固定化的线虫线虫分析神经元内细胞的转运
Summary
线虫线虫(线虫) 是研究轴突和细胞内转运的良好模型。在这里, 我描述了一个协议的在体内记录和分析轴突和 intraflagellar 运输在C. 线虫。
Abstract
轴突运输和 intraflagellar 转运 (IFT) 是轴索和纤毛形态发生和功能的关键。驱动超家族蛋白和蛋白是分别调节顺和逆行转运的分子马达。这些马达使用微管网络作为路轨。线虫线虫(线虫) 是研究轴突转运和 IFT的一个强有力的模型有机体。在这里, 我描述了一个协议, 以观察轴突运输和 IFT 在生活中的C. 线虫。运输货物可以通过使用荧光蛋白 (如绿色荧光蛋白 (GFP)) 标记货物蛋白质来可视化。C. 线虫是透明的, 而 GFP 标记的货物蛋白可以在特定细胞的细胞特异促进剂下表达。活蠕虫可以通过微10% 琼脂糖凝胶固定, 而不会杀死或麻醉蠕虫。在这种情况下, 货物的运动可以直接观察到的轴突和纤毛的生活线虫没有解剖。这种方法可用于观察任何细胞的任何货物分子通过修改目标蛋白和/或他们所表达的细胞。大多数基本的蛋白质, 如分子马达和适配器蛋白, 参与轴突运输和 IFT 是保存在C. 线虫。与其他模型生物体相比, 突变体可以在C. 线虫中更容易获得和维护。将此方法与各种C. 线虫突变体结合, 可以阐明轴突转运和 IFT 的分子机制。
Introduction
活细胞成像是分析细胞内转运的重要工具。在神经元细胞生物学中, 用活细胞成像分析轴突转运是了解神经元功能和形态发生1的必要条件。轴突运输缺陷的基础上几个神经退行性疾病2。驱动超家族蛋白和蛋白进行轴突转运 anterogradely 和逆行, 分别为1,2。
纤毛是另一个细胞室, 其中微管网络和贩卖机械是高度发达的3。蛋白质合成机械没有在纤毛中定位, 这意味着纤毛蛋白必须从细胞质输送到纤毛的尖端。纤毛特异性驱动和蛋白, 分别称为 kinesin-2 和细胞质 dynein-2, 运输的成分纤毛4, 在一个现象称为 intraflagellar 运输 (IFT)5。IFT 的损害导致一系列疾病称为 ciliopathies6。因此, 需要对 IFT 机制进行活体细胞成像分析, 以了解纤毛形成的基本机制和发病机理。
线虫线虫(线虫) 是研究轴突传输和 IFT7、8、9的好模型。为了观察 IFT,衣藻已被广泛用作模型生物体56。由于衣藻是一个单细胞生物体, IFT 与衰老、神经元功能和行为的关系将难以分析。此外, 诸如 CRISPR/Cas9 等基本遗传技术尚未应用到衣藻。在高模型生物体中, 如小鼠和果蝇, 轴突转运和 IFT 的中断经常导致致命的表型, 因为轴突转运和 IFT 对动物的形态发生和稳态至关重要10, 11。在小鼠的情况下, 细胞培养和转染一般需要观察轴突运输和 IFT, 这需要很多技能和大量的时间12,13。此外, 许多重要的生理环境可能会失去在培养细胞和细胞系。因为神经系统对于蠕虫的生存来说不是必需的, 所以轴突运输或 IFT 被破坏的线虫突变体通常不是致命的7、9、14。轴突运输和 IFT 可以直接观察在体内没有解剖, 因为C. 线虫是透明的, 因此很容易观察 GFP 标记标记。
有几种协议可以固定C. 线虫, 例如使用微流体设备15、带有麻醉的琼脂糖垫16或微17。麻醉的纳入可能抑制神经元的贩运事件15。微流控装置方法的一个明显的缺点是, 准备一台流控装置并不总是容易的。相反, 用琼脂糖垫和微固定是一种方便和简单的方法来执行时间推移成像在C. 线虫。在这里, 我描述这个基本的协议, 以固定的线虫和可视化轴突运输和 IFT在体内在线虫.与其他方法相比, 这里所描述的方法不需要特殊设备, 而且更便宜、更容易执行。
Protocol
Representative Results
Discussion
关于现有方法的限制
本文所描述的方法进行了优化, 以观察快速事件, 如轴突运输和 IFT。因此, 固定化比较长的潜伏期更有优先权。虽然我们已经能够观察贩运活动至少20分钟没有明显的摄动, 这种方法可能并不总是适合观察慢事件需要更长的观察, 如轴突伸长和细胞迁移。对于较长的观测, 需要通过减少琼脂糖的百分比 (即, 增加水以避免干燥和减少可能造成损坏的压力) 来优?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者对 Dr. 麻子杉本 (东北大学) 的有益讨论深表谢意。wyIs251是 Dr. 康沈 (斯坦福大学) 的一份慷慨礼物。mnIs17由 CGC 提供, 由 NIH 研究基础设施项目办公室 (P40 OD010440) 出资。这项工作得到了 jsp KAKENHI 赠款 #17H05010 和 #16H06536 和第一三基金会, 脑科学基金会和内基金会的支持。
Materials
| Slideglass (76 x 26 mm) | Matsunami | S1111 | |
| Coverglass (22 x 40 mm) | Matsunami | C024401 | |
| Agarose | Wako | 318-01195 | |
| Polystylene microbeads 0.1 micron | Polysciences | #00876 | |
| Heat block | TAITEC | 0063288-000 | CTU-mini |
| Microscope | Olympus | IX-71 | widefield microscope |
| Spinning disk Scanner | Yokogawa | CSU-X1 | spinning disc confocal scanner |
| Digital CCD camera | Hamamatsu Photonics | C10600-10B | ORCA-R2 degital CCD camera |
| Objective lens (x100, NA1.4) | Olympus | UPLSAPO 100XO | |
| Pasteur pipette (5 inch) | IWAKI | IK-PAS-5P | |
| Glass tube (1.5 cm diameter x 10.5 cm) | IWAKI | 9820TST15-105NP | |
| TV9211: wyIs251 | Laboratory of Kang Shen | N/A | |
| OTL11: mnIs17 | Ref. 27, 29 | N/A | SP2101 was backcrossed with wild type for 6 times |
| Stereo microscope | Carl Zeiss | 435064-9000-000 | STEMI 508 |
| Mirror transillumination unit | Carl Zeiss | 435425-9010-000 | |
| platinum wire (0.2 mm) | Nilaco Corporation | m78483501 | |
| 60 mm plastic dish | Falcon | #351007 | |
| Fiji | N/A | N/A | https://fiji.sc/ |
| nematode growth medium (NGM) | 1.7% (w/v) agarose, 50mM NaCl, 0.25% (w/v) Peptone, 1 mM CaCl2, 5 mg/mL Cholesterol, 25 mM KH2PO4, 1 mM MgSO4 |
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