可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输
Summary
在这里，我们描述了一种体外活成像方法，用于可视化细胞器的细胞内传输和在小鼠星形细胞中贩运血浆膜蛋白。该协议还提出了一种图像分析方法，以确定货物运输路线和动力学。
Abstract
星形细胞是成人大脑中最丰富的细胞类型之一，它们在多种功能中起着关键作用。作为大脑平衡的中心角色，星形细胞为神经元提供重要的代谢物和缓冲细胞外水、离子和谷氨酸。星形细胞是”三部分”突触的组成部分，在突触的形成、修剪、维护和调制中也至关重要。为了启用这些高度交互的功能，星形细胞通过包括细胞在内的大量专用膜蛋白，相互之间以及与其他胶质细胞、神经元、脑血管和细胞外环境进行通信粘附分子、水孢素、孔通道、神经递质输送机和间隙结分子。为了支持这种动态通量，星形细胞，像神经元一样，依赖于紧密协调和有效的细胞内传输。与神经元不同，细胞内贩运被广泛划定，星形细胞中基于微管的迁移研究较少。然而，细胞膜蛋白的外泌和内分细胞贩运和细胞内细胞内传输协调星形细胞的正常生物学，这些过程往往在疾病或对损伤的反应中受到影响。在这里，我们提出一个直接的协议，培养高品质的鼠群细胞，荧光标记星形蛋白和感兴趣的细胞器，并记录其细胞内传输动力学使用时移共聚焦显微镜。我们还演示了如何使用可用的图像分析软件（即 ImageJ/FIJI）插件从获取的电影中提取和量化相关的传输参数。
Introduction
星形细胞是成人中枢神经系统中最丰富的细胞，它们在其中执行独特的发育和静默功能1。星形细胞调节突触发育，通过直接接触前和后发触终端作为三部分突触的一部分，其中包含神经递质受体，传输器和细胞粘附分子，促进突触形成和神经元-天体细胞通信2。此外，星形细胞通过快速去除突触裂口的兴奋神经递质，回收神经递质，并参与突触修剪3，积极控制突触传播，防止神经元兴奋性中毒。,4,5,6.为了启用这些高度交互的功能，星形细胞通过专门的膜蛋白（包括细胞粘附分子、水孢素、离子电池通道）相互之间、与其他胶质细胞和神经元之间通信，神经递质传输器和间隙结分子。星形细胞积极改变这些蛋白质的表面水平，以响应其细胞内和细胞外环境的波动7。此外，线粒体、脂液滴和降解和再循环细胞器的水平和分布的变化调节了能量供应、代谢物可用性和细胞清除过程，这些对星形细胞功能和生存。
膜蛋白和细胞器贩运和星形细胞的定位的动态变化是由运动蛋白和适配器的协同功能促进货物运动8，9。同样，膜蛋白的表面水平是通过内化和回收事件10来调节的。这些货物通过一个复杂的网络，由行为蛋白、微管和可能的中间细丝轨道8进行运输。基于最终结合蛋白1（EB1）的免疫荧光染色的研究表明，在星形细胞束的微管中，微管从围核外辐射，并将其加端向外围11.然而，仍然缺乏对微管和其他细胞骨骼元素使用活细胞成像的组织和极性的全面检查。虽然细胞器和膜蛋白动力学背后的许多机制已经在神经元和其他细胞类型中进行了广泛的研究，但星形细胞中的货运活力却不太了解。我们目前关于星形细胞中蛋白质和细胞器分布变化的大部分知识是基于传统的基于抗体的固定制备标签，这排除了对货物动力学进行精确的空间和时间检查7， 12.
