这项工作是由澳大利亚研究理事会资助DP0986937的支持。

选择合适的控制酵母菌株检测依赖于实验者的视觉评估酵母液泡中积累的红色荧光。由于这样的实验是主观的，依赖于选择合适的对照菌株与生长条件。野生型控制是用来表明自噬一般预期的水平。作为阴性对照为核心的自体吞噬基因（例如，ATG3）的表达应变空是合适的，这种菌株可以不提供的材料（如线粒体，细胞核）液泡 1。酵母细胞的转化质粒DNA 酵母细胞可以很容易地改造主管LiAcetate治疗。可以购买商业试剂盒（例如，EasyComp，Invitrogen公司）公布的协议提供 13 。 在这个协议中，我们使用的野生型菌株BY4741（ 垫的his3Δ1leu2Δ0met15Δ0ura3Δ0），和一个缺失突变株，从它派生的（ 例如 ，atg3Δ），每一个不同的非必需基因的表达缺乏全面库的一些成员。缺失株库代表一个调查使用罗塞拉 - 基于探针的自体吞噬宝贵的的工具。记者在这个协议中采用的是MT -罗塞拉线粒体（ 图2A），由pAS1NB编码：MT -罗塞拉12。 下面的步骤是基于使用的细胞制成主管使用EasyComp转换套件和冷冻保存于-80 ° C，方便使用。 <ol><li>平衡解决方案三（EasyComp转换套件）室温30分钟，再加入酵母细胞。 </li><li> 2-2.5μL的质粒DNA编码记者（pAS1NB：MT -罗塞拉）（〜100毫微克/μL）添加到单元帽无菌1.7毫升塑料离心管。 </li><li>添加5μL刚解冻的主管酵母细胞的悬浮和轻柔吹打混合。 </li><li>添加50μL溶液III（EasyComp转换套件）和旋涡混合。 1.5）或手动用手指轻弹管，每15分钟使用旋涡混合器混合大力孵育1小时在28-30 ° C的转化反应。 </li><li>仔细转移转化混合物单一YMM琼脂生长板为辅组氨酸，蛋氨酸和尿嘧啶，然后轻轻地分发给周围的琼脂表面的细胞，用无菌玻璃吊具。酵母细胞在这个阶段是脆弱的和温柔的处理，可以增加产量的转化。 </li><li>孵育2〜4天或在28-30 ° C，直到菌落直径约2-3毫米，出现生长板。 </li></ol> 2。确认罗塞拉在酵母细胞中的表达正确的定位和高效表达罗塞拉开展详细的实验之前应得到证实。 <ol><li>用无菌牙签选择每一株5改造板，并在单独的无菌1.7毫升单元盖塑料离心管50μL无菌水悬浮细胞单菌落。 </li><li>将10μL的每一个细胞悬液到单独的标记玻璃显微镜载玻片（76 × 26毫米）和盖一个正方形的盖玻片（22 × 22毫米）的细胞悬液。 </li><li>立即研究荧光的细胞，利用荧光显微镜（例如，奥林巴斯Fluoview FV500）。寻找强烈的绿色和红色荧光和标签典型的线粒体定位（图2B和 2C） 。 </li><li>这样荧光检查每个殖民地的其余部分应接种到一个新的YMM生长板。 </li><li>在28-30 ° C孵育板为2天，直到殖民地直径约2-3毫米。 </li></ol> 3。细胞生长和诱导自体吞噬使用一些不同的实验条件，包括进入固定相，中碳的来源，管理雷帕霉素，或氮饥饿变化可诱发细胞自噬。我们经常使用的方法诱导mitophagy氮饥饿。 <ol><li>接种单菌落到10毫升增长含有的乙醇介质和适当的营养缺陷型补充。 </li><li>在28-30 ° C培养酵母的文化与轨道摇晃约48小时（200转）。细胞应在日志中增长阶段。 </li><li>重复1毫升单元盖无菌1.7毫升塑料离心2分钟在室温下在2000 XG离心管样品的文化沉淀细胞。小心取出上清液丢弃，而不会干扰细胞沉淀。 </li><li> 1毫升无菌蒸馏水重新悬浮离心水清洗细胞3次，以去除残留的米edium。 </li><li>第三洗净后悬浮细胞在生长介质或氮饥饿中的100毫升。 </li><li>重新悬浮细胞接种到10毫升新鲜生长培养基或10毫升的氮饥饿中等。孵育6-12小时的轨道晃动，让mitophagy感应。准备后，饥饿的细胞，荧光显微镜下观看。 </li></ol>注：标记与CMAC -精氨酸的液泡当第一次建立的检测是非常有用的确认，荧光显微镜下的罗塞拉运送到液泡。虽然酵母液泡是一个大型的细胞器，其位置也往往容易被透射光图像，液泡在某些菌株的酵母和一些生长条件下，可以分散和难以找到确定。 液泡可以很容易地使用香豆素基于CMAC -精氨酸（7 - 氨基- 4 - chloromethylcoumarin，L -精氨酸酰胺），如液泡染料标记。液泡蛋白酶的液泡的明亮的蓝色荧光标记染料的结果采取行动。蓝光发射，可以很容易地分辨罗塞拉11红色和绿色的排放。 CMAC -精氨酸标签并不需要经常进行，但它是为那些不熟悉的位置和酵母液泡外观建议。 4。液泡的标签与CMAC -精氨酸<ol><li> CMAC -精氨酸（终浓度为100毫米）的生长或氮饥饿的细胞成像之前。 CMAC -精氨酸溶液可以提前做好准备，但作为解决方案是光敏感，它需要保护暴露在光线下。 </li><li>在28-30 ° C孵育30分钟（200转）的轨道晃动。 </li><li>在2000 XG为2分钟，在室温下离心沉淀细胞。 </li><li>上清液。 </li><li>清洗细胞用无菌蒸馏水再悬浮离心去除残留的培养基和多余的CMAC -精氨酸染料的三倍。 </li><li>山为细胞成像，如下所述。 </li></ol> 5。安装激光扫描共聚焦显微镜活酵母细胞（CLSM） <ol><li>熔体中生长中期和氮饥饿中的单独的1毫升分装在70 ° C孵育2毫克低熔点琼脂糖熔化的琼脂糖是保持在70 ° C。 </li><li>轻轻颗粒细胞在每个细胞培养1毫升分装在2000 XG为2分钟，在室温下离心，丢弃上清液。 </li><li>清洗细胞再悬浮和离心1 ml无菌蒸馏水，在步骤5.2三次。 </li><li>重悬于100μL无菌蒸馏水洗涤细胞。 </li><li>要安装到一个标记的玻璃显微镜幻灯片（76 × 26毫米）的细胞当场熔化琼脂糖20μL。 </li><li>立即当场到琼脂糖床洗细胞悬液10μL。 </li><li>盖上盖玻片（22 × 22毫米）的琼脂糖床。琼脂糖表面细胞将分散在盖玻片下方。 </li><li>为了防止脱水和盖玻片的运动，在成像仔细密封薄膜盖玻片边缘的指甲油。 </li><li>当指甲油完全干燥（10分钟）幻灯片准备荧光显微镜下小心查看：指甲油可能会损坏显微镜物镜。 </li><li>细胞的安装，使用这种技术，可以观察到，安装后​​1小时。 </li></ol> 6。可视化自噬使用CLSM 自体吞噬在细胞的人口水平，定期评估，确定后显示红色的液泡荧光前的细胞数量和诱导自体吞噬。理想情况下，自噬的进展监测诱导自体吞噬（图3）在选定的时间点。通过显微镜双目可视化，这是最好。重要的是，采用正确的控制（例如，Δatg3突变）和显微镜设置保持不变，观察不同的样本（例如，使用中性密度过滤器，过滤器的选择）之间。 酵母细胞都比较小，需要配备适合GFP和/或pHluorin（绿色荧光）（FITC标记的过滤器）和DsRed.T3（红色单独可视化的激发/发射滤​​波器为质量好的荧光显微镜（例如，奥林巴斯Fluoview FV500）荧光）（TRITC过滤器）。 <ol><li>使用60X水客观地看待非饿死的野生型细胞，交替使用FITC和TRITC过滤器。野生型细胞应该表现出一个典型的线粒体细胞分布在红色和绿色荧光的细胞，周边。请注意，这种线粒体的分布依赖的pinhOLE设置的共聚焦显微镜。我们通常使用的125微米针孔价值较低，只允许线粒体作为观察到了一系列本地化在外围的细胞（ 图2C），液泡是不能掩盖 12点。如果一个针孔价值高（200微米），这使得典型的丝状线粒体网络分布在整个细胞（和它经历的变化），可观察到11。应荧光标记的细胞在没有其他结构。 </li><li>细胞转移到饥饿中等顺序查看每一个时间点。可视化的绿色和红色的排放过滤器交替使用。在6小时饥饿时间点红色荧光，但没有相应的绿色排放应有迹可寻在液泡。液泡本地化可以单独使用CMAC -精氨酸（ 图2C）证实。查看〜200细胞计数的数字只显示红色荧光在液泡（即线粒体液泡吸收）​​。 </li><li>计数&gt; 200个细胞，然后情节的细胞呈现空泡积累的百分比所有时间观察点液泡积累。 </li><li>检查的自噬阴性对照细胞表达前饥饿的MT -罗塞拉和6 h饥饿。 </li></ol> 7。代表性的成果： 在这里，我们显示使用MT -罗塞拉，在野生型和突变的细胞Δatg3表示获得的典型结果。与乙醇的生长条件下，作为碳源，1野生型和Δatg3突变的细胞表现出细胞分布的荧光（红色和绿色）在酵母细胞中线粒体的典型。红色和绿色荧光（ 图2B和2C）液泡中没有检测到。当受到氮饥饿6 h和超越的话，除了红色和绿色荧光相应线粒体，野生型细胞也表现出红色的积累，但不是绿色的荧光在空泡流明（图2B，图3） 。然而，在既不红也不绿荧光Δatg3突变的细胞积累在液泡流明（ 图2C，图3）。 <img src="/files/ftp_upload/2779/2779fig1.jpg" alt="图1" /> 图1，在酵母细胞的细胞器和车厢。计划。底部，酵母细胞的液泡为中心的观点是表明不同的细胞器和车厢自噬降解。各种细胞器的特定类型的自体吞噬（例如，mitophagy，nucleophagy）之间的差异包括吞没内容（货物选拔）的特异性，各种细胞内的需求和细胞外的线索（例如，饥饿，损坏）， 特定的信号，ATG基因和时间依赖性。 <img src="/files/ftp_upload/2779/2779fig2a.jpg" alt="图2a" /> <img src="/files/ftp_upload/2779/2779fig2b.jpg" alt="图2b" /> <img src="/files/ftp_upload/2779/2779fig2c.jpg" alt="图2c" /> <img src="/files/ftp_upload/2779/2779fig2d.jpg" alt="图2d" /> 图2。 （一）MT -罗塞拉的示意图。线粒体的前导序列（在这种情况下柠檬酸合成酶）是用于目标罗塞拉传感器线粒体基质。为红色荧光蛋白（DsRed.T3）和绿色荧光蛋白（pHluorin）的激发和发射最大值表示。生物传感器在高pH值（pH值线粒体〜8.2）荧光红色和绿色，然而，在低pH值（液泡pH〜5.5）的荧光只有红色（B）预期结果的示意图，在野生型和 Δatg3突变的细胞表达MT -罗塞拉（三）由CLSM实际得到的结果为承受着越来越大的野生型细胞和氮饥饿条件。从左至右依次为：迪爱生 ​​（对比度的区别），RFP（DsRed.T3）荧光，GFP（pHluorin）荧光，荧光CMAC -精氨酸和合并的图像（四）实际得到的结果Datg3突变的细胞在荧光显微镜生长和氮饥饿条件。从左至右依次为：DIC（对比度的区别），RFP（DsRed.T3）荧光，绿色荧光蛋白（pHluorin）荧光，荧光CMAC -精氨酸和合并图像。 <img src="/files/ftp_upload/2779/2779fig3.jpg" alt="图3" /> 图3。表达MT -罗塞拉和氮饥饿下生长的野生型和Δatg3突变的细胞，在液泡中，超过48小时的时间过程中显示红色荧光的百分比。记录显示红色荧光的积累在液泡的细胞比例为0，6，12，24和48小时后开始氮饥饿。

