Imaging cytokinesis of Drosophila S2 cells.

Abstract

Animal cell cytokinesis proceeds through three successive stages: a contractile ring stage, an intercellular bridge stage, and an abscission stage. Many studies have identified a complex network of key proteins required for successful cytokinesis. While each component interacts with, and depends on, several other components, our understanding of how these proteins cooperate in space and time to ensure faithful progression through the stages of cytokinesis remains incomplete. A full understanding of the complexity of the process and its underlying machinery necessitates experimental systems that allow both genetic manipulation and real-time visualization of the various components throughout the successive stages of cytokinesis. Cultured Drosophila S2 cells provide such a system. They are genetically tractable thanks to their exquisite sensitivity to RNA interference mediated by double-stranded RNAs, which can be generated with ease in the laboratory. Furthermore, S2 cells grow well under normal atmospheric conditions, and stable lines expressing fluorescently tagged proteins can be readily generated, making them ideal for long-term live-cell fluorescence microscopy. Here we describe methodology for exploiting S2 cells for the study of cytokinesis, with an emphasis on live-cell imaging. We describe a variety of fluorescent markers available and their utility for highlighting different structures at different stages of cytokinesis. We describe our experimental setup that forms the basis for live-cell analysis of loss-of-function RNAi experiments, rescue experiments, and structure-function analyses of key regulators of cytokinesis. Finally, we describe the types of phenotypes that one can observe at the different stages of Drosophila S2 cell cytokinesis.