Name: surface spreading and immunostaining of yeast chromosomes
Uploaded: 2015-08-09T14:00:00.000+00:00
Duration: 12 min 6 s
Description: Watch this Scientific Journal Video about Surface Spreading and Immunostaining of Yeast Chromosomes at JoVE.com

This work was supported by NIH grant GM50936 to DKB.

注:以下一些步骤的协议要求在清洁通风橱工作。另外，该方法需要一个酵母四分体解剖显微镜配备一个10X长工作距离物镜并监控原生质球。显微镜应设置在提前罩。取出显微手臂和板夹从范围和放置这些物品在安全的地方，远离工作区。 1.准备原生质球<ol><li>生长所需条件下酵母。使用约2×10 8个细胞的每个样品，如4毫升培养的在OD 600 1.4 = 5×10 7个细胞/ ml。虽然样品的即时处理可以优选在某些情况下，对于大多数应用，细胞等份可以存储在冰上达8小时处理之前。 </li><li>转移细胞悬液至15ml离心管中，自旋在临床离心机中以设置图5（2345转/ 857.5 XG）处理3分钟，以沉淀细胞。 </l我&gt; <li>倒出培养基照顾不从粒料输细胞。轻轻重悬细胞于1ml ZK缓冲，然后添加40微升的1M二硫苏糖醇（DTT）。孵育2分钟，轻轻混合。如前颗粒细胞（见步骤1.2）。 </li><li>重悬粒料在1ml ZK缓冲器。添加酶解酶100T的新鲜制备的溶液已被充分混合5微升。孵育20-30分钟，在30℃以除去细胞壁和产生原生质球。 </li><li>测定在显微镜载玻片上的细胞悬浮液的10微升液滴，以确定是否原生质球是完整。细胞解剖显微镜下观察无盖玻片。细胞应该会出现浮肿和圆形而非略呈长方形，应该裂解以下加入20微升的水。如果细胞不能裂解，返回样品至30℃，并重新测定每10分钟，直到水诱导裂解容易观察到。如前颗粒细胞（见步骤1.2）。 </li><li>轻轻悬浮和洗涤细胞沉淀我ñ2.5毫升冷MES /山梨糖醇缓冲。如前颗粒细胞（见步骤1.2）。轻轻悬浮细胞沉淀在300-400微升冷MES /山梨糖醇缓冲。细胞可以保持在冰上，在此阶段进行数小时。 </li></ol> 2.染色体传播注:玻璃表面在其染色体将被传播，无论是幻灯片或盖玻片，应提前准备时间。每张幻灯片或盖玻片应浸入水中，然后乙醇，然后干燥，抛光用镜头纸。如果染色体将被摊在盖玻片，盖玻片应贴有一个透明胶带或橡皮泥滑。除非另有说明，否则该协议的其余部分将介绍在任幻灯片或传播盖玻片<ol><li>使用P20移液管，移液管20微升细胞悬浮液到一个干净的载玻片的表面上。 </li><li> 3％PFA /蔗糖溶液中使用的是P200的移液器，加入40微升，轻轻混合搜索解决方案N以"纷飞"用手滑动，直到纹影线消失。将滑动显微镜下，确认细胞是关注的焦点。煤灰溶液应该是新制备在使用的当天通风柜。 注意:如果目的蛋白质尚未审查的方法以前，最好是准备使用包含2和4％PFA解决方案，以确定是否保持该蛋白质是固定条件敏感附加幻灯片。 4％PFA /蔗糖溶液中使用的情况下，期望的可视化有关的微管蛋白的微管。较高固定液浓度的缺点是，染色体将是"underspread，"2％PFA中也往往产生"underspread"染色体。 </li><li>采用P200的移液器，加80微升1％LIPSOL的，如前漩涡混合解决方案，尽可能完整地，启动一个定时器。如果LIPSOL不可用，80微升1％NP40或80微升的dh 2 O可能取代（见代表结果，图2）。 </li><li>观看细胞仔细，轻轻地旋转幻灯片每15秒。当细胞大约80％的裂解（消失）停止计时，立即从显微镜取出载玻片上，加80微升的PFA /蔗糖溶液和漩涡混合。裂解应启动定时器之后发生从30至90秒。 注意:如果若干幻灯片显示裂解在大约相同的时间，可以最终省略微观监测用于幻灯片的其余部分，简单地仔细计时加成LIPSOL和另外的固定剂的最终等分试样之间的时期。