我们感谢刘慧卿杰克逊，约书亚蔡德曼，和尼古拉斯·雷恩对软件的意见。这项工作是由GM95733（NM），BBBE 103316和麻省理工学院的启动资金（NM）。

<ol>
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L., Volfson, D., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+dynamics+of+single-cell+gene+expression+over+multiple+cell+cycles.">Monitoring dynamics of single-cell gene expression over multiple cell cycles.</a> Molecular Systems Biology. 1 (1), 2005.0024 (2005).</li><li>Paliwal, S., Iglesias, P. A., Campbell, K., Hilioti, Z., Groisman, A., Levchenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446 (7131), 46-51 (2007).</li><li>Charvin, G., Cross, F. R., Siggia, E. 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Meth. 4 (2), 175-181 (2007).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Trans. Sys. Man. Cyber. 9 (1), 62-66 (1979).</li><li>Goranov, A. I., Cook, 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=The+rate+of+cell+growth+is+governed+by+cell+cycle+stage.">The rate of cell growth is governed by cell cycle stage.</a> Genes &amp; Development. 23 (12), 1408-1422 (2009).</li><li>To, T. -. 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作者宣称，他们有没有竞争经济利益。

上述协议描述了一个简单的方法来获取和分析经验有限，在微流或在软件开发的的荧光时间序列数据。它允许一个取得时移荧光电影单个酵母细胞中提取相关的细胞大小和表达测量;牧师跟踪和血统分配和整个细胞的行为进行分析，随着时间的推移，使用市售的微流体培养装置和一个多功能的图形用户界面（GUI）。实验，分割和跟踪步骤已经以各种方式以前11,13,14接近，上面的程序的目的?...

