We would like to thank Gero Miesenb&#246;ck and Tim Ryan for sharing pHluorin cDNA used to generate reagents in this study, Nathan Wright, Joanna Poprawski and Lydia Nyasae for excellent technical assistance, members of the Wendland lab for helpful discussions, and Michael McCaffery and Erin Pryce at the Integrated Imaging Center (Johns Hopkins) for advice and assistance with microscopy and flow cytometry. This work was supported by grants from the National Institutes of Health (to B.W., GM60979) and the National Science Foundation (to B.W., MCB 1024818). K.W. was supported in part by a training grant from the National Institutes of Health (T32-GM007231). A.F.O. was supported by developmental funds from the Department of Biological Sciences at Duquesne University and National Science Foundation CAREER grant 553143.

1.解决方案和媒体<ol><li>准备以下股票，媒体和板: <ol><li>制备10倍的YNB，溶解67克酵母氮碱在1升水中缺乏氨基酸。通过0.22μm硝酸纤维素过滤器进行过滤灭菌。 </li><li>用2％葡萄糖（从无菌50％w / v的股票）和1x氨基酸/养分混合物制备使用1倍的YNB（从10倍股票）的YNB培养基（从100倍的库存，见步骤1.1.4）。质粒的选择，可以根据需要省略单个氨基酸或营养物。为Mup1表达的诱导，另外从氨基酸/营养混合物省略蛋氨酸。 </li><li>准备使用1X YNB（从10倍股票）2％葡萄糖的YNB平板，（从一个无菌的50％w / v的股票），0.7克/升氨基酸/营养混合物（见步骤1.1.5）和2.5％琼脂。通过高压灭菌25克琼脂和0.7g每860毫升水氨基酸/养分混合物的制备平板培养基。使混合物冷却到55℃，然后加入100 ml 10X鼎贞和40毫升邻°F±50％的葡萄糖。倾注平板（约30毫升，每10cm皿介质的），允许该介质在室温下固化，并存储板在4℃。 </li><li>通过溶解在100毫升的去离子水中的下列制备（用于液体介质）100×氨基酸/营养原液:0.1克L-蛋氨酸，0.3 G各自L-亮氨酸和L-赖氨酸，和0.2g各L组氨酸的，（根据需要为特定的菌株可以包括其他氨基酸和营养物）L-色氨酸，腺嘌呤和尿嘧啶。如果需要的话，以溶解用文火，并通过0.45μm的硝酸纤维素滤膜除菌。在室温下储存避光。对于质粒的选择，准备根据需要缺乏具体的元器件库存解决方案。 </li><li>通过组合制备氨基酸/营养混合物（对于板）以下为0.5g腺嘌呤，4-克L-亮氨酸，和2克每L-组氨酸，L-赖氨酸，L-蛋氨酸，L-色氨酸，L-酪氨酸和尿嘧啶。用研钵和研杵研磨，并与亲存储tection避光。质粒的选择，制备缺乏特定组件根据需要的混合物，并且使用0.7克每升平板培养基（见步骤1.1.3）的氨基酸的混合物。 </li></ol></li><li>通过用刀豆蛋白A（ConA）的滑动的表面涂覆制备用于显微镜8孔腔室载玻片。到室幻灯片的各孔中，添加25μl的2mg / ml的刀豆溶解在水中。用移液管尖端，传播刀豆横跨孔的底部表面，然后允许在室温下将载玻片风干至少4-8小时。