Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health in C. elegans.
Pluronic gel-based burrowing assay for rapid assessment of neuromuscular health in C. elegans.
复制标题
基于 Pluronic 凝胶的洞穴测定，用于快速评估线虫的神经肌肉健康状况。
DOI：
10.1038/s41598-019-51608-9
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发表时间：
2019
影响因子：
4.6
通讯作者：
Vanapalli,SivaA
中科院分区：
文献类型：
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作者：
Lesanpezeshki,Leila;Hewitt,JenniferE;Laranjeiro,Ricardo;Antebi,Adam;Driscoll,Monica;Szewczyk,NathanielJ;Blawzdziewicz,Jerzy;Lacerda,CarlaMR;Vanapalli,SivaA
全生物体表型测定是评估模式生物（如线虫）神经肌肉功能和健康的核心。优雅在这项研究中，我们报告了一种新的测定格式的cancellingC。elegansin穴居，使线虫神经肌肉健康的快速评估。与对C.为了提高动物的穴居能力，我们在这里使用光学透明且生物相容的Pluronic F-127凝胶，该凝胶可在室温下从液体转变为凝胶，从而方便安全地处理动物。穴居测定方法包括将动物装载在孔板的底部，将Pluronic的液相浇铸在顶部，其通过适度的温度升高而固化，引诱动物通过对食物的趋化性到达表面，并量化动物在到达化学引诱物方面的相对成功。我们研究了Pluronic浓度、凝胶高度和化学引诱剂选择的影响，以优化测定性能。为了证明检测工作流程的简单性和多功能性，我们展示了其在多个领域的新应用，包括（i）评估致密体和/或M线缺陷的肌肉突变体（pfn-3，atn-1，uig-1，dyc-1，zyx-1，unc-95和tln-1），（i i）调整测定条件以揭示muplogel-8的变化，（iii）在遗传上均匀的野生型群体中分选快速钻洞动物，用于随后定量其不同的肌肉基因表达，以及（iv）测试亨廷顿病和帕金森病的蛋白毒性动物模型。我们的研究结果表明，刺激动物在一个密集的环境中导航，提供三维运动的机械阻力挑战神经肌肉系统的方式不同于标准的爬行和颠簸测定。我们的简单和高通量的穴居试验可以提供深入了解维持神经肌肉健康的分子机制，并有助于筛选治疗靶点。
Whole-organism phenotypic assays are central to the assessment of neuromuscular function and health in model organisms such as the nematodeC. elegans. In this study, we report a new assay format for engagingC. elegansin burrowing that enables rapid assessment of nematode neuromuscular health. In contrast to agar environments that pose specific drawbacks for characterization ofC. elegansburrowing ability, here we use the optically transparent and biocompatible Pluronic F-127 gel that transitions from liquid to gel at room temperature, enabling convenient and safe handling of animals. The burrowing assay methodology involves loading animals at the bottom of well plates, casting a liquid-phase of Pluronic on top that solidifies via a modest temperature upshift, enticing animals to reach the surface via chemotaxis to food, and quantifying the relative success animals have in reaching the chemoattractant. We study the influence of Pluronic concentration, gel height and chemoattractant choice to optimize assay performance. To demonstrate the simplicity of the assay workflow and versatility, we show its novel application in multiple areas including (i) evaluating muscle mutants with defects in dense bodies and/or M-lines (pfn-3, atn-1, uig-1, dyc-1, zyx-1, unc-95andtln-1), (ii) tuning assay conditions to reveal changes in the mutantgei-8, (iii) sorting of fast burrowers in a genetically-uniform wild-type population for later quantitation of their distinct muscle gene expression, and (iv) testing proteotoxic animal models of Huntington and Parkinson’s disease. Results from our studies show that stimulating animals to navigate in a dense environment that offers mechanical resistance to three-dimensional locomotion challenges the neuromuscular system in a manner distinct from standard crawling and thrashing assays. Our simple and high throughput burrowing assay can provide insight into molecular mechanisms for maintenance of neuromuscular health and facilitate screening for therapeutic targets.