AV是由EMBO长期博士后奖学金支持。研究马丁实验室由ERC启动赠款（GeometryCellCycle）和瑞士国家科学基金会资助（31003A_155944），以SGM资助。

裂殖酵母的有性繁殖的显微镜分析 1.媒体准备<ol><li>葡萄糖:10克/升，KH 2 PO 4:1克/升，氯化钠:通过混合以下组分制备的最小孢子形成培养基（MSL-N），15为0.1g / L时，用MgSO 4·7H 2 O 0.2克/ L.添加痕量元素（10000）:100微升/升，维生素（1000倍）1毫升/升，和0.1M的CaCl 2 &#160;毫升/升。过滤消毒用0.22微米孔径的过滤器，并储存于室温（RT）。 <ol><li>使用下面的维生素（1000倍）的股票:泛酸钙1克/升，烟酸:10克/升，肌醇:10克/升，生物素:10毫克/升。过滤消毒用0.22微米孔径的过滤器和储存在4℃。 </li><li>使用下面的微量元素（10000）股票:硼酸5克/ L，硫酸锰4:4克/升，硫酸锌4·7H 2 O:4克/升，氯化铁2·6H 2 O:2克/升，的MoO 3:0.4克/升，碘化钾1克/升， 硫酸铜·5H 2 O:0.4克/升，柠檬酸:10克/升。过滤消毒用0.22微米孔径的过滤器和储存在4℃。 </li></ol></li><li> 10毫升MSL-N与0.2克琼脂糖结合准备MSL-N琼脂糖2％（用来制造的琼脂糖凝胶垫室）。熔融一种用于在微波炉中于高功率〜2分钟，直到琼脂糖溶解并等份0.5毫升在微量离心管中。存放在室温下。 </li><li>制备用氮气（MSL + N）从MSL-N的最小孢子形成培养基中加入（NH 4）2 SO 4:1克/升，亮氨酸:0.225克/升，腺嘌呤:0.225克/ L尿嘧啶:0.225克/升。过滤消毒使用0.22微米孔径的过滤器。存放在室温下。 注意:亮氨酸，腺嘌呤和尿嘧啶加入到该培养基中，以允许营养缺陷型菌株的生长。附加氨基酸补充应包括如果所使用的菌株携带不同的营养缺陷型（例如his3Δ）。请注意具有完全养型菌株的工作建议，因为只有这种菌株将允许高效率的交配。 </li><li>准备200毫升VALAP为室的封闭。在烧杯中添加羊毛脂，凡士林（或其它凡士林）和石蜡的相等权重。在低温热混合并搅拌，直到偶尔充分混合。分装组合成几个小的培养皿中。存放在室温下。 </li></ol> 2.培养裂殖酵母的菌株进行交配实验（图1）。 <ol><li>每日1次，晚上: 从接种固体培养基新鲜的划线株到装有3毫升的MSL + N培养管。使用约2.5厘米直径的平底管。使用其他培养管或瓶用适合培养体积。孵育过夜（O / N），在25℃，200rpm振荡。如果有异宗株工作，分别接种。 <ol><li>稀释细胞悬液媒体第二天早晨，确保文化都有光密度在晚上的0.4-0.8 600纳米（OD 600）测量。 注意:OD 600测量的细胞浓度为裂殖酵母的代理都在大约0.1-1.0的范围内呈线性关系。对于我们的分光光度计OD 600 = 0.1对应于每毫升1.4×10 6个细胞。因此，如果初始OD 600读取该范围外的样品需要稀释/浓缩对可靠的测量。 </li></ol></li><li>第2天，晚上: 稀释细胞在20ml的MSL + N媒体来OD 600 = 0.025在100毫升的烧瓶中。株孵育O / N在30℃，200转。 注:野生型细胞培养应达到OD 600〜0.8第二天早上（15小时后）。如果用更长的生成时间株的工作进行相应调整的稀释。 </li><li> 3日，上午: 测量外径600来验证的文化有外径600〜0.8的细胞密度。如果有异宗株工作，混合伴侣细胞的数目相等。沉淀的细胞以1000 xg离心并转移到1.5ml的管。洗涤细胞三次在1ml MSL-N培养基。重悬的细胞在3ml MSL-N培养基中并稀释细胞至OD 600 = 1.5。 <ol><li>为了监测妹妹细胞之间的交配直接安装细胞成像（见协议第2.4节）。使用同宗配合H90株，其中氮剥夺后出现的有丝分裂过程中交配型开关会发生。在琼脂糖垫细胞直接安装确保了细胞分裂后，姐妹细胞保持彼此相邻。 </li><li>监测细胞极化（探索性动力学和什穆）孵育1-3毫升细胞培养物在30℃，200rpm下为3-4安装细胞之前用于成像小时。在这些3-4小时，最后有丝分裂会时有发生。一些细胞会已经开始两极分化，但大部分将只是开始他们探索的极化动态。 </li><li>监测细胞 - 细胞融合孵育1-3毫升细胞培养物在30℃，200rpm下4-6小时PRI或安装细胞成像。 </li><li>监视 融合后事件孵育在30℃下1-3毫升细胞培养物，200转用于安装细胞成像前8小时。 </li><li>并监控响应于特定的信息素浓度（仅用于异宗株）孵育3毫升细胞培养物在30℃，200rpm下3-4小时之前安装细胞成像。 <ol><li>请从95℃加热块含有MSL-N-离心管（见协议2.4.1节。下同）。在所需的浓度直接在熔融MSL-N琼脂糖添加的P-因子或M-因子。通过直接垫前的准备涡旋拌匀。 </li><li>电池安装后，在25℃反应垫15-30分钟成像之前。 P-因子和M-因子信息素是从1毫克/毫升在甲醇中的储备液使用。 注意:如果用突变型工作菌株的培养时间可以变化。在液态间延长温育后，可能会发生细胞凝集EDIA并可能在某些突变株特别强。成群细胞不能在单层点样到琼脂糖垫，因此很难进行自动对焦和图像。在广泛的细胞聚集的情况下，安装在琼脂糖垫细胞洗涤之后立即以及在氮缺乏媒体重新悬浮（如在协议第2.3.1。），并在30℃下成像之前孵育它们。 </li></ol></li></ol></li><li>安装成像单元格: <ol><li>通过在95℃加热块熔化的等分试样10-15分钟，加入由隔离物（ 图1B）分隔开的两个载玻片之间将200μl制备的MSL-N琼脂糖垫16。使用间隔物的厚度约0.5毫米。使间隔件，使用条带切出的纸板，如用限制性酶递送。 </li><li>沉淀100μl的细胞在1,000×g离心1分钟，除去上清液并重新悬浮于剩余的2-4微升培养基的细胞。在新鲜培养基不要再暂停，因为它延缓交配。 </li><li> 2-3分钟后小心地取出垫片和顶部的玻璃幻灯片。如果几株要安装，准备准备琼脂糖垫前的细胞集中。 </li><li>加入1μl细胞对垫，等待下跌始于盖玻片覆盖，并与VALAP密封前干燥（约1分钟）。 </li><li>让垫在25℃或RT坐成像前至少30分钟。 </li><li>以获得在交配的不同阶段的细胞的混合物，使一个垫立即在MSL-N（见2.3节）。洗涤后，在18°的CO / N（〜15小时）下孵育。这种方法是在具有高时间分辨率和样品信号弱的不同阶段交配细胞成像有用​​。 </li></ol></li></ol>交配的酵母细胞3.活细胞成像<ol><li>调整图像采集设置。 注意:这里介绍的图像采集设置定制奥林巴斯IX-71组成的DeltaVision平台进行了优化倒置显微镜载脂蛋白计划60X / 1.42 NA或U计划阿婆100X / 1.4 NA目标，一个CoolSNAP HQ2照相机和洞察SSI 7种颜色组合单元发光。