理想情况下，用刀豆涂层的24小时之内玻片。 </li><li>生成与在帧使用聚合酶链式反应（PCR）在Longtine 等人描述的基于积分法GFP或pHluorin的融合到感兴趣的内吞货物酵母菌株。和Goldstein和国琛25,26。 注:含pHluorin标记与抗性基因的卡那霉素（KANMX6）和诺尔丝菌素磁带质粒（ <em&gt; NATMX4）以前20描述。 <ol><li>扩增含有与含有特定的基因组整合20,25的部位附加序列的引物的选择标记GFP或pHluorin盒。 </li><li>变换所得到的PCR产物进用锂醋酸盐法27所需的酵母菌株。 </li><li>通过在含有适当的药物的YPD平板上生长的细胞中选择的盒的整合。为了制备板，高压釜10g酵母提取物，20g蛋白胨，0.1g的色氨酸和960毫升水中25克琼脂。浇注板之前，允许该混合物冷却到55℃，再加入毫升40 50％葡萄糖，要么G418至200微克/毫升为KANMX6选择的最终浓度，或诺尔丝菌素至100微克/毫升的最终浓度NATMX4选择。 </li><li>通过PCR确认在单个菌落的GFP或pHluorin标签的集成和/或使用抗GFP抗体Western印迹，以及通过检测标记蛋白的荧光显微镜20。 注意:特定酵母菌株，并在此研究中使用的质粒在表1和2分别列出。 </li></ol></li></ol> 2.端点检测方法的组成性内在化吞货的稳态荧光<ol><li>接种细胞（〜3小菌落）在5毫升的YNB培养基中缺乏所需质粒维护（如果适用的话）氨基酸或营养物。生长细胞为16-24小时在轨道摇动培养箱中于30℃，用250rpm振荡。可替代地，细胞的条纹一〜1-2毫米菌落到缺乏氨基酸或质粒选择的营养物质，一个YNB板，并在30℃下生长的细胞过夜。 </li><li>测量使用分光光度计各过夜培养物的密度（OD 600）。准备在YNB中0.35-0.4 OD 600 /毫升5毫升稀释缺乏解放军氨基酸或营养SMID维护和生长约3小时，在30℃振荡。如果使用电池放在盘子里挑染，跳过此步骤，步骤2.4（见下文）进行。 </li><li>测量细胞密度（OD 600），它们现在应该0.6-1.0的OD 600 /毫升之间。转移1.5毫升培养到微量离心管中，并沉淀细胞以8,000rpm（6800 xg离心）2分钟。除去1475微升上清液。 </li><li>带来的细胞的荧光显微镜。每个样品前成像，在剩余的培养基重悬细胞准备的幻灯片，并安装在盖玻片3微升细胞悬液。可替代地，如在协议3如下所述，使用的YNB培养基以适合质粒的选择制备8-孔腔式载玻片。理想的情况下，用100X，1.4或更高数值孔径（NA）油浸物镜透镜，12或16位的摄像头，以及激发/发射过滤器图像的细胞的GFP荧光优化。 注意:如果使用细胞裸奔Ø增长呐板，通过将3微升新鲜YNB培养基上盖玻片准备的幻灯片。用移液管尖端，收集细胞的一个小的菌落从平板，分散细胞进入的YNB培养基中，紧接在成像之前安装在载玻片上所得的悬浮液中。 </li><li>图像单元为每个条件的4-6无规领域，使用相同的采集参数（ 即 ，过滤器组和曝光时间）为一个实验中的所有图片。 注:收集明或DIC图像的每个字段可以为那里的pHluorin信号在MVB和空泡车厢淬火细胞有帮助的。 </li><li>量化至少30-50细胞每个条件的荧光强度（见协议5）。 </li></ol> 3.动力学检定为货物的内吞作用的定量离岗管制吞<ol><li>在5毫升的YNB培养基中接种酵母细胞（约2毫米每菌落直径）为2-3菌落缺乏甲硫氨酸和添加itional氨基酸或营养物用于维持质粒的选择。成长培养16-24小时，在30℃振荡。 注:本文为研究内吞调节蛋白质的货物将协议利用蛋氨酸通透，Mup1的。该经过调节的内吞作用的其他货物也适合用pHluorin标记（例如，尿嘧啶通透Fur4，其积聚在在不存在尿嘧啶的质膜，并且响应内化于外部施加的尿嘧啶）;对于这些货物，媒体条件应进行修改，以反映该货物的具体表达，诱导和内在条件。 </li><li>成像前约3小时测量每个过夜培养的密度（OD 600）。