单细胞基因表达分析进一步了解基因调控的许多方面。荧光记者表达的静态快照，使用流式细胞仪或显微镜单细胞表达的分布提供了有用的信息，但缺乏需要直接告知基因表达的动态时间序列数据的历史和演变。荧光时间推移显微镜呈现的一种手段，同时获得单细胞测定和他们的历史。各种实验和分析技术已经发展到获取和定量荧光记者表达的电影，从而传授洞察（见检讨1）如细胞与细胞变异细菌的持久化形成4，2,3，转录基因调控功能启动和延伸5，转录6,7爆破，细胞周期依赖性8,9的 ，和遗传10。但是，OBT质量恒常单细胞荧光时间序列，包括培养的单层细胞在一个可控的环境中，并在高通量定量分析所获取的荧光电影的重要的技术挑战。在这里，我们描述一个过程来获取和分析荧光S的电影酵母中没有所需的经验，在细胞培养装置制造或在软件开发中（ 图1）。 首先，我们将详细地，例如，协议产生荧光的时间序列的芽殖酵母表达一种或多种荧光记者的电影。虽然定制的微文化室已建成并成功地采用了先前11-13，我们使用市售的微流体装置从CellAsic（加利福尼亚Hayward）。系统局限单层生长的细胞，并允许灌注环境的持续控制。显微镜协议，我们提出的是一个简单的方法来获得fluoresc芽殖酵母的ENCE电影的，但任何修改过的实验方案（定制培养装置，替代介质条件等 ）的单个酵母细胞产生类似的荧光电影数据可能会被取代。 接下来，我们勾勒出电影使用的图形用户界面（GUI）基于MATLAB软件包（Mathworks公司，马萨诸塞Natic​​k），被称为快速荧光时间​​系列分析的图形用户界面（移植物），提取时间序列数据分析单细胞。移植物有类似的功能，多功能的，开放源码的软件包细胞ID 14分割和跟踪细胞中提取荧光强度和几何信息。然而，移植物提供了重要的额外功能。首先，它提供了简单的交互式编辑分割和跟踪结果来验证数据的准确性，而不是仅仅统计浇注离群区域分析后的痕迹。此外，它扩展了分析，自动生成Ÿ候谱系和细胞周期的芽殖酵母的兴趣点。决定时，母亲和女儿划分，以形成两个独立的单元区域是非常重要的，以确定在整个细胞周期8的全细胞（包括任何连接芽的母亲）测量。该套件包括三个模块来完成这些任务。第一部分单元区域的基础上聚焦和散的明视场图像之间的对比度，并允许用户定义和视觉测试分段参数。第二轨道（布莱尔和克罗克等 IDL例行杜弗兰的MATLAB实现，可在： <a href="http://physics.georgetown.edu/matlab/" target="_blank">http://physics.georgetown.edu/MATLAB/</a> ），通过测量细胞区域自动分配谱系;使视力检查和纠错。一个简单的绘图GUI是来查询单细胞性质。第三个模块赋予现蕾和分工TIMES，和全细胞输出的时间序列数据，以及他们的第一和第二时间的衍生物（9所讨论的）。分析模块输出的数据，为后续研究的统计软件选择作为空间分隔的文本文件。因此，包使用户能够提取高品质的时间序列数据，通过图形界面。我们已经用这种方法在单芽殖酵母细胞的细胞周期9为一个函数估计实时转录率。模块进行了优化，芽殖酵母，参数或，如有必要，可适于其他生物体和图像类型可自由查看的代码。分割，跟踪和谱系分配算法可能是特定类型的成像分配和生物体。现有的算法可以被取代，但仍保留的GUI界面，允许用户友好的可视化分割和跟踪检查和纠正错误总是占据R与任何算法。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Y04C Yeast Perfusion Plate</td>
			<td>CellAsic</td>
			<td>Y04C-02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>ONIX Microfluidic Perfusion Platform</td>
			<td>CellAsic</td>
			<td>EV-262</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Axio Observer.Z1 Microscope</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 63X/1.40 Oil DIC objective</td>
			<td>Zeiss</td>
			<td>440762-9904-000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cascade II EMCCD camera</td>
			<td>Photometrics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lumen 200 metal-halide arc lamp</td>
			<td>PRIOR Scientific</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triple-bandpass dichroic filter cube and excitation and emission filter set</td>
			<td>Chroma Technology Corp</td>
			<td>set #89006</td>
			<td>Used for YFP (Venus/Citrine), CFP (Cerulean), RFP (mCherry/tdTomato)</td>
		</tr>
		<tr>
			<td>MAC 5000 controller and filter wheels</td>
			<td>Ludl Electronic Products</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MATLAB R2011a</td>
			<td>Mathworks</td>
			<td>&nbsp;</td>
			<td>64-bit version handles large data files better than 32-bit</td>
		</tr>
	</tbody>
</table>

acquiring fluorescence time-lapse movies of budding yeast and analyzing single-cell dynamics using grafts

荧光时间推移显微镜在单细胞水平研究的许多生物过程中已经成为一个强大的工具。特别地，描绘基因表达的时间依赖性的电影提供深入了解其调节的动态，但是，也有许多的技术挑战，以获得和分析单个细胞的荧光电影。我们在这里介绍一个简单的协议，使用市售的微流体培养装置，产生这样的数据，和一个基于MATLAB的图形用户界面（GUI）为基础的软件封装量化荧光图像。的软件段和轨道细胞，使用户能够直观地牧师数据中的错误，并自动分配的血统和分裂次数。 GUI进一步分析时间序列产生整个细胞的痕迹以及他们的第一和第二次衍生物。虽然该软件是专为S。酵母 ，其模块化和通用性，应该允许它吨Ø作为一个平台，研究其他细胞类型的一些修改。

成功完成和分析的实验将产生大多是连续的时间序列与实际分配的芽和分裂次数的单全细胞。作为一个例子，我们履行上述协议ADH1启动子组成，观察增长和全球表现可能会有所不同随着时间的推移在单细胞（ 表4，Y47带动表达天蓝荧光蛋白（CFP）的整合拷贝单倍体酵母菌株）。我们跑了时间序列分析，获得整个细胞（ 图7A）测量随着时间的推移，9音...

