体外和体内检测人类细胞中的 Mitophagy,线虫和小鼠
Summary
Mitophagy, 清除受损线粒体的过程, 是线粒体稳态和健康维护所必需的。本文介绍了人类细胞、线虫线虫和小鼠的一些最新的 mitophagy 检测方法。
Abstract
线粒体是细胞的动力, 以 ATP 的形式产生细胞能量。线粒体功能障碍有助于生物老化和多种疾病, 包括代谢疾病, 过早衰老综合征, 和神经退行性疾病, 如阿尔茨海默氏病 (AD) 和帕金森氏症 (PD)。线粒体健康的维持取决于线粒体生物和有效清除功能性线粒体通过 mitophagy。实验方法, 以准确检测自噬/mitophagy, 特别是在动物模型, 一直是挑战, 以发展。最近在了解 mitophagy 分子机制方面取得的进展, 使新的 mitophagy 检测技术得以发展。在这里, 我们介绍了几种通用的技术来监测人类细胞中的 mitophagy,线虫线虫(如, 罗塞拉和 DCT-1/LGG-1 菌株), 和小鼠 (mt-马)。这些 mitophagy 检测技术的结合, 包括物种评估, 将提高 mitophagy 测量的准确度, 并有助于更好地了解 mitophagy 在健康和疾病中的作用。
Introduction
Mitophagy 对线粒体的维护至关重要。线粒体与多个细胞信号通路相交, 是细胞能量产生、细胞新陈代谢和钙稳态的通用细胞器,1,2,3,4. 线粒体经常面临来自内源性和外部来源的挑战, 如活性氧种 (ROS) 和线粒体毒物, 这些都导致了 “衰老” 和功能失调的线粒体的产生。受损线粒体的积累降低了 ATP 生产的效率, 同时增加了有害活性氧的数量, 并与年龄有关的疾病, 如代谢性疾病, AD 和 PD1,5,6.为了防止线粒体诱导的细胞功能障碍, 细胞需要特别识别受损的线粒体, 并通过细胞过程中的线粒体自噬 (mitophagy) 有效地去除它们。这表明 mitophagy 在健康和疾病中的重要性说明了需要准确和有效的方法来检测 mitophagy 的体外和体内。
Mitophagy 是一个多步过程涉及许多蛋白质和蛋白质复合物5,7,8。简言之, 受损的线粒体首先被 double-membraned phagophore, 它可以来源于细胞膜、内质网、高尔基体、细胞核或线粒体本身9,10。球形 phagophore 拉长, 并最终封闭线粒体内, 构成线粒体 autophagosome (mitophagosome)。mitophagosome 然后与溶的降解, 形成一个 autolysosome, 其中受损的线粒体退化和回收利用7,8。主要的噬蛋白也涉及 mitophagy 包括: 自噬相关的 7 (ATG7) 和 Beclin1 (启动), 微管相关蛋白 1 a/1 b 光链 3 (LC3-II) (LGG-1 在C. elegan) 和 p62 (phagophore 的组成部分), 并溶酶体相关膜糖蛋白 2 (LAMP2)6,7。此外, 有几种基本的蛋白质 mitophagy, 包括 PTEN 诱导的假定激酶 1 (PINK-1), Parkin1, 核点蛋白 52 kDa (NDP52), optineurin, BCL2 交互蛋白3像 (NIX/BNIP3L) (DCT-1 在C. 线虫),在其他5、6、11中。
一种检测自噬水平变化的常用方法是 LC3-II/LC3-I 或 LC3-II/actin 的比值。然而, 这种方法是非特异性的, 因为增加这一比例可能反映了增加启动或受损融合的 mitophagosome 到溶12。另一种方法是评估自噬标记 (例如, LC3) 和线粒体蛋白 (如如, Translocase 外线粒体膜 20 (TOMM20, 可被蛋白酶降解)) 之间的定位。但是, 这只能指示总 mitophagy 级别的变化, 并且不能区分发生阻塞的步骤。这可以通过使用溶酶体抑制剂 (例如, E64d+Pepstatin A, 称为 EP) 来澄清, 从而导致 mitophagosomes 的积累。mitophagosomes 在基线上的数目与 EP mitophagosomes 后的数量之间的差异可以表明 mitophagy。这些限制促使了新的 mitophagy 检测技术的发展。鉴于 mitophagy 在广泛的人类疾病中的相关性日益增强, 我们提出了几种健壮的 mitophagy 检测技术, 这可能对研究人员有用。我们包括体外和体内技术, 并建议结合多种技术来验证 mitophagy 的变化。
Protocol
Representative Results
Discussion
精确测量 mitophagy 是技术上的要求。在这里, 我们提出了一些强有力的技术, 允许定性检测 mitophagy 和量化的 mitophagy 水平在最常见的实验室实验模型。
为了获得可复制的数据, 必须进行至少三生物重复的实验设计。所有参与试验和分析的研究人员都必须对实验组的特性视而不见。此外, 成像场必须在图像采集过程中随机选择。对于细胞培养和线虫研究, 至少应该进行三生?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢 Dr. Atsushi 宫和 Dr. 理查德 j 游乐共享 mt 马质粒和 mt 马联合 Hela 细胞。我们感谢 Raghavendra a. Shamanna 和 Dr. Croteau 对手稿的批判性阅读。这项研究得到了国立卫生研究院 (VAB)、国家老龄研究所的内部研究方案以及2014-2015 和2016-2017 的 intra-laboratory 赠款 (VAB) 的支持。HELSE 支持 RHF (项目编号: 2017056) 和挪威研究理事会 (项目编号: 262175)。
作者投稿:
设计了原稿, 起草了草稿;KP, NS, EMF, RDS, JSK, SAC, YH, 和 ED 写了不同的部分的文件;NT, JP, VAB 修改了手稿, 并提供了专门知识。
Materials
| mt-Keima mouse | Jackson Laboratory | ||
| Lipofectamine 2000 DNA transfection reagent | Thermofisher | #11668027 | |
| Opti-MEM medium (Gibco) | Thermofisher | #31985062 | serum-free medium |
| mtKemia plasmid: pCHAC-mt-mKeima | addgene | #72342 | |
| COXII antibody (mouse) | abcam | #ab110258 | |
| LAMP2 antibody (rabbit) | NOVUS | #CD107b | |
| goat-anti-rabbit with wavelength 568 nm of red fluorescent protein (RFP) | Thermofisher | #Z25306 | Alexa Fluor 568 dye |
| goat-anti-mouse with wavelength 488 nm of green fluorescent protein (GFP) | Thermofisher | #Z25002 | Alexa Fluor 488 dye |
| prolong gold antifade with DAPI | Invitrogen | #P36931 | |
| 6-well plate | SIGMA Corning Costar |
#CLS3516 | |
| 4-well chamber slide | THermofisher, Nunc Lab-Tek | #171080 | |