硬件是由softWoRx V4.1.2也使数字自动对焦控制。 <ol><li>确保自动对焦间隔不超过15分钟，因为在Z漂移在更长的时间可以超越该软件自动对焦系统的容量。如果使用基于硬件的自动对焦系统，使用较大的区间。 </li><li>调整摄像间隔。用荧光标记蛋白的第一次工作时拍摄图像每10分钟为〜15小时为起点。这个间隔提供整个交配过程中没有广泛漂白一个很好的概述。 <ol><li>图像用5分钟间隔或更短更精确的时间的事件，如融合物。更短的间隔需要强大的荧光，从而使低曝光时间和小漂白。对于低于1分钟，避免O / N显微镜成像时间间隔而是准备所有配合阶段的混合物作为在协议第2.4.6节详细描述。 </li></ol></li><li>调整图像的曝光时间。 50和300毫秒之间测试的曝光时间。旨在撞击信噪比和漂白之间的平衡作为具有可辨别信号（典型地超过100个），以实现大量的时间点。 </li><li>调整Z-切片。每节0.3微米占地5微米提供了良好的成像深度。注意，Z-切片进一步增加光损伤，这可以通过使用OAI（光轴集成 - 单次扫描采集的总样本深度的）最小化。良好的自动对焦系统，减少了许多Z-部分的需求，并强烈推荐。 </li></ol></li><li>小贴士研究特定型交配行为: <ol><li>为异宗菌株，使用H-和h +细胞表达不同的荧光团，以便两种细胞类型可以区分开来。使用任何基因编码的fluoropho重新表现为胞质版本或融合到一个内源性蛋白，虽然我们曾与sfGFP，GFP 17的快速折叠变种良好的成功。内源性蛋白通常通过同源重组14标记在它们的内源基因组基因座。 </li><li>为同宗配合菌株，表达交配型特异性启动子的控制下的基因编码的胞质荧光蛋白如信息素受体 ​​基因MAP3和mam2的驱动在P和M细胞中的表达，分别。 MAP3和mam2的启动子是营养生长过程中无效，对氮饥饿18,19引起的。构建这些启动子的控制下，驱动胞质GFP或mCherry的表达通常集成在一个基因座（ura4，LEU1）不与性生活周期12的正常进展干扰。 </li></ol></LI&gt; <li>使用交配型特异性胞质荧光团（如在协议第3.2.2节）确定形象化为荧光信号从一个伴侣细胞​​转移到另一个细胞融合的精确定时。 </li></ol> 4.配合和融合效率的量化<ol><li>为了后直接MSL-N量化交配和融合的效率11,12，现货细胞洗到MSL-N琼脂糖片，如上面的步骤2.3.1，并在25℃前成像（ 图1A）放置24小时。使用这种协议的同宗配合野生型菌株交配效率达到〜60％，融合效率99％。为了测试在突变株观察到的这些值的任何降低是否反映终端表型或延迟，重复使用36小时的培养时间的实验。 </li><li>计算交配效率<img alt="公式1" src="/files/ftp_upload/53801/53801eq1.jpg" />交配对包括受精卵，ASCI，并确定了非融合细胞对他们对彼此的地位和增长。 </li><li>计算融合效率<img alt="方程2" src="/files/ftp_upload/53801/53801eq2.jpg" /> 注:熔融接合对由它们如果已知该所研究的突变体不影响芽孢形成孢子的能力来鉴定。如果孢子形成不能被用作读出，融合是通过观察在只有一个配合伙伴表示成两个伙伴胞质标记的扩展来确定。 </li></ol>