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	<li>Levine, B., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+in+the+pathogenesis+of+disease.">Autophagy in the pathogenesis of disease.</a> Cell. 132, 27-42 (2008).</li><li>Mizushima, N., Levine, B., Cuervo, A. M., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+fights+disease+through+cellular+self-digestion.">Autophagy fights disease through cellular self-digestion.</a> Nature. 451, 1069-1075 (2008).</li><li>He, C., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+mechanisms+and+signaling+pathways+of+autophagy.">Regulation mechanisms and signaling pathways of autophagy.</a> Annu. Rev. Genet. 43, 67-93 (2009).</li><li>Kroemer, G., Mari&#241;o, G., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+the+integrated+stress+response.">Autophagy and the integrated stress response.</a> Mol Cell. 40, 280-293 (2010).</li><li>Camougrand, N., Kissov&#225;, I., Salin, B., Devenish, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+mitophagy+in+yeast.">Monitoring mitophagy in yeast.</a> Methods Enzymol. 451, 89-107 (2008).</li><li>Yen, W. L., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+live+long+and+prosper:+autophagy,+mitochondria,+and+aging.">How to live long and prosper: autophagy, mitochondria, and aging.</a> Physiology (Bethesda). 23, 248-262 (2008).</li><li>Tolkovsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy.">Mitophagy.</a> Biochim. Biophys. Acta. 1793, 1508-1515 (2009).</li><li>Bhatia-Kissov&#225;, I., Camougrand, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitophagy+in+yeast:+actors+and+physiological+roles.">Mitophagy in yeast: actors and physiological roles.</a> FEMS Yeast Res. 10, 1023-1034 (2010).</li><li>Kanki, T., Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+mechanism+of+mitochondria+autophagy+in+yeast.">The molecular mechanism of mitochondria autophagy in yeast.</a> Mol. Microbiol. 75, 795-800 (2010).</li><li>Nowikovsky, K., Reipert, S., Devenish, R. J., Schweyen, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mdm38+protein+depletion+causes+loss+of+mitochondrial+K+/H++exchange+activity,+osmotic+swelling+and+mitophagy.">Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy.</a> Cell Death Differ. 14, 1647-1656 (2007).</li><li>Devenish, R. J., Prescott, M., Turcic, K., Mijaljica, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+organelle+turnover+in+yeast+using+fluorescent+protein+tags.">Monitoring organelle turnover in yeast using fluorescent protein tags.</a> Methods Enzymol. 451, 109-131 (2008).</li><li>Rosado, C. J., Mijaljica, D., Hatzinisiriou, I., Prescott, M., Devenish, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosella:+a+fluorescent+pH-biosensor+for+reporting+vacuolar+turnover+of+cytosol+and+organelles+in+yeast.">Rosella: a fluorescent pH-biosensor for reporting vacuolar turnover of cytosol and organelles in yeast.</a> Autophagy. 4, 205-213 (2008).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Methods Enzymol. 350, 87-96 (2002).</li></ol>

