通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用
Summary
Clathrin-mediated endocytosis, a rapid and highly dynamic process internalizes many proteins, including signaling receptors. The protocol described here directly visualizes the kinetics of individual endocytic events. This is essential for understanding how core members of the endocytic machinery coordinate with each other, and how protein cargo influence this process.
Abstract
Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological assays, measuring global changes in endocytosis, have identified over 30 known components participating in CME, and biochemical studies have generated an interaction map of many of these components. It is becoming increasingly clear, however, that CME is a highly dynamic process whose regulation is complex and delicate. In this manuscript, we describe the use of Total Internal Reflection Fluorescence (TIRF) microscopy to directly visualize the dynamics of components of the clathrin-mediated endocytic machinery, in real time in living cells, at the level of individual events that mediate this process. This approach is essential to elucidate the subtle changes that can alter endocytosis without globally blocking it, as is seen with physiological regulation. We will focus on using this technique to analyze an area of emerging interest, the role of cargo composition in modulating the dynamics of distinct clathrin-coated pits (CCPs). This protocol is compatible with a variety of widely available fluorescence probes, and may be applied to visualizing the dynamics of many cargo molecules that are internalized from the cell surface.
Introduction
网格蛋白介导的内吞作用（CME）的过程是依赖于网格蛋白介导的内吞机制的许多组分的良好的定时到来时，收集货物和操纵质膜进入囊泡1-3。 CME是由细胞膜变形，货物适配器蛋白走到了一起，在1内吞作用的新兴网站开始。这些蛋白质招募外壳蛋白网格蛋白，其组装成笼状结构，其形成的笼形蛋白包被的坑（CCP）4。一旦中共完全组装成球形的形状，膜的断裂，主要是通过大量的GTP酶，dynamin上的动作，产生游离网格蛋白包被小泡（校准检验）5,6。此内化触发网格蛋白涂层的快速拆卸，从而可以再次用于多个回合CME的组件。
参与CME蛋白的发现和鉴定已植根于传统biochemical，遗传和显微技术4-6,8。这些实验已经阐明这些元件内吞作用和交互点。虽然用于限定贩卖机的必要成分非常有用，这些测定法是高度限定在捕获CME部件或货物浓度的动态行为。这是一个重要的限制，因为CME是由套蛋白的模块中定义的步骤的编排组件驱动，并且由于在个体内吞事件的动态微小变化可对细胞内吞作用大累积的后果。另外，最近的数据表明，单独的CCP可能不同无论在组成和行为，这表明该过程的生理调节高度空间和时间上的限制9-14。可视个人的内吞的事件，因此，关键是要理解为什么有参与CME的多个冗余的蛋白质，以及这些蛋白可能被控制bŸ生理信号来调节货物的内在。
在这里，我们描述了使用全内反射荧光显微镜（TIRFM）的个体的CCP的活细胞中的动态水平观察CME。 TIRFM依赖于玻璃盖玻片和细胞15,16的流体环境之间折射率的差异。当激发光被导向所述细胞在超过临界角，它是在内部反射，创建维护薄照明的领域延伸的盖玻片上约100nm的渐逝波。这确保了只有在这个狭窄领域的荧光分子被激发。实际上，这允许上或附近的细胞膜荧光分子的激发，并从该单元的内部部件最小化荧光。这提供了一个显著更高的信号 – 噪声比，并沿z轴的分辨率来可视化在质膜，共同事件mpared更常用的模式，诸如传统的表面荧光或共聚焦荧光显微术。我们还描述，在一个介绍和实际操作层面，采用了常用的图像分析平台，分析和定量简单的形态特征和个别货物吞事件的动态。
Protocol
Representative Results
Discussion
在这里，我们描述了使用TIRFM的可视化网格蛋白介导的内吞作用（CME）以各自的CCP的电平在实时活细胞。 CME是由许多在空间和时间上分开的单个事件的累积效应介导的快速和高度动态的事件。使用表面生物素化或配体结合，流式细胞术或固定的细胞测定法测定内化的蛋白质，或蛋白质的电子显微镜定位的量目前使用最测定法，如内在的生物化学测量，监视在固定的时间点集合的变化在蛋白质定位?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to thank Drs. C. Szalinski, H. Teng, and M. Bruchez for help with Imaris, R. Vistein, and D. Shiwarski for technical help and advice, and Dr. M von Zastrow, Dr. T Kirchhausen, Dr. D Drubin, and Dr. W Almers for reagents and helpful discussion. Funding provided by T32 grant NS007433 to SLB and NIH DA024698 and DA036086 to MAP.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| DMEM/High Glucose with L-glutamine and sodium pyruvate | Fisher Scientific | SH3024301 | |
| Dulbecco's Phosphate Buffered Saline (DPBS), no calcium, no magnesium | Gibco, by Life Technologies | 21600-010 | |
| EDTA Free Acid | Amresco | 0322-500G | |
| Fetal Bovine Serum | Gibco, by Life Technologies | 10437-028 | |
| Leibovitz's L-15 Medium, no phenol red | Gibco, by Life Technologies | 21083-027 | |
| Opti-MEM | Gibco, by Life Technologies | ||
| HEPES | CellPURE by Fisher Scientific | BP2937-100 | |
| Effectene | Qiagen | 301425 | Transfection reagent |
| 25 millimeter coverglass | Fisher Scientific | 12-545-86 | |
| Corning cell culture treated flasks, 25cm2 | Fisher Scientific | 10-126-28 | |
| Cell culture 6-well plate | Greiner Bio-One, by VWR | 82050-896 | |
| Monoclonal ANTI-FLAG M1 | Sigma Aldrich | F3040-5MG | |
| [D-Ala2, N-Me-Phe4, Gly5-ol]-Enkephalin acetate salt (DAMGO) | Sigma Aldrich | E7384-5MG | |
| Alexa Fluor 647 Protein Labeling Kit | Life Technologies | A20173 | |
| ImageJ | NIH | http://rsb.info.nih.gov/ij/ | |
| Imaris Image analysis software | BitPLane | http://www.bitplane.com/imaris/imaris, for automated analysis | |
| Nikon Eclipse Ti inverted microscope and required accessories including filter cubes and filters | Nikon | ||
| Nikon TIRF arm with required adapters for Nikon Eclipse Ti | Nikon | For adjusting angle of incidence | |
| iXon+ EMCCD camera and adapters | Andor |
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