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The authors declare that they have no competing financial interests.

环境条件和养分有效性，特别是强烈地影响裂殖酵母生理学。氮饥饿是必要的承诺有性生殖，最初导致在有丝分裂细胞周期进展引人注目的变化（参考文献21和图2）。在从指数增长的人口脱氮，在分裂细胞体积迅速降低（ 图2C）和大部分细胞有丝分裂逮捕比进展新生成倍增长的细胞（参考文献21和图2D）的长度短。由于对面交配类型的细胞有丝分裂逮捕进展他们搞交配的合作伙伴，并继续形成受精卵，孢子（ 图2B，2E，2F）。在野生型细胞中的二维单层，用H90的细胞的百分之六十的情况下，将接合的合作伙伴，以及几乎所有将进行细胞融合（ 图2G </strong&gt;的）。交配效率是在异宗菌株的混合物稍低，并且取决于细胞的密度和它们的相对位置（ 图2G）。一项所述的协议，以获得高交配效率的最关键方面是确保细胞在整个保持所指示的密度范围内。 如图2的细胞的大小和交配效率参数监测然后提供细胞被有效地进入有性生殖的简单控制。 我们注意到，由于交配培养基缺乏任何氮源（即使是低量的氨基酸和氮碱被排除在外），高交配效率，如在图2E和2G呈现仅与完全原养型菌株得到。营养缺陷型菌株可使用，但需要额外的小心，以确保细胞在任何时候都保持在协议规定的细胞密度范围内。即使照顾，只下交配效率会被观察到。交配效率也受温度的影响，在升高的温度下观察到的低效率，在交配方法，该方法可以限制使用温度敏感突变体等位基因。 这里介绍的方法主要用于荧光报告长期成像。因为成像在较长的时间段进行的，裂殖酵母交配成功成像是高度依赖于可用的显微镜​​设置。对于这一点，在显微镜设置，它的灵敏度和获得自动聚焦系统的稳定性是至关重要的。或者基于软件的或内置的硬件自动对焦系统是可能的。另外，为了提高吞吐量，电动载物台使点对多点采集是另一个重要的质量。 成像质量是由感兴趣的荧光团的性质的限制。在Protoco详细的液体MSL-N中的不同的孵化时间L截面的2.3旨在优化信息素诱发的标记蛋白的荧光和成像的兴趣只交配阶段避免不必要的漂白。虽然丰富的或高度富有针对性的荧光标记的蛋白质可以采集数百个时间点在整个生命周期中的性（ 电影S3）的过程中，其他蛋白质在如此长的时间，由于光漂白具有挑战性的形象。图像质量还取决于所选择的荧光团，典型地为绿色荧光蛋白衍生物。我们多次指出，sfGFP 17（GFP超折叠），这与快速动力学褶皱，在交配过程中提供出色的信号，可能是因为强大的自噬诱导快速蛋白质周转。重要的是，我们没有监测必要荧光蛋白成熟17,22氧气供应。因此，建议在使用荧光测量来量化细胞后表达的蛋白质的水平是mounte谨慎ð到幻灯片，或使用替代透氧微流体装置进行这样的实验。此外，琼脂糖垫发现在晚上细胞，并保持在18ºCO / N一直到图像弱记者非常有用，因为这样的衬垫含有细胞的混合物在有性生殖的所有阶段。 配合在裂殖酵母不呈现同步和细胞可容易地看出发起什穆与其他细胞已形成孢子（ 影S1，S2）。例如人口动态呈现古典，散装生化技术很难使用，使得这里所描述的选择的方法，单细胞显微镜。所有基于显微镜的办法原则上可以应用于与此处描述的方案制备的细胞。重要的是，这些方法可以监控显示出交配型特异性动力学诸如通过Dudin和同事12所描述的细胞-细胞融合过程。为了监测不对称，交配型记者已经是特别有用的（ 图3A）。两个配合类型区分采用不同的荧光标记的H-蛋白质和H +细胞株异宗工作时，很容易实现。然而，这不是对同宗配合菌株是可行的。开发分化H90细胞的交配型的一种方法是表达交配型特异性启动子的控制下胞质荧光蛋白。特别是，使用了信息素受体 ​​基因MAP3和mam2的启动子分别驱动P中GFP或mCherry蛋白和M细胞中的表达。此外，这些标记允许的细胞 - 细胞融合的精确定时，可视荧光信号从一个伴侣细胞​​转移到另一个。 交配信息素是通过有性繁殖的不同阶段酵母11进步的关键因素，23，不同水平的合成信息素导致在裂殖酵母不同细胞的偏振状态（ 图3B，3C和参考文献11）。上面包括该协议允许不同的评价外源性信息素的水平如何影响细胞生理学。由于信息素蛋白酶Sxa2快速降解的P-因子，异宗突变株，其中所述蛋白酶被删除用于获得可再现的结果。然而，在相反交配型，不仅水平，而且由一配合伙伴发射信息素的空间分布的细胞的混合物，都可能是重要的。在分析细胞的反应信息素的空间分布的影响将需要更复杂的设置，如对芽殖酵母24,25采用微流控设备的开发。 总之，我们提出了裂殖​​酵母的性生命周期的长期显微镜的基本方法。该协议的小幅调整允许researCH专注于性生命周期的不同阶段的细胞反应。结合单细胞生物化学工具和微流体器件这种做法将进一步推动裂殖酵母的有性繁殖作为一个强大的模型系统。

