评估溶酶体碱化在活的秀丽线虫中的肠道
Summary
用 pH 敏感的生命染料 5 (6)-羧基-2 ‘, 7 ‘-dichlorofluorescein 双乙酸 (cDCFDA) 研究了在线虫的肠道中溶酶体酸度损失的分步指南
Abstract
线虫秀丽线虫(C. 线虫) 是一种广泛用于研究长寿和发育途径的模型系统。这种研究通过动物的透明性、向前和反向基因检测的能力、产生荧光标记蛋白的相对容易性以及荧光染料的使用来促进, 它们可以杏仁到早期胚胎或纳入其食物 (大肠杆菌菌株 OP50) 标签细胞器 (例如9-二乙基-5 H 苯并 (a) phenoxazine-5-一和 (3-{2-[(1 H, 1 ‘ H-22 ‘-bipyrrol 5-基 kappaN (1)) methylidene]-2 H-咯-5-基 kappaN}-N-[2-(二甲基氨基) 乙基] propanamidato) (difluoro) 硼)。在这里, 我们提出使用荧光 pH 敏感染料, 染色肠道溶酶体, 提供了动态, 生理变化的溶酶体酸度在活蠕虫的视觉读数。该协议不测量溶酶体 pH 值, 而是旨在建立一个可靠的方法来评估溶酶酸度的生理相关变化。cDCFDA 是一种细胞 permeant 化合物, 转化为荧光荧光 5-(和-6) 羧基-2 ‘, 7 ‘-dichlorofluorescein (cDCF) 水解后, 细胞内酯。质子内溶酶体陷阱 cDCF 在这些细胞器中, 在那里积聚。该染料由于其4.8 的低碱性, 已被用作酵母中的 pH 传感器。在这里, 我们描述了使用 cDCFDA 作为食物补充, 以评估肠道溶酶体的酸度在线虫.这种技术允许检测活动物中的碱化溶酶体,并具有广泛的实验应用, 包括对衰老、自噬和溶酶合成的研究。
Introduction
蛋白质团聚体的出现被广泛认为是真核细胞老化的标志1,2,3, 和其形成是细胞衰老的原则驱动因素4,5,6,7. 越来越多的证据表明, 随着细胞老化, 蛋白质分解代谢被削弱, 导致蛋白质聚集的增加。衰老细胞蛋白质水解的崩溃涉及自噬8的损伤以及蛋白酶介导的蛋白质降解9。最后, 在旧细胞中增加了不可逆转的蛋白质氧化, 进一步损害了蛋白质的分解代谢10。
自噬最初被认为是一个非选择性的过程, 以大量降解受损的蛋白质, 但最近的研究表明, 自噬是高度选择性的分解代谢的蛋白质聚集和功能失调的细胞器, 不可通过其他蛋白质清除机制11进行降解。在自噬过程中, 受损和聚集的蛋白质被隔离成一个叫做 autophagosome 的双膜囊泡。这个 autophagosome 然后与称为溶酶体的酸性细胞器融合, 导致 autophagosome 货物的降解12。溶酶体代表自噬性通路的终点, 参与不同的细胞过程, 如膜修复、转录控制和养分传感;突出它们在细胞稳态中的集中作用 (在 ref 13 中复习)。一些研究表明, 年龄依赖性的溶酶体功能下降与各种神经退行性疾病的相关性为13。一贯地, 在旧的细胞中恢复溶酶函数可以延缓衰老相关表型的出现,14,15。对 intralumen 环境组成的研究表明, 早期细胞溶酶酶功能的崩溃不是由于溶酶酶蛋白酶16的产生而减少的。另外, 有人建议, intralysosomal 酸度的损失, 它的酶活性的一个关键要求, 可能是在溶酶介导的蛋白质降解17的下降。为了能够探索这一假说, 必须开发试剂和协议, 以一种可复制和一致的方式来探测活细胞中溶酶体 pH 值的动态变化。
线虫的肠道是蠕虫的主要代谢组织, 是系统性稳态和寿命的重要调节因子。我们已经开发了测定蠕虫肠道溶酶体的酸性变化的检测方法, 以确定溶酶介导的蛋白质水解如何导致衰老。虽然以前在线虫中使用了 pH 敏感的显影标记肠道溶酶体, 但还没有努力建立一个成功的协议, 可以检测溶酶体 pH体内的小增加18.在这里, 我们提供了一种协议, 可用于检测C. 线虫肠道细胞中溶酶体酸度的损失, 使用一种简单而方便的喂养协议, 将 pH 敏感的荧光 (cDCFDA) 纳入 OP50 食品中。
Protocol
Representative Results
Discussion
各种细胞和分子事件导致衰老, 受生命史特征和遗传因素的影响。我们最近的研究22表明, 生殖周期通过调节溶酶体 pH 动态, 在控制躯体健康方面起着重要作用。我们表明, 溶酶介导的蛋白质水解是促进, 而动物积极繁殖的 v-atp 酶转录上调, 这反过来确保酸性溶酶。繁殖结束后, v-atp 酶表达下降, 溶酶体 alkalinize, 蛋白质聚集积累在这些细胞。
损害溶酶体酸化的?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们要感谢秀丽遗传学中心的菌株, 自然科学和工程研究理事会 (NSERC) 和加拿大创新基金会 (CFI) 供资。我们要感谢关丽珍博士 (细胞系统和解剖学部, UT 健康圣安东尼奥) 允许不受限制地使用她的实验室设施为所有C. 线虫实验和 Exing 博士 (副主任, 光学成像设备, UT 健康圣安东尼奥) 为协助与共焦显微术。此外, 我们亦多谢何议员提供支持和鼓励, 以协助拍摄影片。
Materials
| OP50 (E. coli) | Caenorhabditis Genetics Center | Order online at https://cgc.umn.edu/strain/OP50 | |
| 5(6)-carboxy-2’,7’-dichlorofluorescein diacetate | ThermoFisher | C369 | Commonly known as cDCFDA |
| 9-diethylamino-5H-benzo(a)phenoxazine-5-one and (3-{2-[(1H,1'H-2,2'-bipyrrol-5-yl-kappaN(1))methylidene]-2H-pyrrol-5-yl-kappaN}-N-[2-(dimethylamino)ethyl]propanamidato)(difluoro)boron | ThermoFisher | L7528 | Commonly known as Lysotracker Red |
| Confocal microscope (e.g. Zeiss LSM 510) | |||
| ImageJ | Download for free from https://imagej.nih.gov/ij/download.html | ||
| LB Broth powder | ThermoFisher | 22700041 | |
| Bacto Agar | Sigma | A5306-1KG | |
| NaCl | Sigma | S9888 | |
| Bacto Peptone | Fisher Scientific | S71604 | |
| Cholesterol powder | Sigma | C3045 | |
| CaCl2 | Sigma | 449709 | |
| MgSO4 | Sigma | M7506 | |
| K3PO4 | Sigma | P5629 | |
| Sodium Azide | Sigma | S2002 | |
| DMSO | Sigma | D8418 | |
| Microscope Slides | VWR | 48311-703 | |
| Cover Slips | ThermoFisher | 3406 | |
| Agarose | Sigma | A6013 | |
| Incubator | |||
| Mirror or other smooth flat surface |
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