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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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+and+characterization+of+a+superfolder+green+fluorescent+protein.">Engineering and characterization of a superfolder green fluorescent protein.</a> Nat Biotechnol. 24 (1), 79-88 (2006).</li><li>Kitamura, K., Shimoda, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Schizosaccharomyces+pombe+mam2+gene+encodes+a+putative+pheromone+receptor+which+has+a+significant+homology+with+the+Saccharomyces+cerevisiae+Ste2+protein.">The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has a significant homology with the Saccharomyces cerevisiae Ste2 protein.</a> EMBO J. 10 (12), 3743-3751 (1991).</li><li>Tanaka, K., Davey, J., Imai, Y., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schizosaccharomyces+pombe+map3++encodes+the+putative+M-factor+receptor.">Schizosaccharomyces pombe map3+ encodes the putative M-factor receptor.</a> Mol Cell Biol. 13 (1), 80-88 (1993).</li><li>Motegi, F., Arai, R., Mabuchi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+two+type+V+myosins+in+fission+yeast,+one+of+which+functions+in+polarized+cell+growth+and+moves+rapidly+in+the+cell.">Identification of two type V myosins in fission yeast, one of which functions in polarized cell growth and moves rapidly in the cell.</a> Mol Biol Cell. 12 (5), 1367-1380 (2001).</li><li>Sajiki, K., Pluskal, T., Shimanuki, M., Yanagida, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomic+analysis+of+fission+yeast+at+the+onset+of+nitrogen+starvation.">Metabolomic analysis of fission yeast at the onset of nitrogen starvation.</a> Metabolites. 3 (4), 1118-1129 (2013).</li><li>Miyawaki, A., Nagai, T., Mizuno, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+protein+fluorophore+formation+and+engineering.">Mechanisms of protein fluorophore formation and engineering.</a> Curr Opin Chem Biol. 7 (5), 557-562 (2003).</li><li>Dyer, J. 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=Tracking+shallow+chemical+gradients+by+actin-driven+wandering+of+the+polarization+site.">Tracking shallow chemical gradients by actin-driven wandering of the polarization site.</a> Curr Biol. 23 (1), 32-41 (2013).</li><li>Beta, C., Bodenschatz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+tools+for+quantitative+studies+of+eukaryotic+chemotaxis.">Microfluidic tools for quantitative studies of eukaryotic chemotaxis.</a> Eur J Cell Biol. 90 (10), 811-816 (2011).</li><li>Lee, S. S., 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=Quantitative+and+dynamic+assay+of+single+cell+chemotaxis.">Quantitative and dynamic assay of single cell chemotaxis.</a> Integr Biol (Camb). 4 (4), 381-390 (2012).</li></ol>

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?...

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。

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			<td height="17" style="height:17px;width:219px;">Glucose</td>
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			<td>Sigma-Aldrich</td>
			<td>1.05108.0050</td>
			<td></td>
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			<td>Sigma-Aldrich</td>
			<td>71381</td>
			<td></td>
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			<td>Sigma-Aldrich</td>
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			<td>AppliChem</td>
			<td>A1038,0250</td>
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			<td>Sigma-Aldrich</td>
			<td>69850</td>
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			<td>AppliChem</td>
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			<td>AppliChem</td>
			<td>A2344,0500</td>
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			<td>Promega</td>
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			<td>Merck</td>
			<td>1.01217.1000</td>
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			<td height="17" style="height:17px;">L-Leucine</td>
			<td>Sigma-Aldrich</td>
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			<td>Reactolab</td>
			<td>92045-74-4</td>
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			<td>Reactolab</td>
			<td>7005600</td>
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</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。...

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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14.4K 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

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

科研

生物学

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 ...

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 ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

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 ...

Sexual Development and Ascospore Discharge in Fusarium graminearum

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces fruiting bodies, called perithecia, on carrot agar. Perithecia contain numerous tissue types, produced at specific stages of perithecium development. These include (in order of appearance) formation of the perithecium initials (which give rise to the ascogenous hyphae), the outer wall, paraphyses (sterile mycelia which occupy the center of the perithecium until the asci develop), the asci, and the ascospores within the asci14. The development of each of these tissues is separated by approximately 24 hours and has been the basis of transcriptomic studies during sexual development12,8. Refer to Hallen et al. (2007) for a more thorough description of development, including photographs of each stage. Here, we present the methods for generating and harvesting synchronously developing lawns of perithecia for temporal studies of gene regulation, development, and physiological processes. Although these methods are written specifically to be used with F. graminearum, the techniques can be used for a variety of other fungi, provided that fruiting can be induced in culture and there is some synchrony to development. We have recently adapted this protocol to study the sexual development of F. verticillioides. Although individual perithecia must be hand picked in this species, because a lawn of developing perithecia could not be induced, the process worked well for studying development (Sikhakolli and Trail, unpublished).
The most important function of fungal fruiting bodies is the dispersal of spores. In many of the species of Ascomycota (ascus producing fungi), spores are shot from the ascus, due to the generation of turgor pressure within the ascus, driving ejection of spores (and epiplasmic fluid) through the pore in the ascus tip2,7. Our studies of forcible ascospore discharge have resulted in development of a "spore discharge assay", which we use to screen for mutations in the process. Here we present the details of this assay.
F. graminearum is homothallic, and thus can form fruiting bodies in the absence of a compatible partner. The advantage of homothallism is that crossing is not necessary to generate offspring homozygous for a particular trait, a facet that has facilitated the study of sexual development in this species14,7. However, heterothallic strains have been generated that can be used for crossing5,9. It is also possible to cross homothallic strains to obtain mutants for several genes in one strain1. This is done by coinoculating one Petri dish with 2 strains. Along the meeting point, the majority of perithecia will be recombinant (provided a mutation in one of the parent strains does not inhibit outcrossing). As perithecia age, they exude ascospores en masse instead of forcibly discharging them. The resulting spore exudate (called a cirrhus) sits at the tip of the perithecium and can easily be removed for recovery of individual spores. Here we present a protocol to facilitate the identification of recombinant perithecia and the recovery of recombinant progeny.

Sexual Development and Ascospore Discharge in Fusarium graminearum

Sexual crosses and isolation of recombinant progeny are important research tools for the filamentous fungus, Fusarium graminearum, The techniques necessary successfully carry out these processes are presented.

性发展和有性放电禾谷镰刀菌</em

Fusarium graminearum has become a model system for studies in development and pathogenicity of filamentous fungi. F. graminearum most easily produces ...

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.

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 ...

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 ...