Pereira António J, Matos Irina, Lince-Faria Mariana, Maiato Helder
Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal.
Methods Mol Biol. 2009;545:145-64. doi: 10.1007/978-1-60327-993-2_9.
Progress from our present understanding of the mechanisms behind mitosis has been compromised by the fact that model systems that were ideal for molecular and genetic studies (such as yeasts, C. elegans, or Drosophila) were not suitable for intracellular micromanipulation. Unfortunately, those systems that were appropriate for micromanipulation (such as newt lung cells, PtK1 cells, or insect spermatocytes) are not amenable for molecular studies. We believe that we can significantly broaden this scenario by developing high-resolution live cell microscopy tools in a system where micromanipulation studies could be combined with modern gene-interference techniques. Here we describe a series of methodologies for the functional dissection of mitosis by the use of simultaneous live cell microscopy and state-of-the-art laser microsurgery, combined with RNA interference (RNAi) in Drosophila cell lines stably expressing fluorescent markers. This technological synergism allows the specific targeting and manipulation of several structural components of the mitotic apparatus in different genetic backgrounds, at the highest spatial and temporal resolution. Finally, we demonstrate the successful adaptation of agar overlay flattening techniques to human HeLa cells and discuss the advantages of its use for laser micromanipulation and molecular studies of mitosis in mammals.
目前我们对有丝分裂背后机制的理解进展受到了阻碍，原因在于那些对分子和遗传研究来说理想的模式生物（如酵母、秀丽隐杆线虫或果蝇）并不适合细胞内显微操作。不幸的是，那些适合显微操作的生物（如新蝾螈肺细胞、PtK1细胞或昆虫精母细胞）却不适用于分子研究。我们相信，通过在一个能将显微操作研究与现代基因干扰技术相结合的系统中开发高分辨率活细胞显微镜工具，我们能够显著拓宽这种局面。在此，我们描述了一系列方法，通过在稳定表达荧光标记的果蝇细胞系中同时使用活细胞显微镜和最先进的激光显微手术，并结合RNA干扰（RNAi），来对有丝分裂进行功能剖析。这种技术协同作用能够在不同遗传背景下，以最高的空间和时间分辨率对有丝分裂装置的多个结构成分进行特异性靶向和操作。最后，我们展示了琼脂覆盖平铺技术成功应用于人类HeLa细胞，并讨论了其在哺乳动物有丝分裂的激光显微操作和分子研究中的应用优势。