虽然两个小区之间的基因交换是有性繁殖中心事件，它依赖于促进细胞分化，允许合作伙伴的选择，进行细胞 - 细胞融合和维持基因组稳定性的事件链。因而性生命周期呈现本身作为一个模型系统以研究了许多关于发育开关生物学问题，响应于外在刺激，质膜融合，染色体分离等探索裂殖酵母性周期来研究这些现象带来的好处模型系统强大的基因，建立了完善的高通量的方法和先进的显微镜。性裂殖酵母是P细胞和不同交配类型的M-细胞之间的异型事件。两种细胞类型中差异表达许多基因1,2-包括那些用于生产分泌P-的和M-外激素，信息素受体​​MAP3和Mam2以及信息素山龙眼的SES Sxa1和Sxa2。同宗配合菌株，如常用的H90株，携带两种交配型的遗传信息在单个基因组和细胞经历的交配型开关在整个分裂周期（在文献3中综述）的复杂模式。异宗裂殖酵母的多个分离物很少或从不切换交配型也是常用的4，最突出的是H + N（P型）和h -S（M型）的菌株。 在裂殖酵母，进入生命周期中的性正在严格的营养调控。只有氮饥饿裂殖酵母细胞有丝分裂逮捕繁殖和产生扩散信息素信号交配伴侣的存在和推广性周期（参考文献5审查）的进一步措施。氮剥夺脱阻抑交配Ste11的关键转录调节充当发育开关并促进È交配特定基因，包括信息素受体 ​​和信息素产生的基因6,7的表达。信息素受体 ​​参与激活受体偶联蛋白G-alpha和下游MAPK信号进一步增强Ste11转录活性8-10，从而增加交配伙伴之间的正反馈信息素的生产。信息素水平是通过调节细胞极性，卢家族GTP酶Cdc42的11的主组织者诱发不同的细胞极化状态至关重要。在暴露于低信息素浓度，活性的Cdc42被可视动态补丁探索细胞外围，并且没有细胞生长在此阶段观察到。增加信息素水平促进Cdc42活性的稳定到一个区和一个偏振投影的生长，称为SHMOO，这使伴侣细胞在接触。随后，两人单倍体交配伴侣融合形成二倍体合子。最近的研究表明日Ë存在一个新颖的肌动蛋白结构融合基本是由配合诱导formin FUS1 12组装的。这种融合焦点集中型-V的肌球蛋白依赖性过程并定位细胞壁降解机械，从而使细胞壁重塑，以允许无细胞裂解12质膜接触。在细胞 - 细胞融合，核所接触并进行核融合。受精卵（马尾巴的运动）的内部核心的一个突出动力蛋白依赖的背部和往复运动进而促进染色体同源13,14，随后是减数分裂的配对。最后，四款产品减数分裂的孢子时打包成单个孢子。 由于其复杂性和所涉及的许多步骤中，交配的详细监测已具有挑战性。两个显着的困难是，整个过程需要以及超过十五小时，使细胞难以同步。这些ðifficulties由单细胞显微镜方法规避。在这里，一般的协议，调查裂殖酵母的性周期呈现。用小的调整，此协议允许的所有不同的步骤的过程中，配合基因产物，交配型切换之后姐妹细胞非姐姐伙伴之间和之间细胞极化和配对，细胞 - 细胞融合的即感应的研究，和后融合的马尾巴的运动，减数分裂和孢子。此方法允许1）轻松地可视化荧光标记的蛋白随着时间的推移售前，售中和融合后; 2）鉴别相反交配型的细胞的行为;和3）测量和量化参数，如什穆，交配，融合或孢子形成的效率。

<table><tbody><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G8270-10KG</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>1.05108.0050</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>71381</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>63140</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>12095</td><td></td></tr><tr><td>Pantothenate</td><td>AppliChem</td><td>A2088,0025</td><td></td></tr><tr><td>Nicotinic Acid</td><td>AppliChem</td><td>A0963,0100</td><td></td></tr><tr><td>Inositol</td><td>AppliChem</td><td>A1716,0100</td><td></td></tr><tr><td>Biotin</td><td>AppliChem</td><td>A0967,0250</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B6768-1KG</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;</td><td>AppliChem</td><td>A1038,0250</td><td></td></tr><tr><td>ZnSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Z4750</td><td></td></tr><tr><td>FeCl&lt;sub&gt;2&lt;/sub&gt;&amp;#8226;6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>AppliChem</td><td>A3514,0250</td><td></td></tr><tr><td>Molybdenum oxide (VI) (MoO&lt;sub&gt;3)&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>69850</td><td></td></tr><tr><td>KI</td><td>AppliChem</td><td>A3872,0100</td><td></td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>AppliChem</td><td>A1034,0500</td><td></td></tr><tr><td>Citric Acid&amp;nbsp;</td><td>AppliChem</td><td>A2344,0500</td><td></td></tr><tr><td>Agarose</td><td>Promega</td><td>V3125</td><td></td></tr><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Merck</td><td>1.01217.1000</td><td></td></tr><tr><td>L-Leucine</td><td>Sigma-Aldrich</td><td>L8000-100G</td><td></td></tr><tr><td>Adenine Hemisulfat Salt, mini 99%</td><td>Sigma-Aldrich</td><td>A9126-100G</td><td></td></tr><tr><td>Uracil&amp;nbsp;</td><td>Sigma-Aldrich</td><td>U0750</td><td></td></tr><tr><td>Lanolin</td><td>Sigma-Aldrich</td><td>L7387</td><td></td></tr><tr><td>Vaseline</td><td>Reactolab</td><td>92045-74-4</td><td></td></tr><tr><td>Paraffin</td><td>Reactolab</td><td>7005600</td><td></td></tr></tbody></table>

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.