在图提出的代表图像。 2B和2C它可以清楚地看到，使用特定细胞器荧光记者和荧光显微镜的增长和氮饥饿细胞中的线粒体定位/分布提供的证据，并允许mitophagy进行可视化。 它强调的是，这种方法不仅限于以下mitophagy。合适的标签可能会被监控的其他细胞成分或细胞器（如核糖体，脂滴，过氧化物酶体，内质网和核），细胞自噬。罗塞拉生物传感器的构造，细胞培养条件和细胞核内的营业额（nucleophagy）的典型结果的详情已报11,12，说明了该方法的更广泛的应用。 监测自噬细胞器特定的荧光记者的需要使用构造设计的几个注意事项要牢记。这些措施包括：（1）选择和融合，以适当的定位信号，实现细胞器的具体目标;（2）选择一个合适的荧光蛋白（S）;（3）较强的荧光发射;（4）正确的细胞器的本地化和/或细胞内的分布。一旦特定的细胞器记者已成功地表达在选择酵母主机应变的考虑成为第二组：（1）（2）酵母的生长条件;细胞样品制备;（3）成像参数，因为它是重要的选择参数CLSM（例如，激光强度，扫描速度和模式，针孔值，缩放值，目标使用或CCD相机曝光）保持一个确保可以控制生长的细胞和饿死样品之间的比较试验;（ 3）自噬诱导和成功交付的记者到液泡。 总体而言，这种荧光显微镜方法是容易的，比较快，具有潜在的应用高通量筛选新的药物，抑制或增强吞噬，也为基因调节或调节自体吞噬。

