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><tbody><tr><td>Adenine</td><td>Sigma</td><td>A8626-25G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Bacto-agar</td><td>Fisher</td><td>BP1423-2</td><td>Use for preparation of plate media</td></tr><tr><td>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><td>Concanavalin A</td><td>Sigma</td><td>C5275-5MG</td><td>Use for coating of chamber slides</td></tr><tr><td>Dextrose</td><td>Fisher</td><td>BP350-1</td><td>Use for preparation of liquid and plate media</td></tr><tr><td>L-Histidine</td><td>Fisher</td><td>BP382-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Leucine</td><td>Acros</td><td>125121000</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Lysine</td><td>Fisher</td><td>BP386-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Methionine</td><td>Fisher</td><td>BP388-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tryptophan</td><td>Fisher</td><td>BP395-100</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>L-Tyrosine</td><td>Acros</td><td>140641000</td><td>Use for preparation of amino acid mixture</td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass (8-well)</td><td>Thermo Scientific</td><td>155411</td><td>Use for kinetic and endpoint assays of Mup1-pHluorin internalization</td></tr><tr><td>Uracil</td><td>Sigma</td><td>U0750-100G</td><td>Use for preparation of amino acid mixture and stock solution</td></tr><tr><td>Axiovert 200 inverted microscope</td><td>Carl Zeiss</td><td>Custom Build</td><td></td></tr><tr><td>100X/1.4 Plan-Apochromat Oil Immersion Objective Lens</td><td>Carl Zeiss</td><td></td><td>Objective should be 100X, 1.4NA or higher</td></tr><tr><td>Sensicam</td><td>Cooke Corporation</td><td></td><td>Camera should have 12-bit or higher dynamic range</td></tr><tr><td>X-Cite 120PC Q Illumination Source</td><td>Excelitas Technologies</td><td></td><td></td></tr><tr><td>Slidebook 5 software</td><td>Intelligent Imaging Innovations</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health</td><td></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

Universidad de Cartagena UDEC

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

Use of pHluorin to Assess the Dynamics of Axon Guidance Receptors in Cell Culture and in the Chick Embryo

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.

We describe here the use of a pH-sensitive green fluorescent protein variant, pHluorin, to study the spatio-temporal dynamics of axon guidance receptors trafficking at the cell surface.&#160;The pHluorin-tagged receptor is expressed both in cell culture and in vivo, using electroporation of the chick embryo.

使用普洛林来评估阿克森指导受体在细胞培养和小鸡胚胎中的动力学

During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation ...

科研

神经科学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

生物学

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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 ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

液泡和胞质pH值的测量<em&gt;在体内</em在酵母细胞悬浮液

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

获取荧光时间推移电影的芽殖酵母和分析单细胞动力学移植物

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Utilizing pHluorin-tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins.

Labeling the extracellular domain of a membrane protein with a pH sensitive fluorophore, superecliptic pHluorin (SEP), allows subcellular localization, expression, and trafficking to be determined. Imaging SEP-labeled proteins with total internal reflection fluorescence microscopy (TIRFM) enables the quantification of protein levels in the peripheral ER and plasma membrane.

利用pHluorin标记的受体监视亚细胞定位和贩运

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular ...

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 ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

酵母的4D显微镜

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...