在YNB培养基0.35-0.4 OD 600 / ml的缺乏甲硫氨酸和另外的氨基酸或营养素质粒保持准备5毫升稀释，并在30℃振荡生长。 </li><li>预平衡的万分之一步3.2的3小时孵化过程中应付的环境室或加热阶段（如果可用）至30℃。 </li><li>传送1 ml的细胞培养至微量离心管中，并以8,000rpm（6800 xg离心）2分钟沉淀细胞。取出975微升上清液。 </li><li>准备成像（协议1.2）一个刀豆处理，8孔玻璃底室幻灯片。 </li><li>添加YNB培养基的200微升缺乏甲硫氨酸和另外的氨基酸或营养物进行质粒选择到每个孔中。悬浮来自步骤3.4中剩余的上清液中的细胞沉淀，并下降2.5微升细胞悬浮液到每个的中心孔。分散在整个井的表面细胞吹打上下，并允许细胞沉降5-10分钟。 </li><li>放在平衡至30°C（见步骤3.3），倒置荧光显微镜室幻灯片。找到使用明照明细胞领域。 </li><li>添加YNB培养基50微升含有100微克/毫升的甲硫氨酸（预热至30℃）至200微升培养基中的井，以获得20微克/ ml的终蛋氨酸浓度。避免破坏在井中介质以防止细胞从底部浮动。 </li><li>允许开始成像之前将细胞平衡2分钟。立即捕捉第一图像之前，调整显微镜到所需焦平面（通常，赤道焦平面效果最好）。 </li><li>获得在5分钟的间隔GFP和明（或DIC）图像45-60分钟，使用相同的采集参数为一个时间序列中的所有图像（例如，100X 1.4 NA油浸物镜和500毫秒采集时间使用GFP的滤波器，100使用DIC过滤毫秒采集时间）。 注:如果在显微镜的阶段和成像软件不允许焦平面漂移的自动修正，它可能需要手动重新调整前各摄像时刻的焦点。此外，收购时间将显微镜之间有所不同，因为在照明和过滤器的差异，和最佳条件应该确定之前手动开始实验。如果使用比Mup1其他的内吞货物，可能有必要修改的采集时间和成像时间间隔，以及在实验的持续时间，以反映该货物的内化动力学。 </li><li>量化在每个时间点的单个细胞的荧光强度（见协议5），并表达值作为第一图像的初始强度的百分比。 </li></ol> 4.端点检测方法的吞货物的定量的离岗管制吞<ol><li>在协议3所述准备成像细胞（步骤3.1到3.6）。 </li><li>图片立即添加蛋氨酸，使用明和GFP过滤器集之前细胞每个条件的4-5个随机领域。 </li><li>添加YNB培养基50微升含有100微克/毫升甲硫氨酸（战前MED至30℃）至200微升培养基中的井，以获得20微克/ ml的终蛋氨酸浓度。避免破坏在井中介质以防止细胞从底部浮动。 </li><li>加入甲硫氨酸后图像4-5细胞的随机字段30分钟，用相同的采集参数为0分钟的时间点（步骤4.2）。 </li><li>量化至少30-50细胞对每个时间点的荧光强度（见协议5）。快递值Mup1-pHluorin后，用公式30分钟内在的百分比:[0分钟时平均强度 - 在30分钟平均强度] / [平均强度为0分]×100％。 注意:可以将通过错开每个测定的开始时间同时执行多达八个端点测定表3提供了八个同时测定的一例的工作流程。 </li></ol> 5.收购后分析<ol><li>从采集软件导出图像为16位的标记图像文件格式（.TIF或.TIFF格式）。 注:其他的文件格式也可以是合适的，并且最好应该使用无损压缩算法。 8位图像一般不适合用于定量目的。 </li><li>使用ImageJ软件（免费提供的"打开"选项，在"文件"菜单中打开文件。使用荧光图像进行定量和明/ DIC图像作为参考，以确定细胞的位置，以排除死细胞（如果存在的话），这似乎比当由DIC查看和用GFP激发/发射滤光片观察时都自身荧光活细胞暗。 </li><li>执行荧光图像上的背景减除。对于具有不均匀的背景信号图像，请使用"Process菜单"中的"背景减法"的算法。使用默认设置（轧制的50像素球半径），这通常是足够的用于定量目的。 注:另一种backgro与统一的背景图像UND减法步骤5.3.1.-5.3.2描述;然而，上述方法也适用于统一的背景。 <ol><li>为具有均匀的背景图像（ 即 ，背景强度是在所有的边缘，并在图像的中心大致恒定），执行手动背景扣除。