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Summary
Abstract
Introduction
Protocol
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Neurociência

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

Published: November 20, 2021
doi:
10.3791/62744
,
1DBT-National Brain Research Centre

Summary

提出了一种使用荧光标记的末端结合蛋白对 体内 动态微管进行成像的协议。我们描述了标记、图像和分析 C.elegans后脊柱（PLM）神经元中的动态微管的方法。

Abstract

在神经元中，微管方向一直是识别轴突的关键评估器，轴突具有加端的微管和树突，通常具有混合方向。在这里，我们描述标记、图像和分析 C. elegans 中触摸神经元发育和再生过程中的微管动力学和生长的方法。我们使用微管尖的基因编码荧光记者，对轴状微管进行成像。使用此协议可以量化在轴切术后启动轴素再生的微管行为的局部变化。这种测定适应其他神经元和遗传背景，以研究微管动力学在各种细胞过程中的调节。

Introduction

神经元有一个精心设计的结构，具有专门的隔间，如树突、细胞体、轴突和突触。神经元细胞骨骼由微管、微丝和神经丝组成，其独特的组织在结构上和功能上支持神经元隔间1、2、3、4、5、6、7、8、9、10 .多年来，微管组织被确定为神经元极性和功能的关键决定因素。当神经元在发育或再生过程中进行结构改造时，微管动力学和方向决定各种神经元隔间7的身份、偏振传输、生长和发展。因此，评估体内的微管动力学和方向与神经元重塑过程相关是当务之急。

微管由α和β图布林异质体的原体组成，具有动态加端和相对稳定的负端11，12。加尖复合蛋白和相关端结合蛋白的发现，使一个平台能够评估微管组织13。端结合蛋白（EBP）与微管的生长增益端短暂关联，其关联动力学与微管蛋白14、15的生长相关。由于加尖复合物与微管的频繁关联和分离，GFP 标记的 EBP 的点传播功能在计时电影15、16中显示为”彗星”。自从在哺乳动物神经元16中率先观察以来，用荧光蛋白标记的端结合蛋白已经用于确定不同模型系统和神经元类型17、18、19、20、21、22、23的微管动力学。

由于其简单的神经系统和透明的身体 ，C.elegans 已被证明是一个优秀的模型系统，研究神经元改造期间的发展和再生 在体内。在这里，我们描述标记、图像和分析 C. elegans 中触摸神经元发育和再生过程中的微管动力学和生长的方法。使用基因编码的EBP-2：GFP，我们成像PLM神经元的微管，这使我们能够确定微管的极性在这个神经元24的两种不同的神经质。这种方法允许观察和量化EBP彗星作为测量不同细胞环境中的微管动力学，例如，在轴切术后启动轴素再生的微管行为的局部变化可以使用我们的协议进行评估。此检测可以适应不同细胞类型和遗传背景中不同细胞过程中微管动力学的调控。

Protocol

1. 记者应变：文化与维护 注：为了测量PLM神经元的微管动力学和方向，我们使用蠕虫菌株表达EBP-2：GFP下触摸神经元特异性促进物mec-4（juIs338等位基因）18，25，26。我们使用标准蠕虫培养和维护方法为这个菌株27。 在水中准备线虫生…

Representative Results

作为一个具有代表性的例子，我们在对EBP彗星的体内观察中描述了PLM神经元的稳定状态和再生轴突。PLM神经元位于蠕虫的尾部区域，具有很长的前过程，形成突触和短后过程。PLM 神经元生长在靠近表皮的前后方向，并负责蠕虫的温和触觉。由于其简化的结构，以及成像和显微外科的便利性，PLM神经元被广泛调查为其微管细胞骨骼29，轴向运输30，31，再生</su…

