Long-Term Imaging of DNA Damage and Cell Cycle Progression in Budding Yeast Using Spinning Disk Confocal Microscopy
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Live cell imaging can monitor biological processes in time and space by providing quantitative measurements of cell behavior on a single-cell basis and in live conditions. However the illumination required to visualize fluorescently tagged endogenous proteins often perturbs cellular physiology, a problem particularly acute for yeast cells that are small, highly photosensitive and with scarce protein content. Analyzing the activation of the DNA damage response (DDR) in various yeast mutants or growth conditions, as well as its consequences for cell cycle progression and cell viability over extended periods of time therefore requires a special microscopy setup that does not by itself create DNA damage or perturb cell growth. Here, we provide a quick guide, strains and advice for imaging the DDR in S. cerevisiae for extended time (3–12 h) using spinning-disk confocal microscopy in conditions of limited photobleaching and photodamage. DDR is a conserved mechanism that allows the cell to respond to various stresses, especially those altering DNA integrity or topology. Acquiring time-lapse images of the DDR at high temporal and spatial resolution is of great interest, in particular when studying the effects of mutations or drugs which compromise genomic stability and cell cycle progression.
Key wordsS. cerevisiae Yeast Spinning disk microscopy Yokogama CSU-X1 DNA damage response Rad52-GFP mCherry-Tub1 Microscopy Recombination foci Phototoxicity
We thank D. Tamborrini and S. Piatti (CRBM, Montpellier) for the initial set-up of yeast live cell imaging, V. Georget (BioCampus MRI Imaging Facility) for endless support, as well as R. Rothstein (Columbia U., New York) and E. Schiebel (ZMBH, Heidelberg) for providing yeast strains and constructs. RM was a Master student from University Milan-Bicocca (Italy) holding an Erasmus+ stipend from the EU. ES thanks Association pour la Recherche contre le Cancer (ARC) and Agence Nationale de la Recherche (ANR-14-CE11-0007-03) for funding.
Wild-type strain (E3416) containing Rad52-GFP (green) and mCherry-tubulin (red) grown at 30 °C on a FluoroDish and imaged every 2 min for 6 h under low illumination conditions (laser GFP 1%, Cherry 3%, 11 z-planes of 300 ms each). Note that cells divide rapidly without showing persistent Rad52-GFP foci (MP4 2662 kb)
Wild-type strain (E3416) containing Rad52-GFP (green) and mCherry-tubulin (red) grown at 30 °C on a FluoroDish and imaged every min for 3 h under higher illumination conditions (laser GFP 3%, Cherry 3%, 11 z-planes of 300 ms each). Due to the three times stronger GFP illumination and increased frequency (every min), most cells show persistent Rad52-GFP foci in G2/metaphase but still divide, albeit more slowly. Note that Movies 1 and 2 were performed on the same day with comparable laser power output (MP4 2269 kb)
Wild-type strain (E3416) containing Rad52-GFP (green) and mCherry-Tub1 (red) grown at 30 °C in the presence of 20 μM camptothecin (CPT, Topoisomerase I inhibitor) and imaged every 2 min for 12 h with 488 nm (5%) and 561 nm (6.5%) channels to visualize Rad52-GFP foci and mitotic spindles (mCherry-Tub1), respectively. Note that the cell cycle is severely extended with cells showing persistent Rad52-GFP foci from the second cell cycle (MP4 4360 kb)
mec1Δ sml1Δ strain (E5357) containing Rad52-GFP (green) and mCherry-Tub1 (red) grown at 30 °C with 20 μM camptothecin and imaged every 2 min for 12 h with 488 nm (5%) and 561 nm (6.5%) fluorescence excitation. Note that cells show Rad52 foci in the 1st cycle but divide and rebud rapidly without dismantling their Rad52-GFP foci. This will lead to cell death a few cycles later. Movies 3 and 4 were performed on successive days with comparable laser output, but after laser power was calibrated down compared to Movies 1 and 2 (MP4 10291 kb)