<table border="1"><tbody><tr><th>试剂/设备名称</th><th>公司</th><th>目录编号</th></tr><tr><td> 酿酒酵母EasyComp转换套件</td><td> Invitrogen公司</td><td> K5050 - 01 </td></tr><tr><td>酵母无氮基氨基酸和硫酸铵</td><td> BD DIFCO </td><td> 200-0030 </td></tr><tr><td>低熔化琼脂糖</td><td> PROGEN生物科学</td><td> 200-0030 </td></tr><tr><td>显微镜载玻片（76 × 26毫米） </td><td>门泽尔，格拉泽</td><td></td></tr><tr><td>显微镜盖玻片（22 × 22毫米） </td><td>门泽尔，格拉泽</td><td> BB022022A1 </td></tr><tr><td>共聚焦激光扫描显微镜Fluoview FV500 </td><td>奥林巴斯</td><td></td></tr><tr><td> CMAC -精氨酸</td><td> Moleular探头，Invitrogen公司</td><td> Y - 7531 </td></tr></tbody></table>所有给出的浓度W / V，除非另有说明。 酵母培养基（YMM）：0.67％酵母无氮基氨基酸，2％葡萄糖，1.5％琼脂（只添加固体介质时）和任何所需的营养缺陷型补充剂（0.01％）]。 酿酒盐（SS）的：（NH 4）2 S0 4，0.12％; KH 2 PO 4，0.10％; MgC1 2 .6 H2O，0.07％; NAC1，0.05％;氯化钙，0.01％; FEC1 3，0.0005％。 生长培养基：SS和2％的乙醇（V / V）和补充适当的营养缺陷型。 氮饥饿介质：0.17％酵母无氮基氨基酸和硫酸铵（DIFCO），2％的乙醇（V / V）。

a fluorescence microscopy assay for monitoring mitophagy in the yeast saccharomyces cerevisiae

自噬是细胞成分的营业额重要的，各种不同的条件下。它提供一个重要的自我平衡功能，以及质量控制的机制，它可以的目标，并选择性地降解细胞的物质，包括organelles1 4。例如，损坏或多余的线粒体（图1），不处置的吞噬，可以代表一个细胞稳态和细胞生存的威胁。在酵母， 酿酒酵母 ，营养匮乏（例如，氮饥饿）或损坏，可促进自体吞噬的过程中被称为 mitophagy 5-9线粒体选择性的营业额。 我们描述了一个简单的荧光显微镜方法，以评估自噬。为了清楚起见，我们限制我们的描述，这里显示的方法可以用来监测在酵母细胞mitophagy。检测使用一种荧光记者，罗塞拉，这是一个双发射的传感器，包括与pH值敏感的绿色荧光蛋白的一个相对稳定的pH值，红色荧光蛋白。记者的运作依赖于液泡中（pH值〜5.0-5.5）和线粒体（pH〜8.2的pH值之间的差异）在活细胞。野生型细胞的生长条件下，表现出一个线粒体的方式特征分布的红色和绿色荧光。荧光发射是不相关的液泡。当受到氮饥饿，这种情况除了红色和绿色荧光标记的线粒体，诱导mitophagy，细胞呈现红色的积累，但没有绿色荧光，在酸性液泡流明代表提供线粒体空泡。评分与红细胞，但不是绿色的荧光空泡可以用来作为衡量细胞5,10-12 mitophagic活性。

Watch this Scientific Journal Video about A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae at JoVE.com

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

一个强大的方法来监视传递到细胞器的降解和回收的酵母液泡酸性流明描述。该方法依赖一种基因编码的双发射荧光pH值传感器，和可视化的单个细胞，用荧光显微镜对特定目标细胞器的标签。

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 ...

a-fluorescence-microscopy-assay-for-monitoring-mitophagy-yeast

Research

JoVE Journal

Biology

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

25.3K 次观看.  Monash University. 一个强大的方法来监视传递到细胞器的降解和回收的酵母液泡酸性流明描述。该方法依赖一种基因编码的双发射荧光pH值传感器，和可视化的单个细胞，用荧光显微镜对特定目标细胞器的标签。

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

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Time-lapse Imaging of Mitosis After siRNA Transfection

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

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

有丝分裂的siRNA转染后的时间推移成像

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

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 and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

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

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

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

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).