点击在主ImageJ的窗口中找到的"徒手选择"按钮，并且不包含细胞的图像内选择3-5随机区域。 </li><li>打开位于"分析"菜单中的"设置测量"功能，选择"平均灰度值&#39;参数，并测量使用"测量"功能（在"分析"菜单中找到）的强度值。计算从3-5测量区的平均值，并从该图像中的所有后续测量中减去。 </li></ol></li><li>对于动力学测定法量化（协议3），生成用于添一个单一的，堆叠的图像E系列。从时间顺序每个时间点打开荧光图像，并生成使用"图像叠"功能栈（在"图像"菜单中的栈子菜单下）。 </li><li>外形采用徒手选择工具电池，确保包括细胞尽可能之外的整个单元格，但尽可能少的区域。 </li><li>测量以下参数:&#39;区，集成密度"和"平均灰度值"。使用"设置测量"功能，位于"分析"菜单中选择参数。 注:集成密度对应于所有的像素强度的总和所选择的区域内，和平均灰度值对应于[集成密度/区]，它校正强度值为细胞大小。 </li><li>打开"工具"子菜单下的"投资回报率经理"位于"分析"菜单。在投资回报率管理器窗口中，单击"添加"按钮，进入高亮显示的区域选择性n作为感兴趣区域（ROI）的新区域。 </li><li>重复步骤5.4至5.6为图像中的剩余的细胞。排除所评定明/ DIC和荧光（死细胞出现在DIC的图像更暗，并具有用GFP激发/发射观看时自身荧光细胞质信号;参见图2F）的任何死细胞，并且触摸所述图像的边缘的任何细胞。通过点击"投资回报率管理"窗口中的"更多"按钮保存所选的ROI为.zip文件，所有的测量导出到电子表格或统计分析软件。 </li><li>对于动力学测定法量化（协议3），轮廓和如在步骤5.4和5.5中所述测量从初始时间点的细胞。使用相同的投资回报率在实验中所有后续时间点的测量。 </li><li>端点测定（协议2和4），测量的最小30-50细胞为每个条件的。为至少2-3个图像执行量化，并且包括在每所有的细胞图像符合在步骤5.8在分析中列出的标准。 </li><li>评估使用单向ANOVA，接着Tukey多重比较检验事后分析样品之间的统计显着性。 </li></ol>通过流式细胞仪Mup1-pHluorin内吞6.人口分析<ol><li>在0.5ml缺乏甲硫氨酸和另外的氨基酸或营养物用于维持质粒选择的YNB培养基中接种酵母细胞（约2毫米每菌落直径）2-3菌落。生长细胞在5ml圆底试管16-24小时，在30℃的辊筒。 </li><li>添加1.5毫升YNB培养基缺乏甲硫氨酸和另外的氨基酸或营养质粒保养，并在30℃下生长在辊筒额外的3小时。 </li><li>传送1萃取池的每一个由两个5毫升的圆底管中。添加0.25毫升的YNB -Met介质与所述第一管（-Met条件），和0.25的YNB培养基中含有100μg/ ml的甲硫氨酸至第二管，得到的20微克/毫升（+蛋氨酸条件）的终蛋氨酸浓度。孵育细胞在30℃下在转鼓45分钟。 </li><li>分析由流动池术，选择前向散射（FS），为细胞大小的量度，并从异硫氰酸荧光素（FITC）滤波器作为货物内化的量度Mup1-pHluorin荧光强度。 </li><li>使用滑块按钮调整FS和FITC通道的电压，以优化检测酵母细胞的尺寸范围和荧光强度使用（对于这些实验中，使用的设置是为FS的50伏和400 V代表FITC）。 注:电压为两个通道将根据流式细胞仪中使用的流变化。此步骤应之前或甲硫氨酸（步骤6.3）的45分钟处理期间进行。 </li><li>测量FS和荧光强度为从每个条件10,000个细胞，使用高流量设定。产生FS对萤光灯的散点图rescence强度:点击"添加图形"，选择FITC（X轴）和FS（Y轴）和图1000点（该软件将1000单元格显示值，即使测定10,000个细胞）。 注:对于最大的一致性，分析细胞加入甲硫氨酸后45-50分钟;涉及大量样品的实验中，错开加入甲硫氨酸以适应流式细胞分析所需的时间。 </li><li>包括野生型（WT）细胞作为对照-Met和+蛋氨酸的条件。使用栅函数，分配使用WT +蛋氨酸条件，使得最亮的细胞的约5％下降到门的右侧的垂直栅极。使用此门在实验中所有其他条件。 </li></ol>

