Biochemical Reconstitution of Ca2+-Dependent Exosome Secretion in Permeabilized Mammalian Cells

J. Ngo, J. Williams, I. Lehman, Randy Schekman
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Abstract

Exosomes are a subpopulation of the heterogenous pool of extracellular vesicles that are secreted to the extracellular space. Exosomes have been purported to play a role in intercellular communication and have demonstrated utility as biomarkers for a variety of diseases. Despite broad interest in exosome biology, the conditions that regulate their secretion are incompletely understood. The goal of this procedure is to biochemically reconstitute exosome secretion in Streptolysin O (SLO)-permeabilized mammalian cells. This protocol describes the reconstitution of lyophilized SLO, preparation of cytosol and SLO-permeabilized cells, assembly of the biochemical reconstitution reaction, and quantification of exosome secretion using a sensitive luminescence-based assay. This biochemical reconstitution reaction can be utilized to characterize the molecular mechanisms by which different gene products regulate exosome secretion. Key features This protocol establishes a functional in vitro system to reconstitute exosome secretion in permeabilized mammalian cells upon addition of cytosol, ATP, GTP, and calcium (Ca2+).
在渗透稳定的哺乳动物细胞中重建 Ca2+ 依赖性外泌体分泌的生化过程
外泌体是分泌到细胞外空间的异质细胞外囊泡池的一个亚群。外泌体被认为在细胞间通讯中发挥作用,并已被证明是多种疾病的生物标志物。尽管人们对外泌体生物学有广泛的兴趣,但调控其分泌的条件尚不完全清楚。该程序的目的是生化重建链溶素O (SLO)渗透的哺乳动物细胞中的外泌体分泌。该方案描述了冻干SLO的重组,细胞质和SLO渗透细胞的制备,生化重组反应的组装,以及使用敏感的基于发光的测定法定量外泌体分泌。这种生化重组反应可以用来表征不同基因产物调节外泌体分泌的分子机制。该方案建立了一个功能的体外系统,在添加细胞质、ATP、GTP和钙(Ca2+)后,在通透性哺乳动物细胞中重建外泌体分泌。
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