然而，从同一样品准备多张幻灯片时，这个捷径是唯一可靠的。最好是监视各样品的裂解显微镜时不同载玻片从在不同时间采集的样品或来自不同文化的制备。 </li><li>放置幻灯片上清洁的平面上并扩散，液体在整个无结霜面传热e。使用保持低于"水坑"的弯月面，但在滑动本身， 即表面上方的清洁，一次性牧场吸管的侧滑动的。不"耙"与吸管滑动的表面上。不要重复使用吸管在不同的幻灯片，以避免样品的污染。 </li><li>离开幻灯片在通风柜干燥过夜。不溶性的核部件将落户到滑动面和绑定到它。对于大型实验，规划利用空间的干燥步骤是重要的。 </li><li>一旦干燥，最佳结果是通过发展到免疫染色阶段在同一天获得。然而，干燥的蔗糖溶液中嵌入扩频染色体在一个"蜂蜜"，允许样品的冷冻:幻灯片可以被转移到一个塑料载玻片盒，并存储在-20℃多年。 </li></ol> 3.免疫染色<ol><li>浸载玻片在0.2％光弗洛30秒。除去蜂蜜。精益幻灯片上的边缘，以在纸巾边缘休息，以去除残留的光弗洛。 </li><li>浸载玻片在1×TBS中5分钟以洗涤。通过在边缘滑靠在去除多余的液体，但不要让他们干出来的。 </li><li>铺设幻灯片磨砂朝上，吸管300微升TBS / BSA上横跨滑板的无结霜部滑动。 </li><li>孵育在潮湿室中在室温持续15分钟。 （下穿孔金属板或塑料滑梯盒内衬水的纸巾用饱和纸巾大型塑料容器）。 </li><li>由搁在纸巾上滑动的一个短边和靠在一个适当的支持所述滑动件（如一个试管架）漏的幻灯片。不要让表面干燥。 </li><li>立即应用80微升含有适当稀释的第一抗体的TBS / BSA缓冲液。如果抗体限制，使用40微升和22×22毫米盖玻片。对于粗兔血清，这是一个典型的1/50和1/500稀释之间。 注意:执行滴定新抗体制剂。最稀制剂能够产生高信号上述背景选择。为了控制的背景染色的水平，核应从适当缺失突变株和/或重复的载玻片制备应与预免疫血清被染色。 </li><li>将在幻灯片上避免气泡抛光22×50毫米盖玻片。握住拇指和食指沿长边之间的盖玻片在靠近一短边缘位置做到这一点。保持盖玻片，在30°角相对于滑动，从手指的短边最远被降低，直到其位于所述滑动只是"水坑"最接近载片的磨砂区域的边缘内侧。然后慢慢放下盖玻片，在平滑的运动，直到拿着幻灯片触摸板凳手指，然后松开。不要试图一次它的土地调整盖玻片的位置。 注:如果利差染色体上制备贴盖玻片（而不是直接在载玻片的表面上），该样品盖玻片将保持固定到整个染色和洗涤步骤的幻灯片。染色和随后丢弃期间额外盖玻片将被放置在样品上盖玻片均匀分布抗体溶液的顶部。 </li><li>在一个密封的腔潮湿的地方幻灯片和孵化一夜之间在4℃。 </li><li>保持磨砂边缘转移的幻灯片到载玻片染色齿条和淹没在含有TBS一个染色缸。 </li><li>轻轻蘸上下滑动，除去盖玻片。尽量不要用力过猛做到这一点。通过浸没在TB​​S洗载玻片2×10分钟。从机架上卸下幻灯片和通过触摸边纸巾排出多余液体。不要让表面干燥。 </li><li>在柔和的灯光工作，立即加入含1/1000倍稀释荧光标记的二次抗体（或40微升用22×22毫米盖玻片）的80微升TBS / BSA。广告ð盖玻片作为前孵育在4℃下2小时，在湿室中在黑暗中。 </li><li>除去盖玻片和以前一样，漏的幻灯片，并允许表面风干1至2小时在黑暗中。如前纸巾精益幻灯片。 注:如果在安装盖玻片进行传播，干燥分离前从幻灯片盖玻片。如在先前步骤中描述的幻灯片盖玻片，然后干燥。 </li><li>在柔光的工作，加入约30微升含安装DAPI（3个10微升滴）中，然后小心地放下盖玻片。如果利差盖玻片上，将安装介质的液滴在干净的玻片，降低盖玻片，染色体端下来，到幻灯片。 </li><li>仍在柔和的光，等待2分钟的盖玻片定居和密封盖玻片用透明指甲油的边缘。将幻灯片平坦滑动箱。 注:对于STED显微镜，用安装延长黄金。使用30微升延长黄金的没有DAPI并允许治愈过夜。用指甲油密封是不必要的。 </li><li>查看幻灯片由宽视场荧光显微镜用40或100X目的使用适当的设置进行DAPI集中的过滤器。可选择存放在4℃的幻灯片在黑暗中了好几个星期。不要冷冻。 </li></ol>