The authors declare no competing interests.

pHluorin的用于在酵母细胞内吞事件量化应用利用，其中所述荧光标签融合到蛋白质（ 图1A）的胞质尾跨膜货物的。对于此处所描述的测定法中，当pHluorin标签暴露于中性的环境中，但是成为当pHluorin标签遇到酸性条件猝灭货物是明亮的荧光。因此，为pHluorin标签的内吞货物容易在质膜的胞质面和早期内涵体检测，但是当掺入管腔MVB囊泡或在递送到液泡腔变成"暗"。在一般情况下，新合成...

囊泡运输在维持在真核细胞的细胞器的身份和功能中起重要作用，并且是用于调节个体细胞区室的蛋白质和膜组合物的关键机制。在质膜，胞吐泡融合提供了新的蛋白质和膜的细胞的表面上，而通过内吞作用产生小泡从表面用于随后的回收或靶向溶酶体除去膜和蛋白质。因此，内吞作用是养分吸收和用于向细胞外环境的反应很重要。胞吐作用和胞吞作用被平衡以调节质膜表面积，并允许受损的蛋白质的周转。 已在酵母和哺乳动物细胞的研究确定了响应大量涉及内吞作用的蛋白，以及促进特定货物的内在多个内吞途径或该行为的各种连接的vironmental条件。该研究得最好的途径是网格蛋白介导的内吞作用（CME），其中网格蛋白和胞质辅助蛋白组装成一个涂层结构以稳定新生吞囊泡。在芽殖酵母实验已经取得了关键洞察动态和招聘的顺序许多内吞蛋白1-3。值得注意的是，在CME机械高度通过进化保守，使得大多数酵母CME相关蛋白具有人直系同源物;因而，芽殖酵母已经了解了在高等真核生物保守的细胞内吞作用的机制的一个重要工具。例如，使用出芽酵母研究确定CME结构即形成和成熟（在酵母中称为皮质肌动蛋白斑块）涉及许多蛋白质的顺序招募，与网格蛋白和货物结合衔接蛋白开始，接着附加内吞附件招募蛋白质，的Arp2 / 3介导的肌动蛋白聚合的活性，以及参与囊泡断裂1,2-蛋白的募集。 CME机械蛋白质招募到皮层肌动蛋白补丁程序是一个高度有序的和一成不变的过程中，和招聘的许多蛋白质的确切订单已经建立了关于CME机械等组成。重要的是，最近的研究已经证实招募在网格蛋白小窝的CME蛋白在哺乳动物细胞中4类似的顺序。 除了CME，许多细胞类型具有依赖于替代机制，以促进从质膜5-7囊泡形成和内化的一个或多个网格蛋白独立内吞（CIE）的途径。在酵母中，我们最近发现，利用小GTP酶RHO1其激活鸟嘌呤核苷酸交换因子（GEF），ROM1 8,9一CIE途径。 RHO1激活formin Bni1促进肌动蛋白聚合10,11，这是需要在酵母12这种形式的网格蛋白独立的内吞作用的。此外，蛋白质的α休止家族招募泛素连接酶Rsp5通过CME 13-17促进货物泛素化和随后的内化，并且还通过在CIE途径促进货物内化，可能通过直接相互作用与参与CIE 18的蛋白质。各种CIE途径也存在于哺乳动物细胞，包括依赖于RHO1直向同源物，RhoA蛋白9,19网格蛋白独立和吞噬途径。 RhoA蛋白在哺乳动物CIE的作用是知之甚少;因此，在芽殖酵母的研究可能提供额外的机械见解，适用于哺乳动物CIE。 内吞事件的精确量化可以提供关于特定蛋白在调节细胞内吞作用的重要信息，并且可以揭示突变对货物内化或progressio的影响通过内吞途径ñ。为了这个目的，生物化学方法可用于监测配位体的吸收或以测量内吞货物蛋白质的降解的速率。在活细胞中，绿色荧光蛋白（GFP）及其与感兴趣货物变体的融合允许货物运输的直接可视化。但是，GFP标记的货物是用于内吞作用的量化用途有限，因为GFP是耐（在酵母或液泡）在溶酶体降解。此外，GFP荧光只有在pH值的变化部分敏感，并保持液泡腔20,21内检测到。因此，绿色荧光蛋白标记的荧光在液泡持续长后的货物的剩余部分已经退化，以及货物的强度的全细胞为基础的定量可能是不准确的，由于在液泡长期GFP荧光。 为了克服液泡GFP荧光的缺点，我们先前提出的使用superecliptic pH值luorin，GFP的pH敏感的变体，在中性pH明亮荧光，但失去了在酸性环境中的荧光，如液泡/溶酶体20,22,23的内腔中。上内吞货物的胞质尾区放置为pHluorin标签允许在质膜和早期内涵体，其中，pHluorin标签保持暴露到细胞质（ 图1A）的货物的可视化。由于早期内涵体成熟，胞内排序复杂所需的运输（ESCRT）机械封装表面本地化货物成芽进入核内体腔内囊泡，产生多泡体（MVBs）24。对于已纳入内部MVB泡货物时，pHluorin标记朝向囊泡流明。内部MVB囊泡酸化;因此，pHluorin标签的货物失去早期内涵体荧光，因为它们成熟为MVBs 20。与液泡的MVB的后续融合提供了MVB肠腔内容物用于降解和pHluorin标签保持在液泡腔的酸性环境骤冷。 本文提供了使用与酵母细胞质pHluorin标签货物的定量测定内吞的详细说明。表达pHluorin标签的货物的菌株可以在动力学和/或终点测定法中使用，这取决于具体的货物的性质和贩卖行为。此外，一些pHluorin标签的货物是适合于高通量的分析方法，包括流式细胞仪。重要的是，pHluorin标签是研究在活细胞内吞的事件，从而使内吞作用和在野生型和突变株的内吞作用的比较的量化的多功能工具。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adenine</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">A8626-25G</td>
			<td style="width:167px;">Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto-agar</td>
			<td>Fisher</td>
			<td>BP1423-2</td>
			<td>Use for preparation of plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BD Difco Yeast Nitrogen Base (without amino acids)</td>
			<td>BD</td>
			<td>291920</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concanavalin A</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C5275-5MG</td>
			<td>Use for coating of chamber slides</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher</td>
			<td>BP350-1</td>
			<td>Use for preparation of liquid and plate media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td>Fisher</td>
			<td>BP382-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Leucine</td>
			<td>Acros</td>
			<td>125121000</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Lysine</td>
			<td>Fisher</td>
			<td>BP386-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Methionine</td>
			<td>Fisher</td>
			<td>BP388-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td>Fisher</td>
			<td>BP395-100</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tyrosine</td>
			<td>Acros</td>
			<td>140641000</td>
			<td>Use for preparation of amino acid mixture</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc Lab-Tek Chambered Coverglass (8-well)</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:119px;">155411</td>
			<td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>Use for preparation of amino acid mixture and stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axiovert 200 inverted microscope</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;">Custom Build</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td>
			<td style="width:71px;">Carl Zeiss</td>
			<td style="width:119px;"></td>
			<td>Objective should be 100X, 1.4NA or higher</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Sensicam</td>
			<td style="width:71px;">Cooke Corporation</td>
			<td style="width:119px;"></td>
			<td>Camera should have 12-bit or higher dynamic range</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">X-Cite 120PC Q Illumination Source</td>
			<td style="width:71px;">Excelitas Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Slidebook 5 software</td>
			<td style="width:71px;">Intelligent Imaging Innovations</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ImageJ software</td>
			<td style="width:71px;">National Institutes of Health</td>
			<td style="width:119px;"></td>
			<td>http://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

