Shankaran Sunita S, Mackay Douglas R, Ullman Katharine S
Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, USA.
Methods Mol Biol. 2013;931:111-22. doi: 10.1007/978-1-62703-056-4_6.
Real-time imaging coupled with a permeabilized cell system presents a very versatile platform to visualize the dynamic and intricate nature of nuclear envelope breakdown, one of the major morphological changes of mitosis. Here, we describe such a strategy in which the plasma membrane of cells expressing fluorescently tagged nucleoporin POM121 and Histone H2B is permeabilized with digitonin. These cells are then incubated with mitotic Xenopus egg extract to create conditions that recapitulate the major events of mitotic nuclear remodeling seen in live-cell imaging, providing the opportunity to probe mechanisms and pathways that coordinate nuclear disassembly.
实时成像与通透细胞系统相结合，为可视化核膜破裂这一有丝分裂主要形态变化之一的动态和复杂本质提供了一个非常通用的平台。在此，我们描述了这样一种策略：用洋地黄皂苷使表达荧光标记核孔蛋白POM121和组蛋白H2B的细胞的质膜通透化。然后将这些细胞与有丝分裂非洲爪蟾卵提取物一起孵育，以创造出重现活细胞成像中所见有丝分裂核重塑主要事件的条件，从而有机会探究协调核解体的机制和途径。
