Field C M, Pelletier J F, Mitchison T J
Harvard Medical School, Boston, MA, United States; Marine Biological Laboratory, Woods Hole, MA, United States.
Methods Cell Biol. 2017;137:395-435. doi: 10.1016/bs.mcb.2016.04.014. Epub 2016 May 9.
We report optimized methods for preparing actin-intact Xenopus egg extract. This extract is minimally perturbed, undiluted egg cytoplasm where the cell cycle can be experimentally controlled. It contains abundant organelles and glycogen and supports active metabolism and cytoskeletal dynamics that closely mimic egg physiology. The concentration of the most abundant ∼11,000 proteins is known from mass spectrometry. Actin-intact egg extract can be used for analysis of actin dynamics and interaction of actin with other cytoplasmic systems, as well as microtubule organization. It can be spread as thin layers and naturally depletes oxygen though mitochondrial metabolism, which makes it ideal for fluorescence imaging. When combined with artificial lipid bilayers, it allows reconstitution and analysis of the spatially controlled signaling that positions the cleavage furrow during early cytokinesis. Actin-intact extract is generally useful for probing the biochemistry and biophysics of the large Xenopus egg. Protocols are provided for preparation of actin-intact egg extract, control of the cell cycle, fluorescent probes for cytoskeleton and cytoskeleton-dependent signaling, preparation of glass surfaces for imaging experiments, and immunodepletion to probe the role of specific proteins and protein complexes. We also describe methods for adding supported lipid bilayers to mimic the plasma membrane and for confining in microfluidic droplets to explore size scaling issues.
我们报告了制备肌动蛋白完整的非洲爪蟾卵提取物的优化方法。这种提取物是受到最小干扰的、未稀释的卵细胞质，其细胞周期可通过实验进行控制。它含有丰富的细胞器和糖原，并支持活跃的新陈代谢和细胞骨架动力学，能紧密模拟卵的生理状态。通过质谱分析可知最丰富的约11,000种蛋白质的浓度。肌动蛋白完整的卵提取物可用于分析肌动蛋白动力学以及肌动蛋白与其他细胞质系统的相互作用，以及微管组织。它可以铺展成薄层，并通过线粒体代谢自然耗尽氧气，这使其成为荧光成像的理想选择。与人工脂质双层结合时，它可用于重建和分析在早期胞质分裂期间定位分裂沟的空间控制信号。肌动蛋白完整的提取物通常可用于探究大型非洲爪蟾卵的生物化学和生物物理学。本文提供了制备肌动蛋白完整的卵提取物、控制细胞周期、用于细胞骨架和细胞骨架依赖性信号传导的荧光探针、用于成像实验的玻璃表面制备以及免疫去除以探究特定蛋白质和蛋白质复合物作用的实验方案。我们还描述了添加支持的脂质双层以模拟质膜以及限制在微流控液滴中以探索尺寸缩放问题的方法。
