活体和功能性鼠耳蜗毛细胞中的德特贴标签和吸收
Summary
在这里，我们提出了一种方法，用于可视化3 kDa德州红标dextran在听觉毛细胞与功能性干扰诱导通道的吸收。此外，3~10 kDa的分泌器可用于研究毛发和Corti器官支持细胞的内分泌。
Abstract
发细胞调子（MET）通道在听力中起着重要的作用。然而，MET的分子特性和结构信息仍然未知。对毛细胞的电生理学研究表明，MET通道具有较大的导导率，可渗透到相对较大的荧光阳离子分子，包括一些styryl染料和德州红标氨基糖苷抗生素。在此协议中，我们描述了一种可视化和评估 Corti 外植器官毛细胞中荧光分管的产生情况的方法，该方法可用于功能 MET 通道的测定。我们发现，3 kDa德州红标记dextran特别标记功能听觉毛细胞后1⁄2小时孵育。特别是，3 kDa dextran 标记两个较短的立体细胞行，并在存在功能 MET 通道时以漫反射模式在细胞体中累积。在毛细胞和周围支持细胞的细胞体中观察到了一种类似囊泡的标记模式。我们的数据表明，3 kDa德州红dextran可用于可视化和研究细胞染料摄入的两种途径;通过功能MET通道和内分泌的毛细胞特定进入途径，这种模式也可用于更大的dextran。
Introduction
内耳毛细胞是检测声音和隐藏电信号机械刺激的感官细胞，最终由我们的大脑解释。这些细胞有一个楼梯形的捆绑三行基于活性蛋白的细丝，称为立体丝，从它们的锥形区域1，2突出。机械刺激使立体丝向最长的行偏转，并触发机械转导（MET）通道3的打开。MET通道的开放导致阳离子的涌入，使细胞去极化，从而在头发细胞的基础区域发出突触囊泡的释放。
MET通道对听力至关重要的生物物理特性得到了广泛的描述。在其他属性中，这些通道是阳离子选择性的，具有相对较大的电导率（低 Ca2+中为 150±300 pS）4、5、6、7、8、9、10 。值得注意的是，大型荧光分子，如FM1-43和得克萨斯红标记氨胶苷是MET通道的渗透阻滞剂，导致其积累在头发细胞体，可以使用荧光显微镜11，12，13，14。相反，MET通道的分子特性和结构及其渗透途径仍然难以捉摸。越来越多的实验证据表明，跨膜样通道蛋白1（TMC1）是成熟毛细胞15、16、17、18、19中MET通道的组成部分。跨膜状通道1（TMC1）中的突变会改变MET通道特性19、20、21、22，并引起耳聋。此外，TMC1还本地化到MET通道18、23的站点，并与负责将机械力传输到MET通道24、25的尖端链路进行交互。此外，最近的生物信息学分析已经发现TMC蛋白是进化相关的对美甲敏感通道TMEM63/OSCA蛋白质和TMEM16蛋白质，钙活性氯化物通道和脂质scramblass26，27，28。基于这些蛋白质之间关系的TMC1结构模型揭示了在蛋白质-脂质界面27处存在一个大腔。该腔包含两个TMC1突变，导致常染色体显性听力损失（DFNA36）27、29、30、31、32，以及选择性地修饰半胱氨酸突变体，使其在腔内残留物改变MET通道特性28，表明它可以作为MET通道的渗透通路。TMC蛋白质中这种预测腔的较大尺寸可以解释大分子渗透MET通道的能力。为了测试MET通道包含异常大渗透通路的预测，并突破TMC1中观察到的腔体大小的极限，我们开发了一个协议，在Corti外植的器官中用更大的分子进行吸收实验，3 kDa dextran荧光标记为德州红。
Dextran是一种复杂的分枝多糖，由许多D-葡萄糖分子组成，由α-1，6糖碱基结结合。其高溶性在水中，低细胞毒性，和生物不性使其成为研究多种细胞过程的多功能工具。此外，dextran 有多种尺寸可供选择，并带有多种颜色的荧光标记。荧光标记的dextrans通常用于细胞和组织渗透性研究33，34，研究多细胞系统35，36的内分泌，以及神经追踪37，38。在听觉领域，dextran分子也被用来评估细胞-细胞结的中断和听觉感觉上皮完整性的丧失后暴露于Corti39，40的chinchilla器官的强烈噪音。
在这项工作中，我们利用一些最小的（3和10 kDa）荧光除变性器的特性，在鼠内耳毛细胞中进行吸收实验，并探索内耳毛细胞MET通道的渗透通路的大小。此外，我们使用激光扫描共聚焦显微镜（LSM）880配备了Airyscan探测器，以可视化和本地化荧光dextran在立体神经和听觉毛细胞的细胞体。
Protocol
动物护理和实验程序按照《实验室动物护理和使用指南》进行，该准则经国家神经疾病和中风研究所动物护理和使用委员会批准（动物协议#1336到KJS）。 1. 老鼠 设置一对C57BL/6J野生型的繁殖对在动物设施中繁殖，以控制幼崽的出生日期，并跟踪幼崽的年龄。 2. 耳蜗解剖 设置一个靠近立体显微镜的清洁空间以执行解剖（?…
Representative Results
我们观察到，在野生型产后6（P6）小鼠的Corti外体器官孵育2小时后，毛细胞的强健和特殊标记，3 kDa dextran荧光标记有德州红（dextran-TR）（图2A-B）。在Corti器官的基底、中、顶区域（图2B）的内侧和外发细胞（IHC和OHC）中观察到了Dextran标记。 荧光标记的phalloidin用于对抗丝状物（F-actin）和可视化基于功能素…
Discussion