Watch this Scientific Journal Video about Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS at JoVE.com

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

我们提出了一个简单的协议来获得生长的酵母细胞的荧光显微镜的电影，和一个基于GUI的软件程序包，提取单细胞的时间序列数据。该分析包括自动沿袭和集成的跟踪数据的目视检查和手工curation分组表决的时间分配。

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 ...

acquiring-fluorescence-time-lapse-movies-budding-yeast-analyzing

Research

JoVE Journal

Biology

12.7K 次观看.  Massachusetts Institute of Technology. 我们提出了一个简单的协议来获得生长的酵母细胞的荧光显微镜的电影，和一个基于GUI的软件程序包，提取单细胞的时间序列数据。该分析包括自动沿袭和集成的跟踪数据的目视检查和手工curation分组表决的时间分配。

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

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 ...

科研

生物学

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

有丝分裂的siRNA转染后的时间推移成像

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of signals to create complex, dynamic patterns as a response to environmental cues. These patterns are self-organizing and emergent; they cannot be predicted by observing the behavior of the individual cells. Using a time-lapse microcinematography tracking assay, we identified a distinct emergent pattern in M. xanthus called chemotaxis, defined as the directed movement of a swarm up a nutrient gradient toward its source 1.
In order to efficiently characterize chemotaxis via time-lapse microcinematography, we developed a highly modifiable plate complex (Figure 1) and constructed a cluster of 8 microscopes (Figure 2), each capable of capturing time-lapse videos. The assay is rigorous enough to allow consistent replication of quantifiable data, and the resulting videos allow us to observe and track subtle changes in swarm behavior. Once captured, the videos are transferred to an analysis/storage computer with enough memory to process and store thousands of videos. The flexibility of this setup has proven useful to several members of the M. xanthus community.

Recording Multicellular Behavior in Myxococcus xanthus Biofilms using Time-lapse Microcinematography

To study Myxococcus xanthus swarm behavior, we have designed a time-lapse microcinematography protocol that can be modified for different assays. It employs standard growth conditions adapted for microscopy, and yields reproducible results by the use of inexpensive, reusable silicone gaskets. We have used this method to quantify multicellular chemotaxis.

在录制多细胞行为黏细菌生物膜，使用定时Microcinematography

A swarm of the &delta;-proteobacterium Myxococcus xanthus contains millions of cells that act as a collective, coordinating movement through a series of ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

随机使用时移视频显微镜细胞迁移的定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

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 ...

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

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

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

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 ...

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

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single 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 ...

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 ...

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

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 not interfere with cell physiology, and requires minimal experimental handling. The low levels of technical interference enable researchers to study cells across multiple cycles of mitosis and to observe meiosis from beginning to end. Using fluorescent tags such as Green Fluorescent Protein (GFP) and Red Fluorescent Protein (RFP), researchers can analyze different factors whose functions are important for processes like transcription, DNA replication, cohesion, and segregation. Coupled with data analysis using Fiji (a free, optimized ImageJ version), live-cell imaging offers various ways of assessing protein movement, localization, stability, and timing, as well as nuclear dynamics and chromosome segregation. However, as is the case with other microscopy methods, live-cell imaging is limited by the intrinsic properties of light, which put a limit to the resolution power at high magnifications, and is also sensitive to photobleaching or phototoxicity at high wavelength frequencies. However, with some care, investigators can bypass these physical limitations by carefully choosing the right conditions, strains, and fluorescent markers to allow for the appropriate visualization of mitotic and meiotic events.

Here, we present live-cell imaging which is a non-toxic microscopy method that allows researchers to study protein behavior and nuclear dynamics in living fission yeast cells during mitosis and meiosis.

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 ...