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
Summary
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.
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We would like to thank Gero Miesenböck 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.
Materials
| Adenine | Sigma | A8626-25G | Use for preparation of amino acid mixture and stock solution |
| Bacto-agar | Fisher | BP1423-2 | Use for preparation of plate media |
| BD Difco Yeast Nitrogen Base (without amino acids) | BD | 291920 | Use for preparation of liquid and plate media |
| Concanavalin A | Sigma | C5275-5MG | Use for coating of chamber slides |
| Dextrose | Fisher | BP350-1 | Use for preparation of liquid and plate media |
| L-Histidine | Fisher | BP382-100 | Use for preparation of amino acid mixture and stock solution |
| L-Leucine | Acros | 125121000 | Use for preparation of amino acid mixture and stock solution |
| L-Lysine | Fisher | BP386-100 | Use for preparation of amino acid mixture and stock solution |
| L-Methionine | Fisher | BP388-100 | Use for preparation of amino acid mixture and stock solution |
| L-Tryptophan | Fisher | BP395-100 | Use for preparation of amino acid mixture and stock solution |
| L-Tyrosine | Acros | 140641000 | Use for preparation of amino acid mixture |
| Nunc Lab-Tek Chambered Coverglass (8-well) | Thermo Scientific | 155411 | Use for kinetic and endpoint assays of Mup1-pHluorin internalization |
| Uracil | Sigma | U0750-100G | Use for preparation of amino acid mixture and stock solution |
| Axiovert 200 inverted microscope | Carl Zeiss | Custom Build | |
| 100X/1.4 Plan-Apochromat Oil Immersion Objective Lens | Carl Zeiss | Objective should be 100X, 1.4NA or higher | |
| Sensicam | Cooke Corporation | Camera should have 12-bit or higher dynamic range | |
| X-Cite 120PC Q Illumination Source | Excelitas Technologies | ||
| Slidebook 5 software | Intelligent Imaging Innovations | ||
| ImageJ software | National Institutes of Health | http://imagej.nih.gov/ij/ |
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