Quantifying the Lifespan of Saccharomyces cerevisiae Aging Model using Microfluidics

BUSTAMANTE TORRES, MOISES; SALZMAN, VALENTINA; GODAS, MARIA JOSE; BRAVO, JOAQUÍN; ESTRADA, LAURA C.; AGUILAR, PABLO S.

Rosario

Congreso; LIX Reunión Anual de la SAIB; 2023

SAIB

Replicative Lifespan (RLS) is defined as the number of cell divisions that occur before a cell reachessenescence. Using S. cerevisiae as a model organism, RLS is traditionally measured by separatingdaughter cells from mother cells using a manual micromanipulator equipped with a fiber optic needle.This process takes from 3 to 5 weeks, making it a particularly challenging and error-prone endeavor. Toaddress this limitation, we use a microfluidic single-cell analysis chip in conjunction with time-lapsemicroscopy to observe hundreds of individual cells throughout their lifespans. We were able to monitorcell lifespan in real-time of multiple positions over the array were recorded at 8.5 min intervals with a40× objective lens for 96 h. Trapped yeast cells were cultured with continuous flow of SC media andmaintained at 28 °C. Using FIJI software (ImageJ), we manually reconstructed the entire lifespan ofeach trapped cell. This approach allowed us to successfully generate survival curves for both thewell-known Wild-Type (WT) and long-lived tor1Δ strains, highlighting the device's capability forautomated lifespan measurements. Notably, we determined the single-cell generation times for eachstrain throughout the entire experiment. Furthermore, these devices enabled us to assess phenotypicchanges that occurred during aging and correlate them with different lifespan trajectories. Additionally,we employed curve fitting techniques to the experimental data using several parametric survivalLIX Annual Meeting SAIB 2023distributions (Weibull, Gamma, Gompertz). This enabled us to reliably predict our RLS curves even inshorter experiments. To improve the resolution of the RLS quantification we calibrated the agedistribution of initially loaded cells. For this we determined the number of bud scars in recently trappedcells through calcofluor staining by fluorescent microscopy. Consistently, this device allows us tomeasure the RLS of yeast strains and, using fluorescence microscopy, to study multiple changes thatoccur as the cell ages throughout its lifespan. Besides, using our microfluidic device, we found that thedisassembly of plasma membrane nanodomains known as eisosomes extends yeast’s replicativelifespan. These findings are currently unpublished and hold significant implications for ourunderstanding of cell aging and lifespan regulation.