这项工作是支持的生物技术和生物科学研究理事会（批31/C15982），加拿大卫生研究院，加拿大，不列颠哥伦比亚省知识发展基金，创新基金的迈克尔史密斯健康研究基金会（授予民事司法制度改革雄狮），并为视力扑灭。

材料和方法： <ol><li>酵母菌株： <ul><li> ACP1 - GFP（绿色荧光蛋白：）：C -末端标记GFP的ACP1生成一个使用（包含GFP和HIS5）质粒pKT128 PCR介导的同源重组。共聚焦显微镜和菌落PCR确认阳性转化。应变背景BY7043（MAT阿尔法can1Δ：STE2pr lue2lyp1Δhis3Δ1leu2Δ0ura3Δ0met15Δ0）从布恩实验室（塘和Boone，2006年）。 </li><li>缺失突变体阵列（DMA）（XXX：：KanR）：它是一个非必需基因的缺失突变体约5000的数组。所有这些不必要的基因被淘汰的G418的。数组的应变背景BY4741（MATA his3_1 leu2_0 met15_0 ura3_0）。我们的DMA最初是从布恩实验室。 </li></ul></li><li>纳入ACP1 - GFP在DMA： 以下协议GFP的阵列是改编自合成的遗传分析（SGA）的技术与修改（塘和Boone，2006）。所有副本寄托的步骤是通过使用一个歌手转子高密度阵列的机器人系统。另外，手册寄托或液体的高密度组阵机器人可以使用。 缩略语：YPD，酵母提取物，蛋白胨，葡萄糖，SD，合成辍学; LRK，略/精氨酸/赖氨酸辍学媒体; LHRK，略/他/精氨酸/赖氨酸辍学媒体 制备 <ol><li>增长到YPD板浇筑5毫升的通宵ACP1 - GFP的文化ACP1 - GFP细胞的草坪。允许板充分干燥火焰或在生物通风柜。 </li><li>在30˚C过夜孵育板。 </li><li>到1536菌落每盘使用一个机器人，阵列ACP1 - GFP的应变。与此同时，准备一个新鲜的DMA副本，副本寄托YPD + G418的板。 </li><li>在30˚C过夜培养板。 交配和二倍体选择 </li><li>伴侣的副本寄托和铺设YPD板的殖民地ACP1 - GFP与DMA应变。 </li><li>在30˚C过夜培养板。 </li><li>副本板的殖民地上的SD - 他+ G418的板选择二倍体细胞（马塔/ MATalpha）。 </li><li>在30˚C过夜培养板。 Sporulationg和萌发 </li><li>副本板关于加强产孢媒体二倍体殖民地，其中包含碳和氮的来源的水平下降，诱发减数分裂孢子的形成。 </li><li>在25˚C的最少5天的孵育板。 </li><li>发芽副本电镀到LRK媒体的殖民地的马塔减数分裂后代和孵化板块在30˚C为两天。 </li><li>这媒体会诱发单倍体细胞从孢子萌发。由于LUE2开放阅读框（ORF）是集成在特定的马塔STE2发起人，将所有的单倍体发芽MATA。 两个等位基因的选择 </li><li>现在有必要选择的绿色荧光蛋白基因和单个突变的等位基因。而要做到这一点，马塔细胞副本镀上LRK + G418的媒体 ，单倍体细胞，进行基因缺失（XXX：：kanR）选择。 </li><li>在30˚C过夜培养板。 </li><li>最后，MATA减数分裂后代副本镀上LHRK + G418的媒体选择增长的单突变体（XXX：kanR：），也窝藏ACP1 - GFP（绿色荧光蛋白：：他的） 。这些板块可以存储在4˚彗星为3个月。 </li></ol> 以下协议GFP的阵列是改编自合成的遗传分析（SGA）的技术与修改（塘和Boone，2006）。所有副本寄托的步骤是通过使用一个歌手转子高密度阵列的机器人系统。另外，手册寄托或液体的高密度组阵机器人可以使用。 缩略语：YPD，酵母提取物，蛋白胨，葡萄糖，SD，合成辍学; LRK，略/精氨酸/赖氨酸辍学媒体; LHRK，略/他/精氨酸/赖氨酸辍学媒体 制备 </li><li>显微镜： <ol><li>选取所需的突变体（S），表示从盘直接ACP1 - GFP。 </li><li>细胞增长在30 ° C在YPD或SD - 早期日志阶段他的媒体。 </li><li>可视化共聚焦显微镜使用的GFP filterset。预期结果的一个例子是在图1示出。 </li></ol></li></ol> SGA的媒体： 在本议定书中使用的生长介质（YPD）和选择媒体（SD）是经常使用的酵母分子生物学。请参考方法在酵母中的详细说明（安伯格等，2005年）。 LRK，LHRK媒体（400毫升总量） <table border="0"><tbody><tr><td>为了添加</td><td>金额</td></tr><tr><td>硫酸铵酵母氮肥基地</td><td> 2.7摹</td></tr><tr><td>氨基酸组合</td><td> 0.8摹</td></tr><tr><td>洋菜</td><td> 8 g的</td></tr><tr><td> DH 2 O </td><td> 360毫升</td></tr><tr><td colspan="2">高压灭菌器</td></tr><tr><td> 20％葡萄糖</td><td> 40毫升</td></tr><tr><td colspan="2">冷却至65˚彗星</td></tr><tr><td> 100毫克/毫升刀豆</td><td> 0.2毫升</td></tr><tr><td> 100毫克/毫升thialysine </td><td> 0.2毫升</td></tr><tr><td colspan="2">混合，倒入50毫升每盘</td></tr></tbody></table> LRK，LHRK + G418的媒体（400毫升总量） <table border="0"><tbody><tr><td>为了添加</td><td>金额</td></tr><tr><td>没有硫酸铵酵母氮肥基地</td><td> 0.7摹</td></tr><tr><td>氨基酸组合</td><td> 0.8摹</td></tr><tr><td>味精</td><td> 0.4 </td></tr><tr><td>洋菜</td><td> 8 g的</td></tr><tr><td> DH 2 O </td><td> 360毫升</td></tr><tr><td colspan="2">高压灭菌器</td></tr><tr><td> 20％葡萄糖</td><td> 40毫升</td></tr><tr><td colspan="2">冷却至65˚彗星</td></tr><tr><td> 100毫克/毫升刀豆</td><td> 0.2毫升</td></tr><tr><td> 100毫克/毫升thialysine </td><td> 0.2毫升</td></tr><tr><td> 200 mg / ml的G418 </td><td> 0.4毫升</td></tr><tr><td colspan="2">混合，倒入50毫升每盘</td></tr></tbody></table>富含孢子媒体（400毫升总量） <table border="0"><tbody><tr><td>为了添加</td><td>金额</td></tr><tr><td>醋酸钾</td><td> 4摹</td></tr><tr><td>酵母提取物</td><td> 0.4摹</td></tr><tr><td>葡萄糖</td><td> 0.2克</td></tr><tr><td>产孢氨基酸酸混合</td><td> 0.04摹</td></tr><tr><td>洋菜</td><td> 8 g的</td></tr><tr><td> DH 2 O </td><td> 360毫升</td></tr><tr><td colspan="2">高压灭菌器</td></tr><tr><td colspan="2">冷却至65˚彗星</td></tr><tr><td> 200 mg / ml的G418 </td><td> 0.4毫升</td></tr><tr><td colspan="2">混合，倒入50毫升每盘</td></tr></tbody></table>

