Combining Magnetic Sorting of Mother Cells and Fluctuation Tests to Analyze Genome Instability During Mitotic Cell Aging in <em>Saccharomyces cerevisiae</em>

Abstract

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Preferential retrotransposition in aging yeast mother cells is correlated with increased genome instability

“…Magnetic cell sorting to separate biotin-labeled mother cells from their daughter cell populations was performed as previously described [29]. Briefly, cells were grown from glycerol stocks on YPD agar at 30°C, and 10 8 cells per collection point were labeled with NHS-LC-biotin (Thermo Scientific) and inoculated into YPD medium at a density of 5 × 10 6 cells/ml.…”

“…At each collection point, portions of the column eluate (mother cells) and flow through (daughter cells) were retained for analyses, and portions of sorted mother cells were inoculated into YPD liquid medium for additional rounds of growth and sorting to increase replicative age. Cell populations grown overnight and washed with buffer in preparation for sorting were stored on ice for up to one hour, and labeled or sorted cells were stored for up to three hours on ice before inoculation into fresh medium in some instances [29]. Bud scars were stained with an Alexa Fluor 488 conjugate of wheat germ agglutinin (WGA, Life Technologies) to determine replicative age of cells.…”

“…Bud scars were stained with an Alexa Fluor 488 conjugate of wheat germ agglutinin (WGA, Life Technologies) to determine replicative age of cells. Manual bud scar counts obtained from fluorescence microscopy were plotted versus the geometric means of fluorescence intensity of stained cells measured with a BD LSR II flow cytometer and normalized to unstained controls for three trials to obtain a trend line for calculating cell age [29]. Cell ages for populations were then calculated from normalized geometric mean signals from individual trials using unsorted control populations to verify consistent staining and were averaged for multiple trials in a given set of experiments.…”

“…Cell ages for populations were then calculated from normalized geometric mean signals from individual trials using unsorted control populations to verify consistent staining and were averaged for multiple trials in a given set of experiments. Cell viability was determined directly using trypan blue dye exclusion, as previously described [29]. Plating efficiencies were determined from the percent of cells spread onto rich medium that were able to form colonies after incubation at 30°C for five days, and the values reported for mother cells of different ages were normalized to the initial plating efficiencies of the strains.…”

“…Rates were calculated using the Ma-Sandri-Sarkar (MSS) maximum likelihood method [32]. Predicted retromobility frequencies for cells of given ages were calculated as described previously using initial frequencies and retromobility rate values [29]. …”