applications of phluorin for quantitative, kinetic and high-throughput analysis of endocytosis in budding yeast

绿色荧光蛋白（GFP）及其变种被广泛使用的工具用于研究蛋白质的定位和事件，如细胞骨架重构和活细胞囊泡运输的动态。使用嵌合的GFP融合物的定量方法已经被开发用于许多应用;然而，绿色荧光蛋白是蛋白水解有所抗性，因而其荧光在溶酶体/液泡，从而可以在吞途径妨碍货物贩卖定量存在。用于定量的内吞作用和胞吞后贩卖事件的另一种方法是利用superecliptic pHluorin，GFP的pH敏感的变体是在酸性环境中骤冷的。 pHluorin的嵌合融合的跨膜蛋白的货物导致的荧光在货物进入多泡体（MVBs）和交付溶酶体/液泡流明掺入一个阻尼胞质尾。因此，液泡的荧光的猝灭便于quantifi内吞作用和在吞途径的早期事件的阳离子。本文介绍如何使用pHluorin标签的货物通过荧光显微镜内吞作用的量化使用流式细胞仪基于人群的检测方法，以及。

稳态定位，定量的内在组成货物内吞蛋白质STE3的 为了演示在活细胞中，嵌合GFP和pHluorin融合与STE3，在酵母α-因子信息素受体的胞质C-末端尾部的本地化该pHluorin标签的货物可用于细胞内吞作用的定量，进行了比较。 STE3是一个G蛋白偶联受体（GPCR），其组成型转运到质膜，内化，并靶向液泡降解28。因此，?...

Watch this Scientific Journal Video about Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast at JoVE.com

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

pHluorin的申请在芽殖酵母细胞内吞的定量，动力学和高通量分析

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

applications-phluorin-for-quantitative-kinetic-high-throughput

Research

JoVE Journal

Biology

10.8K 次观看.  The Johns Hopkins University. Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

视频: Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

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 ...

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, ...

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 ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

高通量MHC II类分子结合分析抗原表位肽的定量分析

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

恢复成年斑马鱼的心脏的高通量应用

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

高通量筛选转录后调控基因的化学调节剂

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

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 ...