裂殖酵母生长和配套动力一旦氮源的去除 作为氮饥饿是用于裂殖酵母有性生殖的起始的一个先决条件，野生型同宗配合H90菌株在从氮剥夺介质的富氮（ 图2）移位监视，以下在图1中所概述的协议。简言之，将细胞在MSL + N培养基中生长的O / N至指数期（OD 600 = 0.5），收集，洗涤并再悬浮于MSL-N液体培养基至最终OD 600 = 1.5。细胞每2小时等分是calcofluor染色和成像（ 图2A - D）。 Calcofluor染色（ 图2A）表明，在富氮介质〜细胞的21％被septating（N&gt; 300， 图2B），并在迪维该平均气泡长度锡永为15.2±1.4微米（50， 图2C）。非分裂细胞显示细胞长度的宽分布（8-14微米; N&gt; 200， 图2D）。然而，移位到MSL-N介质后两小时有在非分裂细胞的长度分布的急剧变化，细胞是短于9微米（N&gt; 200， 图2D）的60％以上。在9.8±0.8微米（50， 图2A，2C）的减小的长度也被检测到的小区septation。在两个非分裂和分裂细胞的长度进一步减少，也观察到，随着时间在MSL-N增加。事实上，经过8小时的文化septation指数下降2％以下（N&gt; 300）和细胞超过85％的长度小于7微米（N&gt; 200， 图2D）逮捕他们的细胞周期。 细胞开始形成的移位到液体MSL-N之后交配对4小时介质（&lt;1％， 图2A）。接着交配对的数量迅速增加，细胞的移10％后8小时接合的配合伙伴（N&gt; 200， 图2B）。 并监控交配动力学，6小时饥饿诱导后，将细胞的等分试样也安装在用于成像的MSL-N琼脂糖垫。细胞在随后的21小时进行什穆，融合和产孢（ 图2E，2F）没有任何明显的同步性（ 电影S1）。在单层（ 图2G和电影S1）密集设置时的配合效率取决于在视给定的场的相对位置和细胞密度，与细胞的60％以上接合的伙伴。 野生型异宗H +和H-裂殖酵母菌株也进行相同的协议与在除氮的时间混合的H +和H-细胞在一对一的比率的n个附加步骤。观察到类似的饥饿和交配的动态，但交配效率略低（ 图2G，电影S2）。 监测荧光标记蛋白动力学交配裂殖酵母细胞 从本机座表达Myo52-3GFP要监视型-V球蛋白Myo52（参考文献20）的动态交配细胞成倍增长H-细胞H +细胞同样从P表达Myo52-tdTomato，以及胞质GFP涨跌互现β细胞特异性启动子MAP3。将细胞悬浮液洗涤，稀释在MSL-N培养基，并在30°C以上孵育6小时，以200rpm之前安装细胞到用于以10分钟的间隔O / N成像MSL-N琼脂糖垫。 据此前报道11,12 Myo52整个细胞皮质初步形成的动态区域（ 图3A，箭镞和电影S3）。然后Myo52信号被稳定化（ 图3A中，箭头和电影S3）在单个焦点只是融合事件，该事件是由细胞内的GFP信号从H +进入H-细胞转印可视之前（ 图3A和电影S3） 。所述Myo52-tdTomato信号也可以在融合颈部其膨胀期间观察到，但作为受精卵进行到孢子形成（ 图3A和电影S3）没有可辨别信号是显而易见的。 监测异宗裂殖酵母细胞受到外部信息素行为 缺乏Sxa2蛋白酶降解P-因子异宗脱敏H-细胞容易合成P-因素作出反应。一个H- </EM&gt;sxa2Δ应变，表达SCD2-GFP作为Cdc42活性11的标志，是生长在MSL + N媒体，因此所描述的协议。细胞在MSL-N培养基中洗涤，并在30°C以上孵育4小时。含甲醇（数据未示出），0.1微克/毫升（低信息素， 图3B）或1微克/毫升的MSL-N琼脂糖垫（高信息素， 图3C）的P-因子的制备，切割和定位在一个新的幻灯片生成包含不同的信息素量微型垫。 0.5微升细胞悬浮液装在以5分钟的间隔各微型焊盘O / N成像。 在公布业绩11一致性，低P-因子水平提升动态SCD2区域无增长（ 图3B， 电影S4）的形成，而高含量P-因素稳定的单一SCD2区，从而从一个细胞极诱导SHMOO延伸（Figure 3C， 电影S5）。 <img alt="图1" src="/files/ftp_upload/53801/53801fig1.jpg" /> 图1:议定书示意图 （A）用于监测裂殖酵母的性周期和（ 二 ）长期成像准备裂殖酵母样的协议的示意图说明<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/53801fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53801/53801fig2.jpg" /> 图2:从MSL + N移到MSL-N媒体裂殖酵母细胞的生长和配套动力 calcofluor染色的细胞（A）落射荧光显微照片转移到MSL-N媒体所指示的时间周期。 （B）百分比septating的（深蓝色线，正按时间点&gt; 300）和交配（淡蓝色线，正按时间点&gt; 300）细胞转移到MSL-N媒体所指示的时间周期。 septating细胞（C）的平均长度转移到MSL-N媒体的时间所示时间（N = 50％的时间点，除了8小时的时间点，其中n = 20）。 （D）的非分裂细胞的长度分布转移到MSL-N介质的时间的指示的时间段（正&gt; 200每时间点）。 （ 五 ）未融合（深蓝色线）的平均分数，融合（淡蓝色线）和H90野生型酵母的群体形成孢子（黑线）细胞转移到MSL-N媒体所指示的时间周期，在时间点6小时（每时间点N&gt; 900细胞从三个不同的timelapses）安装到琼脂垫。这些细胞被认为在融合伙伴之间没有屈光细胞壁是用来得分SP在DIC显微照片和形成孢子的细胞壁可见orulating细胞。交配细胞（F）DIC显微照片转移到MSL-N媒体所指示的时间周期。 （G）的配合效率细胞密度对琼脂糖垫H90（深蓝色点）的函数和h + / H-（淡蓝色圆点）细胞转移到MSL-N媒体24小时。