Discussion

多年来，了解微管动力学一直是细胞骨骼研究领域的一个关键焦点。微管经历核化和灾难，以及一个连续的动态不稳定过程44，45，46，47。这些信息大部分是通过体外测定获得的，如自由与聚合的tubulin的光散射读数、荧光管蛋白的微管生长检测等。虽然在薄细?…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢金一石和安德鲁·奇斯霍尔姆在研究中给予的最初支持和治疗。细菌菌株OP50由美国国家卫生研究院研究基础设施项目办公室（P40 OD010440）资助，由卡诺哈布迪炎遗传学中心（CGC）提供商业利用。我们还感谢达曼德拉·普里对实验程序的标准化。这项研究由国家大脑研究中心的核心赠款资助（由生物技术部支持， 印度政府）、DBT/Wellcome信托印度联盟早期职业补助金（赠款/E/E/18/1/504331）给S.D.，韦康信托-DBT印度联盟中期赠款（赠款#IA/I/13/1/500874）给A.G.-R，以及科学和工程研究委员会（SERB： CRG/2019/002194） 至 A.G.-R.

Materials

CZ18975 worm strain Yishi Jin lab CZ18975 Generated by Anindya Ghosh-Roy
Agarose Sigma A9539 Mounting worms
Coverslip (18 mm x 18 mm) Zeiss 474030-9010-000 Mounting worms
Dry bath with heating block Neolab Mounting worms
Glass slides (35 mm x 25 mm) Blue Star Mounting worms
Polystyrene bead solution (4.55 x 10^13 particles/ml in aqueous medium with minimal surfactant) Polysciences Inc. 00876 Mounting worms
Test tubes Mounting worms
OP50 bacterial strain Caenorhabditis Genetics Center (CGC) OP50 Worm handling
60mm petri plates Praveen Scientific 20440 Worm handling
Aspirator/Capillary VWR 53432-921 Worm handling
Incubator Panasonic MIR554E Worm handling
Platinum wire Worm handling
Stereomicroscope with fluorescence attachment Leica M165FC Worm handling
0.3% Sodium Chloride Sigma 71376 Nematode Growth Medium
0.25% Peptone T M Media 1506 Nematode Growth Medium
10mg/mL Cholesterol Sigma C8667 Nematode Growth Medium
1mM Calcium chloride dihydrate Sigma 223506 Nematode Growth Medium
1mM Magnesium sulphate heptahydrate Sigma M2773 Nematode Growth Medium
2% Agar T M Media 1202 Nematode Growth Medium
25mM Monobasic Potassium dihydrogen phosphate Sigma P9791 Nematode Growth Medium
0.1M Monobasic Potassium dihydrogen phosphate Sigma P9791 1X M9 buffer
0.04M Sodium chloride Sigma 71376 1X M9 buffer
0.1M Ammonium chloride Fisher Scientific 21405 1X M9 buffer
0.2M Dibasic Disodium hydrogen phosphate heptahydrate Sigma S9390 1X M9 buffer
Glass bottles Borosil Buffer storage
488 nm laser Zeiss Imaging
5X objective Zeiss Imaging
63X objective Zeiss Imaging
Camera Photometrics Evolve 512 Delta Imaging
Computer system for Spinning Disk unit HP Intel ® Xeon CPU E5-2623 3.00GHz Imaging
Epifluorescence microscope Zeiss Observer.Z1 Imaging
Halogen lamp Zeiss Imaging
Mercury Arc Lamp Zeiss Imaging
Spinning Disk Unit Yokogawa CSU-X1 Imaging
ZEN2 software Zeiss Imaging
Image J (Fiji Version) Image analysis and processing
Adobe Creative Cloud Adobe Image analysis and processing
Computer system for Image Analysis Dell Intel ® Core ™ i7-9700 CPU 3.00GHz Image processing/Representation
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Tags

Keywords: Microtubule DynamicsMicrotubule OrientationCaenorhabditis Elegans NeuronsIn Vivo AssessmentCytoskeletonAlpha And Beta TubulinEnd-binding ProteinsEBP-2GFPPLM NeuronsMake-3 PromoterMountingPolystyrene BeadsFluorescence MicroscopySpinning Disk Unit
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Dey, S., Ghosh-Roy, A. In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons. J. Vis. Exp. (177), e62744, doi:10.3791/62744 (2021).
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