现行制度，有几个实际问题严重限制了这些模型的效用微管动力学的精确测量。首先，大量的微管，从几十到上千个细胞，使其难以理直气壮跟踪单个微管随着时间的推移。许多研究通过成像选择性定位于微管的端部，如在端部结合（EB）家族蛋白的蛋白应对这一挑战。然而，这些蛋白质是已知的唯一定位于在后生动物2生长微管的端部。因此，这些蛋白质的效用是有限的，以直接测量生长速率，而只有间接测量动力学的其他方面，诸如灾难的频率。其次，尽管在基因组编辑技术的进步，产生稳定表达荧光标记的微管蛋白或引入突变，以选择性地操纵微管蛋白的微管或调节细胞仍然是一个挑战显著。此外，许多微管蛋白同种型的后生动物存在混淆的突变如何影响个人微管蛋白基因的研究。 芽殖酵母系统提供几个重要的优点用于体内 microtubu测量乐动态。酵母具有简化的微管网络，它允许个别微管的可视化。在酵母中，微管从组织称为纺锤体极体（SPBS），其被嵌入在核膜3中心发出。所述SPBS用作支架用于该成核微管4,5γ微管蛋白小复合物。 SPBS核两类微管，纺锤体微管及微管星体的。纺锤体微管伸入和核质是重要的，用于经由微管的着丝点附连到染色体和用于经由重叠极间微管6稳定主轴。相比之下，微管星体向外项目进入细胞质，并比较少见相比纺锤体微管的密集网络。在有丝分裂，预后期细胞具有1-2只从任一SPB发出星体微管;这些都是普通的Individual微管，而不是作为束7。星体微管的有丝分裂过程中的作用是细胞核和主轴移动到母亲和芽的隔间，被称为芽颈部之间的交界处。这种运动包括良好定义的途径上星体微管生成的力，拉细胞核和主轴朝向并最终进入芽颈部8。 酵母系统的另一个优点是它的遗传学，其可用于研究的微管调节剂和微管蛋白亚单位具有无可比拟的精度的效用。酵母也具有微管蛋白同种型的简化剧目：单β微管蛋白基因（TUB2）和两个α微管蛋白基因（TUB1和TUB3）。突变可以容易地引入这些基因，从而以均匀的微管蛋白人口9,10研究。有许多的广泛对于标签的微管结构可用，而且这些设置可以针对在染色体位点稳定表达的整合（见讨论）。 该方法的总的目标是图像单个微管在活酵母细胞在四个维度（X，Y，Z和T​​）为微管动力学的高分辨率测量。用于荧光标记的微管蛋白在酵母细胞中的组成型，低水平表达整合构建体的方法进行说明。在成像之前，活细胞被安装到已经涂覆了凝集素伴刀豆球蛋白A，以稳定长期成像细胞的滑动室。用于图像采集的最佳参数，以及对数据进行分析的工作流，也有所说明。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DOB (dropout bases)</td>
			<td>Sunrise science&#160;</td>
			<td>1650</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM-Leu</td>
			<td>Sunrise science&#160;</td>
			<td>1005</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Ameresco</td>
			<td>N833</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm polystyrene plates</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>ssDNA (Samon Sperm)&#160; in sterile DiH2O</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>&#160;D7656</td>
			<td>resuspend at 10 mg/mL in DiH2O. Store aliquots at -20 &#186;C</td>
		</tr>
		<tr>
			<td>Synthetic Complete Media&#160;</td>
			<td>Sunrise science&#160;</td>
			<td>1459-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Concanavalin A</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>L7647</td>
			<td>resuspend at 2 mg/mL in DiH2O. Store aliquots at -20 &#186;C</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-541-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-12</td>
			<td>paraffin film</td>
		</tr>
		<tr>
			<td>VALAP (Equal parts of Vaseline, lanolin and paraffin)</td>
			<td></td>
			<td></td>
			<td>melt at 75 &#186;C before use</td>
		</tr>
		<tr>
			<td>forceps&#160;</td>
			<td>Fisher Scientific</td>
			<td>16-100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Poyethylene glycol (PEG) 3350</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>202444</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ti E inverted Perfect Focus microscope</td>
			<td>Nikon Instruments</td>
			<td>MEA53100</td>
			<td></td>
		</tr>
		<tr>
			<td>1.45 NA 100x CFI Plan Apo objective</td>
			<td>Nikon Instruments</td>
			<td>MRD01905</td>
			<td></td>
		</tr>
		<tr>
			<td>Piezo electric stage&#160;</td>
			<td>Physik Instrumente</td>
			<td>P-736</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disk scanner</td>
			<td>Yokogawa</td>
			<td>CSU10</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser combiner module</td>
			<td>Agilent Technologies</td>
			<td>MCL400B</td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor Technology</td>
			<td>iXon Ultra 897&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements software</td>
			<td>Nikon Instruments</td>
			<td>MQS31100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel software</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB software</td>
			<td>MathWorks, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ64</td>
			<td>NIH</td>
			<td></td>
			<td>Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016.</td>
		</tr>
		<tr>
			<td>Bio-Formats Importer plug-in</td>
			<td>Open Microscopy Environment</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pUC19-LEU2::GFP-TUB1</td>
			<td></td>
			<td>pSK1050</td>
			<td>reference: Song, S. and Lee, K. S. A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis. J Cell Biol. 152 (3), 451-469 (2001)</td>
		</tr>
	</tbody>
</table>

high-resolution imaging and analysis of individual astral microtubule dynamics in budding yeast

动态微管是许多细胞过程的根本，微管动力学的精确测量可以提供深入了解细胞如何调节这些过程和突变的影响如何调控基因。微管动力学的后生动物模型的量化有许多相关的挑战，包括高密度微管和限制遗传操作。相比之下，芽殖酵母模型提供了克服这些挑战的优势。这个协议描述来测量在活酵母细胞单个微管的动力学的方法。表达荧光标记的微管蛋白的细胞粘附到组装的滑动室，从而允许稳定的隔时图像采集。用于高速的详细指南，四维图像采集本发明还提供，以及用于在共焦图像栈量化动态微管的性质的协议。该方法中，结合常规酵母遗传学省IDES了一种方法，是唯一适合用于定量评估该改变微管蛋白亚单位的活性的微管调节剂或突变的影响。

在活酵母细胞测量微管动力学提供了令人信服的工具，以评估在编码微管调节剂或微管蛋白基因中的突变如何亚基冲击聚合和解聚率，以及这些状态之间的过渡频率。 图1显示在野生型细胞中的时间序列的星体微管动力学的和β微管蛋白具有突变的突变型细胞（tub2-430Δ）。微管标记有GFP标记的α微管蛋白可视化微管的长度。红色箭头跟踪每个图像?...

Watch this Scientific Journal Video about High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast at JoVE.com

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

出芽酵母是研究体内微管动力学由于其强大的遗传学和其微管骨架的简单性的有利的模型。以下方案描述了如何变换和培养的酵母细胞，获得共焦显微镜图像，和定量分析在活酵母细胞的微管动力学。

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 ...

Research

JoVE Journal

Biology

Measuring Replicative Life Span in the Budding Yeast

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 ...

科研

生物学

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

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 ...

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

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

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 ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

高分辨率成像定时，并在活体人脐静脉内皮细胞微管动力学自动分析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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

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 ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

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

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

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

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

通过旋转磁盘显微镜在单极式斜接主轴中测量微管动力学

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...