细胞长期成像的最合适的密度大约25,000个/ mm 2的实现。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/53801fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53801/53801fig3.jpg" /> 图3:用所示基因型细胞荧光蛋白的动态定位裂殖酵母交配期间 （A）的去卷积单z平面萤光显微照片从MSL + N转向MSL-N媒体6小时和安装到MSL-N琼脂糖垫在指定的时间。箭头指示动态Myo52-3GFP区和箭头融合重点指出Myo52定位。 （B）中，用表示基因型的细胞（C）的去卷积单z平面萤光显微照片从MSL + N转向MSL-N介质4小时，并安装在含有0.1微克/毫升的MSL-N琼脂糖微型垫（B）的或1微​​克的P-因子/毫升（℃）在指定的时间。箭头表示动态的（B）或稳定的（C）SCD2-GFP区域。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/53801fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="剧场1" src="/files/ftp_upload/53801/53801movie1.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie1.mov" target="_blank">补充电影1:</a> 野生型H90裂殖酵母CE的DIC缩时<html> LLS那名氮饥饿成像前6小时。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie1.mov" target="_blank">（右键点击下载）。</a> <img alt="剧场2" src="/files/ftp_upload/53801/53801movie2.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie2.mov" target="_blank">补充电影2:</a> 野生型H +和H-裂殖酵母细胞混合和氮饥饿成像前6小时缩时DIC <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie2.mov" target="_blank">（右键点击下载）。</a> <img alt="MOVIE3" src="/files/ftp_upload/53801/53801movie3.jpg" /> "&gt; 补充电影3: 去卷积单z平面萤光和从天然基因座表达Myo52-3GFP H-细胞DIC缩时，且h +细胞从P-细胞特异性MAP3启动子的天然基因座和胞质表达GFP Myo52-tdTomato混合。细胞在MSL-N中在30ºC生长6小时之前成像。细胞质GFP转移到H-细胞定义了融合的时间。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie3.mov" target="_blank">（右键点击下载）。</a> <img alt="Movie4" src="/files/ftp_upload/53801/53801movie4.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie4.mov" target="_blank">补充电影4:</a> 反褶积单z平面表面荧光和H- sxa2细胞EXP的DIC缩时ressing SCD2-GFP从其天然启动子与0.1微克/毫升的P-因子治疗。细胞在MSL-N介质在成像之前30ºC生长4小时。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie4.mov" target="_blank">（点击下载）。</a> <img alt="Movie5" src="/files/ftp_upload/53801/53801movie5.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie5.mov" target="_blank">补充电影5: 。</a> 去卷积单z平面萤光以及从与1μg/ ml的P-因子细胞治疗其天然启动子表达SCD2-GFP H- sxa2细胞DIC缩时在MSL-N介质在30ºC中生长4小时前成像。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53801/SupplementalMovie5.mov" target="_blank">（点击下载）。</a> <table fo:keep-together.within页="1"&gt; <tbody><tr><td> YSM995 </td><td> H-重量</td></tr><tr><td> YSM1371 </td><td> H +重量</td></tr><tr><td> YSM 1396 </td><td> H90重量</td></tr><tr><td> YSM2534 </td><td> H- myo52-3GFP :: kanMX </td></tr><tr><td> YSM2730 </td><td> H + myo52-tdTomato :: natMX Pmap3:GFP :: ura4 + @ ura4locus </td></tr><tr><td> YSM2731 </td><td> H- SCD2-GFP :: natMXsxa2Δ:: kanMX </td></tr></tbody></table> 表S1:用菌株在这项研究中。