该协议描述了如何在3 kDa dextran德州红的Corti外植植物的鼠器官中执行接受实验。这种方法的目的是测试之前测试的分子是否也能够专门标记听觉毛细胞，并渗透到MET通道。类似的实验方案以前曾用于评估毛细胞与其他荧光染料的渗透性，如FM1-43（0.56 kDa）12、19、20和得克萨斯州红标记的氨糖苷（1.29~1.43 kDa）13、52。<sup c…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢NICHD显微镜和成像核心的文森特·施拉姆协助共聚焦图像采集，以及张青惠在殖民地管理和小鼠护理方面的宝贵帮助。这项研究得到了国家研究所、NIH、贝塞斯达、医学博士、K.J.S.A.B.的内学研究计划的支持，并得到了国立国家研究所内学研究计划的罗伯特·温特霍尔德博士后奖学金的支持。NIDCD。
Materials
| #1.5 glass coverslips 18mm | Warner Instruments | 64-0714 | |
| Alexa Fluor 488 Phalloidin | ThermoFisher | A12379 | |
| Alexa Fluor 594 Phalloidin | ThermoFisher | A12381 | |
| alpha Plan-Apochromat 63X/1.4 Oil Corr M27 objective | Carl Zeiss | 420780-9970-000 | |
| Amiloride hydrochloride | EMD MILLIPORE | 129876 | |
| Benchwaver 3-dimensional Rocker | Benchmarks scientific | B3D5000 | |
| C57BL/6J wild-type mice | strain 000664 | The Jackson Laboratory | |
| Cell impermeant BAPTA tetrapotassium salt | ThermoFisher | B1204 | |
| Dextran, Fluorescein, 10,000 MW, Anionic, Lysine Fixable | ThermoFisher | D1820 | |
| Dextran, Texas Red, 10,000 MW, Lysine Fixable | ThermoFisher | D1863 | |
| Dextran, Texas Red, 3000 MW, Lysine Fixable | ThermoFisher | D3328 | |
| Formaldehyde Aqueous Solution EM Grade | Electron microscopy science | 15710 | |
| HBSS, calcium, magnesium, no phenol red | ThermoFisher | 14025 | |
| HBSS, no calcium, no magnesium, no phenol red | ThermoFisher | 14170 | |
| Image J or FIJI | NIH | http://fiji.sc/ | |
| Immersol 518F oil immersion media | Carl Zeiss | 444970-9000-000 | |
| Leibovitz's L-15 Medium, GlutaMAX Supplement | ThermoFisher | 31415029 | |
| neomycin trisulfate salt hydrate | Sigma | N6386 | |
| PBS (10X), pH 7.4 | ThermoFisher | 70011069 | |
| Phalloidin-CF405M | Biotium | 00034 | |
| ProLong Diamond antifade mounting media | ThermoFisher | P36970 | |
| superfrost plus microscope slide | Fisherbrand | 22-037-246 | |
| Triton X-100 | Sigma | T8787 | |
| Zen Black 2.3 SP1 software | Carl Zeiss | https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html |
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Citer Cet Article
Ballesteros, A., Swartz, K. J. Dextran Labeling and Uptake in Live and Functional Murine Cochlear Hair Cells. J. Vis. Exp. (156), e60769, doi:10.3791/60769 (2020).