Long-Term Imaging of DNA Damage and Cell Cycle Progression in Budding Yeast Using Spinning Disk Confocal Microscopy

Abstract

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.

Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi

“…Responses to such DNA damage were monitored expressing Rad52-GFP (Rad52 is a DNA double strand break repair protein) and mCherry-alpha-tubulin in S. cerevisiae and the persistence of Rad52-GFP fluorescence clearly depended on the 488 nm excitation light dose applying spinning disk confocal microscopy (SDCM). Interestingly, the topoisomerase 1 inhibitor camptothecin resulted in extended cell cycle and persistent Rad52-GFP fluorescence, and a mec1Δ sml1 S. cerevisiae strain defective in DNA damage-elicited cell-cycle arrest showed persistent Rad52-GFP fluorescence in the presence of camptothecin and underwent cell death a few cycles later (Montecchi and Schwob 2018 ).…”