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The authors declare no competing financial interests.

The appearance of spread nuclei critically depends upon the balance between fixation and lysis/detergent treatment. As discussed above, the preservation of varying cellular structures requires the use of different PFA concentrations. For most proteins, 3% PFA is optimal. However, preservation of spindles requires use of 4% PFA. Even with a single set of reagents, the timing of lysis relative to fixation can also affect the quality of spread nuclei. For the most consistent results, the time of lysis should be normalize...

在芽殖酵母提供了许多优势，为生物过程的分子机制，包括控制染色体功能的蛋白质的研究研究。虽然有出芽酵母基因，分子和生物化学研究的公知的优点，蛋白质在细胞核分布的细胞学研究是由它的小尺寸复杂。典型的酵母核的直径小于一微米，这是可见光的只有约5倍的分辨率极限。因此，对核蛋白质，可以从常规的免疫染色或通过使用荧光蛋白标记，如绿色荧光蛋白（GFP）来获得的分配信息的数量是有限的。一个有用的方法来表征蛋白质的亚核的分布是染色体的表面扩散。该方法包括去除细胞壁，扰乱细胞和核膜，和一个llowing核的不溶性内容沉降到显微镜载玻片的表面上。这些不溶性组分包括核基质和染色体。除了允许去除可溶性核内容，从而增强检测的染色质结合的蛋白质的能力，所述染色体扩频方法导致染色体大幅度减压，使得传播核具有的直径约3至5微米的（对于二倍体减数分裂核）和2至3微米为二倍体有丝分裂核。这种减压使检测核亚结构是相对困难或不可能解决在完整的细胞核。 一个明显的缺点，以染色体扩频这样的可能性，即在扩展过程可以部分或完全破坏感兴趣的结构。特别值得关注的是，一个特定的染色体结合蛋白可能会丢失作为扩展过程的结果。这种潜在的并发症寿LD解释数据时必须牢记这一点。的蛋白质的一个例子是于扩展过程敏感的β-微管蛋白。在某些情况下，主轴，其由主要的微管蛋白，被扩频3中保存。主轴的可视化往往是有益的舞台感兴趣的原子核。然而，可视化微管蛋白需要有高浓度的固定剂处理;主轴下述的标准条件下丢失。这个实施例说明，在分析以前未表征的蛋白质的分布时，它改变固定液以确定蛋白质如何敏感是这种变化的浓度是重要的。尽管关于扩频条件对染色体结构的影响的关注，扩展方法的实用性和功率已被证明在许多情况下，具有在有丝分裂的表征，特别是减数分裂细胞4-7广泛的用途。 TWø扩频方法已被广泛使用。第一这些方法，通过修整和吉鲁8研制，避免了使用洗涤剂的，并且可以得到显示时染色的DNA特异性染料DAPI具有相对保存良好染色体形态传播的制剂。然而，这种方法是比较困难的完善和传播核的质量显着地发生变化，当一个滑动件的一个区域被与其他区域相比。这个问题变得更复杂，涉及很多成像细胞核未选定从一个幻灯片，以避免数据采集偏见定量方法。第二种染色体传播方法，通过Loidl和克莱因1发达，要平衡固定由多聚甲醛，用裂解和染色质解由洗涤剂溶液提升。当适当地进行，该方法提供了用更少的区域对区域的变化非常可重复的结果相比，德莱和吉鲁方法。本演讲将重点在modified版，因为它的可靠性和简易Loidl和克莱因的方法，。 染色体扩展并不复杂或费时;高达100幻灯片可以用于免疫染色在一天制备。此外，传播制剂可贮存在冷冻多年之前免疫染色，因此实验室可以开发冷冻染色体差，可以当新的生物问题出现的或新的染色试剂变得可用可使用的存储库。 染色体扩频方法是最常用的与免疫染色和宽场荧光显微镜组合，但是也可能制备载玻片超分辨率光学显微镜方法如受激发射损耗（STED）显微镜。

