Technical Reference #1719

1719.

Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells Anto´ nio J. Pereira; Irina Matos; Mariana Lince-Faria and Helder Maiato, Universidade do Porto, Methods in Molecular Biology; Mitosis, 545(1719), (2009)
Link To Paper

Abstract:
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-theart 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.

Keywords:
Mitosis; Drosophila; S2 cells; HeLa cells; live cell microscopy; RNAi; agar overlay; laser microsurgery.

Materials & Methods:
Rose chambers are a proven cost-effective alternative to reusable chambers for live-cell imaging. We designed our own modified version of Rose chambers taking into account our specific objective features and to allow different assembly options (e.g. open or closed chambers). Nevertheless there are other possible commercial alternatives. We recommend the MatTek glass bottom culture disks (MatTek #P35G-1.5-20-C). The same principles discussed previously regarding coverslip thickness also apply in this case.

Cell Type(s)
Schneider 2, Hela