<ol>
	<li>Amberg, D. C., Burke, D., Strathern, J. 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> Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual. , (2005).</li><li>Tong, A. H., Boone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+genetic+array+analysis+in+Saccharomyces+cerevisiae.">Synthetic genetic array analysis in Saccharomyces cerevisiae.</a> Methods Mol Biol. 313, 171-192 (2006).</li></ol>

这种方法可以帮助有效地纳入各种突变的背景线粒体标记，ACP1 - GFP。它依赖于使用的机器人系统，并且可以很容易地通过使用任何机器人系统。此过程也可用于包含其他类型的标记。例如，可视化ER，我们经常使用的标记Erg11 - GFP。在我国有代表性的图片，一个突变体和野生型与ACP1 - GFP的制造商visualiz海关共聚焦显微镜研究线粒体的形态。扰乱线粒体突变体的表型，因此是一个很好的候选人作进?...

<table><tbody><tr><td>SGA media</td><td></td><td></td><td>The growth media (YPD) and synthetic dropout media (SD) including LRK and LHRK, and enriched sporulation media used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.</td></tr></tbody></table>

a high-throughput method to globally study the organelle morphology in s. cerevisiae

高通量的方法研究蛋白质的本地化或细胞器的形态，是研究蛋白质相互作用的有效工具，并能够帮助实现的分子途径全面了解。在酿酒酵母中，与非必需的基因缺失阵列发展，我们可以在全球范围研究的形态不同的细胞器如内质网（ER）和线粒体使用GFP（或变种）的标记在不同的基因背景。然而，在每一个单一的突变体含有绿色荧光蛋白标记单独是一个劳动力密集的过程。在这里，我们描述了，经常是在我们的实验室中使用的程序。我们可以通过使用一个机器人系统来处理的高密度的酵母阵列和药物筛选技术，大大缩短所需的时间，提高可重复性。总之，我们穿过一个GFP标记的线粒体标记（Apc1 GFP）的机器人副本寄托到4672不必要的基因缺失突变体的高密度阵列。通过二倍体选择，孢子形成，萌发和双标记选择，我们收回这两个等位基因。因此，每个单倍体单突变包含Apc1 - GFP在其基因组位点中。现在，我们可以研究线粒体的形态在所有非必要的突变背景。使用这种高通量的方法，我们可以方便地研究和划定的途径，并在继承和细胞器的形成，在全基因组的设置有关的基因。

Watch this Scientific Journal Video about A high-throughput method to globally study the organelle morphology in S. cerevisiae at JoVE.com

A high-throughput method to globally study the organelle morphology in S. cerevisiae

GFP融合蛋白被广泛应用于共聚焦显微镜可视化的细胞器。然而，筛选突变，影响细胞器的形态一般需要个人的突变，非常耗时。在这里，我们展示了一种方法，同时结合近5000个非必要基因在酵母细胞器- GFP标记。

一种高通量的方法，在全球范围内研究在酿酒酵母中的细胞器形态

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

a-high-throughput-method-to-globally-study-organelle-morphology-s

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.3K 次观看.  University of British Columbia - UBC. GFP融合蛋白被广泛应用于共聚焦显微镜可视化的细胞器。然而，筛选突变，影响细胞器的形态一般需要个人的突变，非常耗时。在这里，我们展示了一种方法，同时结合近5000个非必要基因在酵母细胞器- GFP标记。

视频: A high-throughput method to globally study the organelle morphology in S. cerevisiae

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 ...

科研

生物化学

Visualizing Yeast Organelles with Fluorescent Protein Markers

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have constructed fluorescent protein-based markers for major organelles and endomembrane structures, including the nucleus, endoplasmic reticulum (ER), Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, lipid droplets, and autophagosomes. The protocol presented here describes the procedures for using these markers in yeast, including DNA preparation for yeast transformation, selection and evaluation of transformants, fluorescent microscopic observation, and the expected outcomes. The text is geared toward researchers who are entering the field of yeast organelle study from other backgrounds. Essential steps are covered, as well as technical notes about microscope hardware considerations and several common pitfalls. It provides a starting point for people to observe yeast subcellular entities by live-cell fluorescent microscopy. These tools and methods can be used to identify protein subcellular localization and track organelles of interest in time-lapse imaging.

Here we describe the use of a set of fluorescent protein-based organelle markers in live-cell imaging of the budding yeast, Saccharomyces cerevisiae.

The budding yeast, Saccharomyces cerevisiae, is a classic model system in studying organelle function and dynamics. In our previous works, we have ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

生物学

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

高通量酵母菌株，带基于滴滴的RNA测序

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

生物工程

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

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 ...

High-Throughput Live Imaging of Microcolonies to Measure Heterogeneity in Growth and Gene Expression

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental inputs into stress tolerance, pathogenicity, and other key components of fitness. This manuscript describes a microscope-based assay that tracks approximately 105&#160;Saccharomyces cerevisiae microcolonies per experiment. After automated time-lapse imaging of yeast immobilized in a multiwell plate, microcolony growth rates are easily analyzed with custom image-analysis software. For each microcolony, expression and localization of fluorescent proteins and survival of acute stress can also be monitored. This assay allows precise estimation of strains&#39; average growth rates, as well as comprehensive measurement of heterogeneity in growth, gene expression, and stress tolerance within clonal populations.

Yeast growth phenotypes are precisely measured through highly parallel time-lapse imaging of immobilized cells growing into microcolonies. Simultaneously, stress tolerance, protein expression, and protein localization can be monitored, generating integrated datasets to study how environmental and genetic differences, as well as gene-expression heterogeneity among isogenic cells, modulate growth.

微殖器的高通量实时成像，测量生长和基因表达中的异质性

Precise measurements of between- and within-strain heterogeneity in microbial growth rates are essential for understanding genetic and environmental ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

使用延时显微镜和蛋白质的阶段特异性核耗竭来研究酿酒酵母的减数分裂

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...