使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络
Summary
在这里，我们提出了一个详细的方案来可视化神经肌肉接头和肌肉细胞中的微管网络。结合 黑腹果蝇强大的遗传工具，该方案极大地促进了微管网络调节蛋白在神经系统中的作用的遗传筛选和微管动力学分析。
Abstract
微管网络是神经系统的重要组成部分。许多微管调节蛋白的突变与神经发育障碍和神经系统疾病有关，如微管相关蛋白Tau对神经退行性疾病，微管切断蛋白Spastin和Katanin 60分别引起遗传性痉挛性截瘫和神经发育异常。检测神经元中的微管网络有利于阐明神经系统疾病的发病机制。然而，神经元的小尺寸和轴突微管束的密集排列使得微管网络的可视化具有挑战性。在这项研究中，我们描述了一种解剖幼虫神经肌肉接头和肌肉细胞的方法，以及α-微管蛋白和微管相关蛋白Futsch的免疫染色，以可视化果蝇中的微管网络。神经肌肉接头使我们能够观察突触前和突触后的微管，果蝇幼虫中大尺寸的肌肉细胞可以清晰地观察微管网络。在这里，通过在黑腹果蝇中突变和过表达Katanin 60，然后检查神经肌肉接头和肌肉细胞中的微管网络，我们准确地揭示了Katanin 60在神经发育中的调节作用。因此，结合果蝇黑腹果蝇强大的遗传工具，该方案极大地促进了微管网络调节蛋白在神经系统中的作用的遗传筛选和微管动力学分析。
Introduction
微管（MTs）作为细胞骨架的结构成分之一，在细胞分裂、细胞生长和运动、细胞内运输和维持细胞形状等多种生物过程中发挥着重要作用。微管动力学和功能通过与其他蛋白质的相互作用进行调节，例如 MAP1、MAP2、Tau、Katanin 和驱动蛋白 1,2,3,4,5。
在神经元中，微管对于轴突和树突的发育和维持至关重要。微管异常会导致功能障碍，甚至导致神经元死亡。例如，在阿尔茨海默氏症患者的大脑中，Tau 蛋白过度磷酸化会降低微管网络的稳定性，导致神经系统异常6。因此，检查微管网络将有助于理解神经发育和神经系统疾病的发病机制。
神经肌肉接头（NMJ）是在运动神经元轴突末梢和肌纤维之间形成的外周突触，是研究突触结构和功能的优秀而强大的模型系统7。Futsch 是果蝇中的一种蛋白质，与哺乳动物中发现的微管结合蛋白 MAP1B 同源8。它仅在神经元中表达，并在 NMJ 突触按钮的发育中发挥作用 8,9。在野生型中，通过用抗Futsch进行免疫染色来观察沿NMJ过程中心运行的丝状束。当到达 NMJ 的末端时，该束能够形成由微管组成的环或失去其丝状结构，从而导致弥漫性和点状外观10。微管环与暂停的生长锥有关，这表明微管阵列是稳定的11。因此，我们可以通过 Futsch 染色间接确定 NMJ 中微管的稳定发育。果蝇幼虫中大尺寸的肌肉细胞可以清晰地观察微管网络。通过分析微管的密度和形状，可以发现影响微管网络稳定性的因素。同时，可以将肌肉细胞的微管网络状态与NMJ的结果进行交叉验证，以获得更全面的结论。
许多协议已被用于研究微管的网络和动力学。然而，这些研究通常集中在体外研究12,13,14,15,16。或者，一些体内实验已经使用电子显微镜来检测细胞骨架17。根据荧光标记的抗体或化学染料与蛋白质或DNA的特异性结合，这里介绍的方法允许在体内单个神经元水平上检测NMJ中的微管网络，结果通过肌肉细胞的观察得到证实。当与果蝇黑腹果蝇中可用的强大遗传工具结合使用时，该方案是简单、稳定和可重复的，能够对体内神经系统中微管网络调节蛋白的作用进行各种表型检查和遗传筛选。
Protocol
Representative Results
Discussion
这里描述了用于 果蝇 幼虫神经肌肉接头和肌肉细胞的解剖和免疫染色的方案。有几个要点需要考虑。首先，在解剖过程中，避免观察到的肌肉受伤至关重要。在切除内脏之前，可能值得固定鱼角，以防止镊子和肌肉直接接触。为避免肌肉损伤或与幼虫表皮分离，重要的是要确保在洗涤和孵育过程中振荡器的速度不超过 15 rpm。此外，必须精确控制皮肤延伸，以确保清晰、完整地拍摄 NMJ 形?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢熊莹博士对手稿的讨论和评论。这项工作得到了中国国家科学基金委员会（NSFC）对C.M.（31500839）的资助。
Materials
| Alexa Fluor Plus 405 phalloidin | invitrogen | A30104 | dilute 1:200 |
| Enhanced Antifade Mounting Medium | Beyotime | P0128M | |
| FV10-ASW confocal microscope | Olympus | ||
| Goat anti-Mouse antibody, Alexa Fluor 488 conjugated | Thermo Fisher | A-11001 | dilute 1:1,000 |
| Laser confocal microscope LSM 710 | Zeiss | ||
| Micro Scissors | 66vision | 54138B | |
| Mouse anti-Futsch antibody | Developmental Studies Hybridoma Bank | 22C10 | dilute 1:50 |
| Mouse anti-α-tubulin antibody | Sigma | T5168 | dilute 1:1,000 |
| Paraformaldehyde | Wako | 168-20955 | Final concentration: 4% in PB Buffer |
| Stainless Steel Minutien Pins | Entomoravia | 0.1mm Diam | |
| Stereomicroscope SMZ161 | Motic | ||
| stereoscopic fluorescence microscope BX41 | Olympus | ||
| Texas Red-conjugated goat anti-HRP | Jackson ImmunoResearch | dilute 1:100 | |
| TO-PRO(R) 3 iodide | Invitrogen | T3605 | dilute 1:1,000 |
| Transfer decoloring shaker TS-8 | Kylin-Bell lab instruments | E0018 | |
| TritonX-100 | BioFroxx | 1139 | |
| Tweezers | dumont | 500342 |
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