Dewey Evan B, Parra Amalia S, Johnston Christopher A
Department of Biology, University of New Mexico;
Department of Biology, University of New Mexico.
J Vis Exp. 2019 Aug 23(150). doi: 10.3791/60049.
Drosophila S2 cells are an important tool in studying mitosis in tissue culture, providing molecular insights into this fundamental cellular process in a rapid and high-throughput manner. S2 cells have proven amenable to both fixed- and live-cell imaging applications. Notably, live-cell imaging can yield valuable information about how loss or knockdown of a gene can affect the kinetics and dynamics of key events during cell division, including mitotic spindle assembly, chromosome congression, and segregation, as well as overall cell cycle timing. Here we utilize S2 cells stably transfected with fluorescently tagged mCherry:α-tubulin to mark the mitotic spindle and GFP:CENP-A (referred to as 'CID' gene in Drosophila) to mark the centromere to analyze the effects of key mitotic genes on the timing of cell divisions, from prophase (specifically at Nuclear Envelope Breakdown; NEBD) to the onset of anaphase. This imaging protocol also allows for the visualization of the spindle microtubule and chromosome dynamics throughout mitosis. Herein, we aim to provide a simple yet comprehensive protocol that will allow readers to easily adapt S2 cells for live imaging experiments. Results obtained from such experiments should expand our understanding of genes involved in the cell division by defining their role in several simultaneous and dynamic events. Observations made in this cell culture system can be validated and further investigated in vivo using the impressive toolkit of genetic approaches in flies.
果蝇S2细胞是组织培养中研究有丝分裂的重要工具，能以快速且高通量的方式为这一基本细胞过程提供分子层面的见解。已证明S2细胞适用于固定细胞成像和活细胞成像应用。值得注意的是，活细胞成像能够产生关于基因缺失或敲低如何影响细胞分裂过程中关键事件的动力学和动态变化的有价值信息，这些关键事件包括有丝分裂纺锤体组装、染色体汇聚和分离，以及整个细胞周期的时间安排。在这里，我们利用稳定转染了荧光标记的mCherry:α-微管蛋白的S2细胞来标记有丝分裂纺锤体，并用GFP:CENP-A（在果蝇中称为“CID”基因）来标记着丝粒，以分析关键有丝分裂基因对细胞分裂时间的影响，从前期（特别是在核膜破裂；NEBD时）到后期开始。这个成像方案还能在整个有丝分裂过程中可视化纺锤体微管和染色体动态变化。在此，我们旨在提供一个简单而全面的方案，使读者能够轻松地将S2细胞应用于活细胞成像实验。通过此类实验获得的结果应能通过确定基因在多个同时发生的动态事件中的作用，扩展我们对参与细胞分裂的基因的理解。在这个细胞培养系统中所做的观察结果可以在体内使用果蝇令人印象深刻的遗传方法工具包进行验证和进一步研究。
