基于图像的高通量线粒体DNA合成和分布定量
Summary
描述了使用多孔板格式和自动免疫荧光成像来检测和量化mtDNA合成和分布的细胞中线粒体DNA（mtDNA）代谢动力学的程序。这可以进一步用于研究各种抑制剂，细胞应激和基因沉默对mtDNA代谢的影响。
Abstract
绝大多数细胞过程需要持续的能量供应，其中最常见的载体是ATP分子。真核细胞通过氧化磷酸化在线粒体中产生大部分ATP。线粒体是独特的细胞器，因为它们有自己的基因组，可以复制并传递给下一代细胞。与核基因组相反，细胞中线粒体基因组有多个拷贝。详细研究负责线粒体基因组复制、修复和维持的机制对于了解线粒体和整个细胞在正常和疾病条件下的正常功能至关重要。本文提出了一种允许高通量定量体外 培养的人细胞 中线粒体DNA（mtDNA）的合成和分布的方法。该方法基于对通过5-溴-2′-脱氧尿苷（BrdU）掺入标记的活性合成DNA分子进行免疫荧光检测，以及同时检测具有抗DNA抗体的所有mtDNA分子。此外，线粒体用特定的染料或抗体可视化。以多孔形式培养细胞和利用自动荧光显微镜使得在相对较短的时间内在各种实验条件下研究mtDNA的动力学和线粒体的形态变得更加容易。
Introduction
对于大多数真核细胞来说，线粒体是必不可少的细胞器，因为它们在许多细胞过程中起着至关重要的作用。首先，线粒体是细胞1的主要能量供应商。线粒体还参与调节细胞稳态（例如，细胞内氧化还原2和钙平衡3），细胞信号传导4，5，细胞凋亡6，不同生化化合物的合成7，8和先天免疫反应9。线粒体功能障碍与各种病理状态和人类疾病有关10.
线粒体的功能取决于位于两个独立基因组中的遗传信息：核基因组和线粒体基因组。与核基因组相比，线粒体基因组编码的基因数量很少，但所有mtDNA编码的基因对人类生命都是必不可少的。维持mtDNA所必需的线粒体蛋白机制由nDNA编码。线粒体爬替体的基本成分以及一些线粒体生物发生因子已经被确定（在以前的研究中回顾了11，12）。然而，线粒体DNA的复制和维持机制仍远未被理解。与nDNA相反，线粒体基因组以多个拷贝存在，这为调节线粒体基因表达提供了额外的层。目前对细胞器内mtDNA的分布和分离知之甚少，这些过程在多大程度上受到调节，如果是，涉及哪些蛋白质13。当细胞包含野生型和突变mtDNA的混合群体时，分离模式至关重要。它们的不均匀分布可能导致产生具有有害数量突变mtDNA的细胞。
到目前为止，mtDNA维持所必需的蛋白质因子主要通过生化方法，生物信息学分析或通过疾病相关研究来确定。在这项工作中，为了确保识别以前逃脱识别的因素的高机会，描述了一种不同的策略。该方法基于在复制或修复过程中用5-溴-2′-脱氧尿苷（BrdU）标记mtDNA，BrdU是胸苷的核苷类似物。BrdU在DNA合成过程中很容易掺入新生的DNA链中，并且通常用于监测核DNA14的复制。然而，这里开发的程序已经过优化，可以使用抗BrdU抗体的免疫荧光检测掺入mtDNA中的BrdU。
该方法允许对 体外培养的人类细胞中的mtDNA合成和分布进行高通量定量。需要高通量策略才能在相对较短的时间内在不同的实验条件下进行测试;因此，在协议中建议利用多孔格式进行细胞培养和自动荧光显微镜进行成像。该方案包括用siRNA文库转染人HeLa细胞，以及随后使用BrdU对新合成的DNA进行代谢标记来监测mtDNA的复制或修复。这种方法与抗DNA抗体的帮助下对DNA进行免疫染色相结合。这两个参数都使用定量荧光显微镜进行分析。此外，线粒体用特定的染料可视化。为了证明该方案的特异性，在缺乏mtDNA的细胞（rho0细胞）上测试了BrdU染色，在沉默了众所周知的mtDNA维持因子的HeLa细胞上，以及在用mtDNA复制抑制剂处理后的HeLa细胞上测试了BrdU染色。mtDNA水平也通过一种独立的方法测量，即qPCR。
Protocol
1. siRNA混合物的制备 在实验开始前一天，将细胞（例如HeLa）接种在100毫米的培养皿上，以便第二天达到70%-90%的汇合。注意：所有操作必须在无菌条件下在层流室中进行。 在Opti-MEM培养基中制备稀释至140nM浓度的适量的siRNA（参见 材料表）。96孔板可用作储液器。 向黑色 384 孔细胞培养微孔板的每个孔中加入 5 μL siRNA 溶液（或用于对照样品的 Opti-M…
Representative Results
图1显示了mtDNA合成和分布动力学的高通量研究程序方案。使用多孔板形式可以同时分析许多不同的实验条件，例如使用siRNA文库沉默不同的基因。用于用BrdU标记新合成的DNA分子的条件允许检测HeLa细胞线粒体中的BrdU标记DNA（图2A），但也可以用作建立其他细胞类型测定的起始条件。应根据时程实验为单个细胞系选择BrdU孵育时间（补充图1）?…
Discussion
从历史上看，通过BrdU掺入和抗体检测进行DNA标记已被用于核DNA复制和细胞周期研究14，27，28。到目前为止，所有用于检测BrdU标记DNA的方案都包括DNA变性步骤（酸性或热性）或酶消化（DNase或蛋白酶），以实现表位暴露并促进抗体渗透。这些协议是为紧密包装的核DNA开发的。然而，mtDNA的不同组织使得开发没有变性步骤的程序成…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了波兰国家科学中心的支持（资助/奖励编号：2018/31/D/NZ2/03901）。
Materials
| 2′,3′-Dideoxycytidine (ddC) | Sigma-Aldrich | D5782 | |
| 384 Well Cell Culture Microplates, black | Greiner Bio-One | #781946 | |
| 5-Bromo-2′-deoxyuridine (BrdU) | Sigma-Aldrich | B5002-1G | Dissolve BrdU powder in water to 20 mM stock solution and aliquot. Use 20 µM BrdU solution for labeling. |
| Adhesive sealing film | Nerbe Plus | 04-095-0060 | |
| Alexa Fluor 488 goat anti-mouse IgG1 secondary antibody | Thermo Fisher Scientific | A-21121 | |
