Metz Jeremy, Castro Inês G, Schrader Michael
Biosciences, University of Exeter, Exeter, UK.
Bio Protoc. 2017 Sep 5;7(17). doi: 10.21769/BioProtoc.2536.
Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the β-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp ., 1996; Wiemer ., 1997; Schrader ., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello ., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.
细胞器的运动、分布和相互作用有助于真核细胞的组织。过氧化物酶体是多功能细胞器，对细胞脂质代谢和活性氧稳态有重要作用。它们在哺乳动物细胞中均匀分布，并通过驱动蛋白和动力蛋白沿着微管移动。它们与线粒体和内质网（ER）的代谢合作对于脂肪酸的β-氧化以及髓磷脂脂质和多不饱和脂肪酸的合成至关重要。评估哺乳动物细胞中过氧化物酶体运动性的一个关键检测方法是表达带有过氧化物酶体靶向信号的荧光融合蛋白（如绿色荧光蛋白-过氧化物酶体靶向信号1，GFP-PTS1），该信号靶向过氧化物酶体基质并允许对过氧化物酶体进行活细胞成像。在这里，我们首先介绍一种用带有过氧化物酶体标记物增强型绿色荧光蛋白-丝氨酸-赖氨酸-亮氨酸（EGFP-SKL）转染培养的哺乳动物细胞以在活细胞中观察过氧化物酶体的方法。这种方法揭示了过氧化物酶体不同的运动行为以及对过氧化物酶体膜动力学的新见解（拉普等人，1996年；维默等人，1997年；施拉德等人，2000年）。然后我们介绍一种将活细胞方法与过氧化物酶体运动性测量以及哺乳动物细胞中过氧化物酶体动力学定量相结合的方法。最近，我们使用这种方法证明，当将过氧化物酶体与内质网连接起来的分子系链缺失时，过氧化物酶体的运动性和位移会增加（科斯特洛等人，2017年b）。与内质网定位的囊泡相关膜蛋白B（VAPB）相互作用的过氧化物酶体酰基辅酶A结合结构域蛋白ACBD5的沉默增加了皮肤成纤维细胞中过氧化物酶体的运动，表明膜接触位点可以调节细胞器的分布和运动性。所描述的方法可以适用于其他细胞类型和细胞器，以在不同实验条件下测量和定量细胞器的运动。