<table><tbody><tr><td>Zymolyase</td><td>US Biological&amp;nbsp;</td><td>Z1004</td><td>Prepare 20 mg/mL solution in 50 mM Tris pH 7.5 supplemented with 2% glucose. Prepare fresh each experiment and store at 4&amp;deg;C until ready for use.</td></tr><tr><td>Lipsol&amp;nbsp;</td><td>L.I.P. Ltd&amp;nbsp;</td><td>no longer commercially available</td><td>Prepare 1% (v/v) solution in water. Store on ice.</td></tr><tr><td>NP-40</td><td>USB</td><td>19628</td><td>Prepare 1% (v/v) solution in water. Store on ice.</td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P2287</td><td></td></tr><tr><td>Slides</td><td>Corning</td><td>2948-75x25</td><td></td></tr><tr><td>Standard coverslip</td><td>Fisher</td><td>12-544-E or 12-540-B</td><td></td></tr><tr><td>High resolution coverslips</td><td>Fisher</td><td>12-542-B</td><td></td></tr><tr><td>Photo-Flo 200 solution</td><td>Kodak</td><td>P-7417</td><td>Prepare 0.2% (v/v) solution in water.&amp;nbsp;</td></tr><tr><td>TBS</td><td></td><td></td><td>137 mM NaCl, 2.7 mM KCl, 24.7 mM Tris, pH 8</td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td>Prepare a 1% (w/v) solution in TBS. Store at 4&amp;deg;C for up to a month.</td></tr><tr><td>Primary antibody</td><td></td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Donkey Anti-Rabbit</td><td>Invitrogen</td><td>A-21206</td><td></td></tr><tr><td>IgG (H+L) Antibody</td><td></td><td></td><td></td></tr><tr><td>Vectashield mounting media with DAPI</td><td>Vector&lt;br/&gt; Laboratories</td><td>H-1200</td><td></td></tr><tr><td>ProLong Gold</td><td>Invitrogen</td><td>P36930</td><td></td></tr><tr><td>Plastic slide box</td><td>Fisher</td><td>03-448-1</td><td>Store slides containing dried spreads in slots at -20&amp;deg;C. Also, use as a wet chamber.</td></tr><tr><td>Cardboard slide box</td><td>Fisher</td><td>12-587-10</td><td>Use to conveniently transport stained/sealed slides or store at 4&amp;deg;C.</td></tr><tr><td>Coplin jar</td><td>Fisher</td><td>08-816</td><td>Use as a wash basin for slides.&amp;nbsp;</td></tr></tbody></table>

surface spreading and immunostaining of yeast chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

传播核的外观需要依靠染色体固定和脱压实之间的平衡。即使当试剂被适当平衡，变异在染色体脱压实度可发生在相同的幻灯片之间和/或不同的幻灯片的不同区域。因此，图像被解释之前以幻灯片的给定区域差的质量进行评估。 可使用抗减数分裂重组蛋白来说明的"笼压"和"underspreading"的影响。在图1中 ，减数分裂细胞核双重免疫染色两种真核生物链交换的蛋白质...

Watch this Scientific Journal Video about Surface Spreading and Immunostaining of Yeast Chromosomes at JoVE.com

Surface Spreading and Immunostaining of Yeast Chromosomes

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

表面扩散和酵母染色体免疫染色

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

surface-spreading-and-immunostaining-of-yeast-chromosomes

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.9K 次观看.  University of Chicago. A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

视频: Surface Spreading and Immunostaining of Yeast Chromosomes

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

从斑马鱼精子细胞中培养染色体传播

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

科研

遗传学

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 ...

生物学

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 ...

生物化学

Yeast Colony Embedding Method

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

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

酵母殖民地嵌入方法

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

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

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 ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

使用荧光原位杂交对毛尿胃肠道中的念珠菌的可视化

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

免疫与感染

Microscopy of Fission Yeast Sexual Lifecycle

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

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

裂殖酵母性生命周期的显微镜

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

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 ...

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 ...