Boussardon Clément, Keech Olivier
Department of Plant Physiology, Umeå Plant Science, Umeå University, Umeå, Sweden.
Methods Mol Biol. 2022;2363:13-23. doi: 10.1007/978-1-0716-1653-6_2.
Membrane-bound organelles are unique features of eukaryotic cell structures. Among them, mitochondria host key metabolic functions and pathways, including the aerobic respiration. In plants, several procedures are available to isolate mitochondria from the other cell compartments, as high-quality purified extracts are often necessary for accurate molecular biology or biochemistry investigations. Protocols based on differential centrifugations and subsequent density gradients are an effective way to extract rather pure and intact mitochondria within a few hours. However, while mitochondria from seedlings, large leaves or tubers are relatively easy to extract, tissue-specific isolation of organelles had remained a challenge. This has recently been circumvented, only in transformable plants though, by the use of affinity-tagged mitochondria and their isolation with magnetic beads.We hereby describe a step-by-step protocol for the rapid and tissue-specific isolation of Arabidopsis thaliana mitochondria, a method named IMTACT (Isolation of Mitochondria TAgged in specific Cell Types). Cell-specific biotinylated mitochondria are isolated with streptavidin magnetic beads in less than 30 min from sampling to final extract. Key steps, enrichment, bead size comparison, and mitochondrial depletion in the sample are also reported in order to facilitate the experimental setup of the user.
膜结合细胞器是真核细胞结构的独特特征。其中，线粒体承担着关键的代谢功能和途径，包括有氧呼吸。在植物中，有多种方法可用于从其他细胞区室中分离线粒体，因为高质量的纯化提取物对于准确的分子生物学或生物化学研究通常是必需的。基于差速离心和随后的密度梯度的方案是在数小时内提取相当纯净和完整线粒体的有效方法。然而，虽然从幼苗、大叶或块茎中提取线粒体相对容易，但细胞器的组织特异性分离仍然是一个挑战。最近，仅在可转化植物中，通过使用亲和标记的线粒体及其与磁珠的分离，这一问题才得以解决。我们在此描述了一种用于快速、组织特异性分离拟南芥线粒体的分步方案，该方法名为IMTACT（特定细胞类型中线粒体的分离）。从取样到最终提取物，在不到30分钟的时间内，用链霉亲和素磁珠分离细胞特异性生物素化的线粒体。还报告了关键步骤、富集、磁珠大小比较以及样品中的线粒体消耗情况，以方便用户进行实验设置。
