Use of <em>Drosophila</em> S2 Cells for Live Imaging of Cell Division

Abstract

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.

Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

“…Live-cell imaging and movie rendering was done according to our previously published protocol 46 . Briefly, movies were acquired using S2 cells stably expressing an inducible GFP:CID (a generous gift from Dr. Gary Karpen, UC Berkeley), which we subsequently stably transfected with inducible mCherry-α-tubulin (selected for using puromycin resistance) to generate a stable double transgenic S2 cell line.…”