| Alexa Fluor 555 goat anti-mouse IgM secondary antibody | Thermo Fisher Scientific | A-21426 | |
| BioTek 405 LS microplate washer | Agilent | ||
| Bovine Serum Albumin (BSA) | Sigma-Aldrich | A4503 | |
| Cell counting chamber Thoma | Heinz Herenz | REF:1080339 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Cytiva | SH30243.01 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Thermo Fisher Scientific | 41965-062 | |
| Fetal Bovine Serum (FBS) | Thermo Fisher Scientific | 10270-106 | |
| Formaldehyde solution | Sigma-Aldrich | F1635 | Formaldehyde is toxic; please read the safety data sheet carefully. |
| Hoechst 33342 | Thermo Fisher Scientific | H3570 | |
| IgG1 mouse monoclonal anti-BrdU (IIB5) primary antibody | Santa Cruz Biotechnology | sc-32323 | |
| IgM mouse monoclonal anti-DNA (AC-30-10) primary antibody | Progen | #61014 | |
| LightCycler 480 System | Roche | ||
| Lipofectamine RNAiMAX Transfection Reagent | Thermo Fisher Scientific | #13778150 | |
| MitoTracker Deep Red FM | Thermo Fisher Scientific | M22426 | Mitochondria tracking dye |
| Multidrop Combi Reagent Dispenser | Thermo Fisher Scientific | ||
| Opti-MEM | Thermo Fisher Scientific | 51985-042 | |
| Orca-R2 (C10600) CCD Camera | Hamamatsu | ||
| Penicillin-Streptomycin | Sigma-Aldrich | P0781-100ML | |
| Phosphate buffered saline (PBS) | Sigma-Aldrich | P4417-100TAB | |
| PowerUp SYBR Green Master Mix | Thermo Fisher Scientific | A25742 | |
| qPCR primer Fw B2M (reference) | CAGGTACTCCAAAGATTCAGG | ||
| qPCR primer Fw GPI (reference gene) | GACCTTTACTACCCAGGAGA | ||
| qPCR primer Fw MT-ND1 | TAGCAGAGACCAACCGAACC | ||
| qPCR primer Fw POLG | TGGAAGGCAGGCATGGTCAAACC | ||
| qPCR primer Fw TFAM | GATGAGTTCTGCCTGCTTTAT | ||
| qPCR primer Fw TWNK | GCCATGTGACACTGGTCATT | ||
| qPCR primer Rev B2M (reference) | GTCAACTTCAATGTCGGATGG | ||
| qPCR primer Rev GPI (reference gene) | AGTAGACAGGGCAACAAAGT | ||
| qPCR primer Rev MT-ND1 | ATGAAGAATAGGGCGAAGGG | ||
| qPCR primer Rev POLG | GGAGTCAGAACACCTGGCTTTGG | ||
| qPCR primer Rev TFAM | GGACTTCTGCCAGCATAATA | ||
| qPCR primer Rev TWNK | AACATTGTCTGCTTCCTGGC | ||
| ScanR microscope | Olympus | ||
| siRNA Ctrl | Dharmacon | D-001810-10-5 | |
| siRNA POLG | Invitrogen | POLGHSS108223 | |
| siRNA TFAM | Invitrogen | TFAMHSS144252 | |
| siRNA TWNK | Invitrogen | C10orf2HSS125597 | |
| Suction device | NeoLab | 2-9335 | Suction device for cell culture |
| Triton X-100 | Sigma-Aldrich | T9284-500ML | |
| Trypsin | Biowest | L0931-500 | |
| UPlanSApo 20x 0.75 NA objective | Olympus |
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Citer Cet Article
Borowski, L. S., Kasztelan, K., Czerwinska-Kostrzewska, J., Szczesny, R. J. High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution. J. Vis. Exp. (195), e65236, doi:10.3791/65236 (2023).