Watch this Scientific Journal Video about Microscopy of Fission Yeast Sexual Lifecycle at JoVE.com

裂殖酵母性生命周期的显微镜观察|生物学 |视频

Microscopy of Fission Yeast Sexual Lifecycle

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 ...

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14.5K 次观看.  University of Lausanne. 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. 

视频: Microscopy of Fission Yeast Sexual Lifecycle

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

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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.

<meta charset="utf8"></head><body>使用荧光蛋白标记物可视化酵母细胞器

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

使用荧光蛋白标记物可视化酵母细胞器

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

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 ...

Spatiotemporal Analysis of Cytokinetic Events in Fission Yeast

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and development. Cytokinesis involves a series of events that are well coordinated in time and space. Cytokinesis involves the formation of an actomyosin ring at the division site, followed by ring constriction, membrane furrow formation and extra cellular matrix remodeling. The fission yeast, Schizosaccharomyces pombe (S.&#160;pombe)&#160;is a well-studied model system that has revealed with substantial clarity the initial events in cytokinesis. However, we do not understand clearly how different cytokinetic events are coordinated spatiotemporally. To determine this, one needs to analyze the different cytokinetic events in great details in both time and in space. Here we describe a microscopy approach to examine different cytokinetic events in live cells. With this approach it is possible to time different cytokinetic events and determine the time of recruitment of different proteins during cytokinesis. In addition, we describe protocols to compare protein localization, and distribution at the site of cell division. This is a basic protocol to study cytokinesis in fission yeast and can also be used for other yeasts and fungal systems.

The fission yeast, Schizosaccharomyces pombe&#160;is an excellent model system to study cytokinesis, the final stage in cell division. Here we describe a microscopy approach to analyze different cytokinetic events in live fission yeast cells.

在裂殖酵母中Cytokinetic活动的时空分析

Cytokinesis, the final step in cell division is critical for maintaining genome integrity. Proper cytokinesis is important for cell differentiation and ...

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 ...

Rapid Freezing using Sandwich Freezing Device for Good Ultrastructural Preservation of Biological Specimens in Electron Microscopy

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ultrastructure of cells; artifacts and extraction of cell contents are usually observed. Rapid freezing is a better alternative for the preservation of cell structure. Sandwich freezing of living yeast or bacteria followed by freeze-substitution has been used for observing the exquisite natural ultrastructure of cells. Recently, sandwich freezing of glutaraldehyde-fixed cultured cells or human tissues has also been used to reveal the ultrastructure of cells and tissues. 
These studies have thus far been carried out with a handmade sandwich freezing device, and applications to studies in other laboratories have been limited. A new sandwich freezing device has recently been fabricated and is now commercially available. The present paper shows how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. Also shown is the preparation of specimens for ultrathin sectioning after rapid freezing and procedures for freeze-substitution, resin embedding, trimming of blocks, cutting of ultrathin sections, recovering of sections, staining, and covering of grids with support films.

Here we show how to use the sandwich freezing device for rapid freezing of biological specimens, including bacteria, yeast, cultured cells, isolated cells, animal and human tissues, and viruses. We also show how to prepare specimens for ultrathin sectioning after rapid freezing.

使用夹层冷冻装置快速冷冻，在电子显微镜下对生物标本进行良好的超微结构保存

Chemical fixation has been used for observing the ultrastructure of cells and tissues. However, this method does not adequately preserve the ...

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 ...

裂殖酵母性生命周期的显微镜

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