| Nunc F 96-well plate | Thermofisher | #152038 | |
| LC3B antibody rabbit | NOVUS | #NB100-2220 | |
| DNA antibody | Progen Biotechnik | #anti-DNA mouse monoclonal, AC-30-10 | |
| DAPI | Thermofisher | #D1306 | antifade mounting medium with DAPI |
| IN Cell analyzer (fluorescent reader ) | GE Healthcare Life Sciences | #IN Cell analyzer 2200 | |
| Eclipse TE-2000e confocal microscope | Nikon | #TE-2000e | |
| Colocalization software | Volocity | #Volocity 6.3 | alternative Zeiss ZEN 2012 software |
| IN Cell Investigator Software | GE Healthcare Life Sciences | #28408974 | |
| cell culture medium | Thermofisher | #DMEM–Dulbecco's Modified Eagle Medium | |
| Penicillin-Streptomycin (10,000 U/mL) | Thermofisher | #15140122 | |
| Fetal Bovine Serum | Sigma-Aldrich | #12003C-1000ML | |
| Cell culture Incubator | Thermofisher | #Thermo Forma 3110 CO2 Water Jacketed Incubator | |
| epifluorescence microscope | Zeiss | Zeiss Axio Imager Z2 | |
| camera | Olympus | Olympus DP71 | |
| confocal microscope | Zeiss | Zeiss Axio Observer Z1 | |
| confocal software | Zeiss | ZEN 2012 | |
| image analysis software | Image J | colocalization analysis, etc | https://imagej.nih.gov/ij/ |
| statistical analysis software | GraphPad Software Inc., San Diego, USA | GraphPad Prism software package | |
| material to make a worm pick | Surepure Chemetals | #4655 | The pick is made of 30 gauge 90% platinum 10% iridium wire |
| IR: N2;Ex[pmyo-3 TOMM-20::Rosella] | Material inquiry to Tavernarakis Nektarios | Maintain transgenic animals at 20 °C | |
| IR: N2; Ex[pdct-1 DCT-1::GFP; pmyo-3 DsRed::LGG-1] | Material inquiry to Tavernarakis Nektarios | ||
| pmyo-3 TOMM-20::Rosella | Material inquiry to Tavernarakis Nektarios | ||
| pdct-1 DCT-1::GFP | Material inquiry to Tavernarakis Nektarios | ||
| pmyo-3 DsRed::LGG-1 | Material inquiry to Tavernarakis Nektarios | ||
| Paraquat solution | see supplementary data for preparation | ||
| M9 buffer | see supplementary data for preparation | ||
| M9-levamisole buffer | see supplementary data for preparation | ||
| Glass Microscope Slides and Coverslips | Fisher Scientific | #B9992000 | |
| Surgical forceps | STERIS Animal Health | 19 Piece Canine Spay Pack Economy | |
| Surgical scissors | STERIS Animal Health | 19 Piece Canine Spay Pack Economy | |
| 1x PBS | Thermofisher | #AM9625 | 10x PBS needs to be diluted to 1x PBS by using ddH2O |
| shaker | Fisher Scientific | #11-676-178 | Thermo Scientific MaxQ HP Tabletop Orbital Small Open Air Platform Shaker Package A |
| 2% agarose pad | see supplementary data for preparation | ||
| Vibroslice blades | World precision instruments | #BLADES-2 | single-edge blade |
| metal plate | MSC | #78803988 | 0.012 in thick x 6 in wide x 12 in long, 430 Stainless Steel Sheet |
| Triton X-100 | detergent | ||
| Methyl viologen dichloride hydrate | Sigma-Aldrich | #856177 | paraquat |
| Incubator for nematodes | AQUALYTIC | Incubator to maintain 20 °C | |
| Dissecting stereomicroscope | Olympus | SMZ645 | |
| Confocal microscope | Zeiss | AxioObserver Z1 | For nematodes (step 2) |
| epifluorescence microscope | Zeiss | AxioImager Z2 | For nematodes (step 2) |
| UV crosslinker | Vilber Lourmat | BIO-LINK – BLX-E365 | UV light source; 356 nm |
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