研究卡埃纳哈布迪炎细胞结构和细胞形态学的定量方法
Summary
本研究概述了使用免费提供的图像处理工具对C. elegans的突触大小和定位、肌肉形态和线粒体形状的定量测量。这种方法允许将来在C.elegans的研究定量比较由于基因突变而导致的组织和细胞器结构变化的程度。
Abstract
定义疾病背后的细胞机制对于开发新的治疗方法至关重要。经常用来解开这些机制的策略是引入候选基因的突变,并定性地描述组织和细胞细胞器的形态变化。然而,定性描述可能无法捕捉细微的型值差异,可能歪曲人群中个体的型型变化,并且经常被主观地评估。本文介绍了定量方法,利用激光扫描共聚焦显微镜与市售的生物图像处理软件相结合,研究线虫卡埃纳哈布迪炎的组织和细胞器的形态。对影响突触完整性(大小和综合荧光水平)、肌肉发育(肌肉细胞大小和肌苷丝长度)和线粒体形态(循环和大小)的表型进行了定量分析,以了解基因突变对这些细胞结构的影响。这些定量方法并不限于此处描述的应用,因为它们可以很容易地用于定量评估线虫中其他组织和细胞器的形态,以及其他模型生物体中的形态。
Introduction
线虫卡埃内沙布迪炎(C.elegans)越来越多地被用作一个模型系统,以揭示人类疾病所涉及的生物和分子过程。成年线虫的体长略高于1毫米,可以产生多达300个鸡蛋的大卵1。孵化后,C.elegans只需3-4天才能成年,并存活2至3周。由于其易于培养,C. elegans是目前最抢手的体内动物模型之一,用于进行经济高效、快速的药物筛选,以确定人类疾病的治疗药物。此外,其遗传保护,明确界定的行为范式,透明的身体荧光或光显微镜,以及易于基因操作,使研究细胞和分子后果的基因突变很容易实现3. C. elegans基因组与人类基因共享大约 60-80% 的正畸,其中约 40% 与疾病相关。在C.elegans中建模和研究的一些人类疾病包括神经退行性疾病(阿尔茨海默氏病、帕金森病、肌萎缩性侧索硬化症、Charcot-Marie-Tooth 病)、肌肉相关疾病(杜琴肌肉萎缩症),和代谢性疾病(高血糖)2,4。在大多数人类疾病中,疾病引起的细胞和细胞器定位和形态变化发生,这很容易在线虫模型中评估。
荧光标记已广泛用于标记组织和细胞器,用于显微镜下的动态可视化。然而,在C.elegans中,评估由于基因突变引起的形态不规则性的传统方法主要依靠视觉描述。虽然定性评估可以涵盖更广泛的表型描述范围(突触形态学、GFP结块、特定斧头形状、肌肉纤维厚度等),并提供形态变化的鸟瞰图,但它们不太适合比较不同组之间的小差异。此外,定性评估以视觉、主观评估为基础,这可能导致对形态异常的高估或低估。最后,定性观测结果也可能因个人而异,给数据复制造成困难。
近年来,已经开发出一些用户友好、现成的计算算法,可以定量分析图像。然而,这种图像分析软件的利用,在一些形态学研究,特别是有关身体壁肌和线粒体的研究,在C.elegans的研究已经滞后。为了改进C.elegans的基本结构分析,试验了一些现成的开源图像分析软件,以定量比较基因突变对肌肉线粒体、身体壁肌和突触的影响形态。这些实验程序详细概述了这些程序(Fiji、ilastik、CellProfiler、SQUASSH)如何用于评估突触大小和突触蛋白定位、体壁肌肉面积和纤维长度以及线粒体尺寸和线粒体尺寸的变化。由于线虫的基因突变而循环。
Protocol
Representative Results
Discussion
形态变异经常通过手工计算明显差异或使用任意阈值来确定与野生型表型相比的缺陷来评估。然而,最近,定量方法被用于形态学的比较研究,以无偏见的方式准确测量和描述细胞和亚细胞水平上的变化。识别表型之间微妙但与生物学相关差异的能力是了解控制由环境因素或遗传疾病引起的形态变化的基本分子机制的有力手段。计算能力和显微镜成像分辨率的不断提高,加上C. elegans遗传学的易?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Neumann 实验室的成员进行了宝贵的讨论和投入。一些菌株由CGC提供,由NIH研究基础设施项目办公室(P40 OD010440)资助。作者感谢WormBase提供了丰富的关于C.elegans的信息,并感谢莫纳什大学的莫纳什微成像提供了仪器、培训和技术支持。这项工作得到了CMTAA研究资助(2015年和2018年)的支持,NHMRC项目赠款1101974和1099690授予了B.N.
Materials
| Agar-agar | Merck | 1.01614.1000 | |
| Agarose | Invitrogen | 16500-500 | |
| Confocal microscope | Leica | TCS SP8 | Inverted platform |
| Fluorescence microscope | Carl Zeiss AG | Zeiss Axio Imager M2 | |
| Glass coverslips #1 | Thermo scientifique | MENCS22221GP | |
| Glass coverslips #1.5 | Zeiss | 474030-9000-000 | Made by SCHOTT |
| Glass slides | Thermo scientifique | MENS41104A/40 | |
| Light LED | Schott | KL 300 LED | |
| Stereo Microscope | Olympus | SZ51 | |
| Tryptone (Peptone from casein) | Merck | 107213 | Ingredients for Lysogeny Broth (LB) medium |
| Yeast Extract | Merck | 103753 | Ingredients for Lysogeny Broth (LB) medium |
| Sodium chloride | Merck | 106404 | Ingredients for Lysogeny Broth (LB) medium |
| Peptone (Peptone from meat) | Merck | 107214 | Ingredients for Nematode Growth Media (NGM) agar |
| Agar | Sigma | A1296 | Ingredients for Nematode Growth Media (NGM) agar |
| Sodium chloride | Merck | 106404 | Ingredients for Nematode Growth Media (NGM) agar |
| Cholesterol | Sigma | C8667-25G | Ingredients for Nematode Growth Media (NGM) agar |
| Calcium chloride | Merck | 102382 | Ingredients for Nematode Growth Media (NGM) agar |
| Magnesium sulfate | Merck | 105886 | Ingredients for Nematode Growth Media (NGM) agar |
| Dipotassium phosphate | Merck | 105101 | Ingredients for Nematode Growth Media (NGM) agar |
| Potassium dihydrogen phosphate | Merck | 104873 | Ingredients for Nematode Growth Media (NGM) agar |
| Disodium phosphate | Merck | 106586 | Ingredients for M9 buffer |
| Sodium chloride | Merck | 106404 | Ingredients for M9 buffer |
| Potassium dihydrogen phosphate | Merck | 104873 | Ingredients for M9 buffer |
| Magnesium sulfate | Merck | 105886 | Ingredients for M9 buffer |
| Pasteur pipette | Corning | CLS7095D5X-200EA | |
| Petri dishes | Corning | CLS430589-500EA | |
| Platinum wire | Sigma | 267201-2G | |
| Spatula | Met-app | 2616 | |
| Tetramisole hydrochloride | Sigma | L9756-5G |
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