在这里，我们描述了一种在高纯度原鼠星形细胞培养中为活成像标记膜蛋白和细胞器的方法。使用该协议，我们提供了示例，其中我们跟踪绿色荧光蛋白 （GFP） 标记膜蛋白在转染星细胞中的动态定位，包括间隙结蛋白 Connexin 43 （Cx43-GFP） 和兴奋氨基酸运输器 1 （EAAT1-GFP）。我们还描述了使用荧光酸度探针来可视化酸性细胞器，并跟踪其在活星形细胞中的贩运动态。最后，我们演示了如何分析延时数据，以提取和评估单个货物的运输参数。
Protocol
Representative Results
Discussion
在这里，我们描述了一种实验性方法，用于在高纯度原生小鼠皮质MD星形细胞中使用延时视频显微镜来表达、可视化和跟踪荧光标记的细胞器和膜蛋白。我们还概述了测量粒子动力学的方法。在原星形细胞中直接显示蛋白质和细胞器动力学，为研究这些细胞体内细胞内迁移的调节提供了一个有力的工具。
上述建立小鼠MD星形细胞培养的方法结合了其他已公布的协议13、14、15的?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
DNL作为西蒙斯学者得到北卡罗来纳大学教堂山分校（UNC）医学院的支持。TWR得到了UNCPREP赠款R25 GM089569的支持。使用UNC神经科学中心显微镜核心设施的工作部分得到了NIH-NINDS神经科学中心支持赠款P30 NS045892和NIH-NICHD智力和发育障碍研究中心支持赠款U54的支持支持。HD079124。
Materials
| 2.5% Trypsin (10x) | Gibco | 15090-046 | |
| Benchtop Centrifuge | Thermo Scientific | 75-203-637 | Sorvall ST8 Centrifuge |
| Cell Culture Grade Water | Gen Clone | 25-511 | |
| Cell Culture Microscope | Zeiss | WSN-AXIOVERT A1 | Vert.A1 inverted scope, Inverted tissue culture microscope with fluorescent capabilities |
| Cytosine Arabinoside | Sigma | C1768-100MG | (AraC) Dissolve lyophilized powder in sterile cell culture grade water to make a 10mM stock, aliquot and freeze for long term storage |
| DAPI | Sigma | D9542-5MG | Dissolve lyophilized powder in deionized water to a maximum concentration of 20 mg/ml, heat or sonicate as necessary. Use at 300 nM for counterstaining. |
| Dissecting Microscope | Zeiss | Stemi 305 | |
| Dissecting Scissors | F.S.T | 14558-09 | |
| Dulbecco's Modified Eagle Medium | Gen Clone | 25-500 | |
| Fetal Bovine Serum | Gemini | 100-106 | Heat-Inactivated |
| FIJI (Fiji is Just Image J) | NIH | Version 1.52i | |
| Fine Tip Tweezers | F.S.T | 11254-20 | Style #5 |
| Fluorescence light source | Excelitas | 012-63000 | X-Cite 120Q |
| GFAP antibody | Cell Signaling | 3670S | GFAP (GA5) mouse monoclonal antibody |
| Glass Bottom Dishes | Mattek corporation | P35G-1.5-14-C | 35 mm Dish | No. 1.5 Coverslip | 14 mm Glass Diameter | Uncoated |
| Graefe Forceps | F.S.T | 11054-10 | Graefe Iris Forceps with curved tips |
| Green fluorescent dye that stains acidic compartments (late endosomes and lysosomes) | Life Technologies | L7526 | LysoTracker Green DND 26. Pre-dissolved in DMSOS to a 1mM stock solution. Dilute to the final working concentration in the growth medium or buffer of choice. |
| Hank's Balanced Salt Solution (10x) | Gibco | 14065-056 | Magnesium and calcium free |
| Imaging Media | Life Technologies | A14291DJ | Live Cell Imaging Solution |
| Inverted Confocal Microscope | Zeiss | LSM 780 | |
| KymoToolBox | https://github.com/fabricecordelieres/IJ_KymoToolBox | ||
| Lipofection Enhancer Reagent | Life Technologies | 11514015 | Plus Reagent |
| Lipofection Reagent | Life Technologies | 15338100 | Lipofectamine LTX reagent |
| Orbital shaking incubator | New Brunswick Scientific | 8261-30-1008 | Innova 4230 , orbital shaking incubator with temperature and speed control |
| Penicillin/Strepomycin solution (100x) | Gen Clone | 25-512 | |
| Phosphate Buffered Saline (10x) | Gen Clone | 25-507x | |
| Poly-D-Lysine Hydrobromide | Sigma | P7405 | Dissolve in Tris buffer, pH 8.5, at 1mg/mL. Freeze for long term storage. Avoid cycles of freezing and thawing |
| Reduced serum medium | Gibco | 31985-062 | OPTI-MEM |
| Tissue Culture Flasks | Olympus Plastics | 25-209 | 75 cm^2. 100% angled neck access, 0.22um hydrophobic vented filter cap |
| Tissue culture incubator | Thermo Scientific | 51030285 | HERAcell VIOS 160i, tissue culture incubator with temperature, humidity, and CO2 control |
| Tris-Base | Sigma | T1503 | 8.402 g dissolved to one liter in water with 4.829 g Tris HCl to make 0.1 M Tris buffer, pH 8.5 |
| Tris-HCl | Sigma | T3253 | 4.829 g dissolved to one liter in water with 8.402 g Tris Base to make 0.1 M Tris buffer, pH 8.5 |
| Trypsin-EDTA (0.25%), phenol red | Gibco | 25200072 | |
| Vacuum-Driven Filter Systems | Olympus Plastics | 25-227 | 500 ml, PES membrane, 0.22 µm |
| Vannas scissors straight | Roboz | RS-5620 |
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