Exploring Calcium Dynamics in Yeast Cells During Pheromone Response: A Single-Cell Perspective

TARKOWSKI, NAHUEL; PONCE DAWSON, SILVINA; AGUILAR, PABLO S.

Rosario

Congreso; LIX Reunión Anual de la SAIB; 2023

Yeast cells, Saccharomyces cerevisiae, represent an ideal system for investigating cellular signalingmechanisms. In particular, haploid cells of S. cerevisiae offer a context of special interest, as theyrespond specifically to the sexual pheromone released by cells of the opposite sexual type, triggering asequence of events that can be observed with the naked eye. Among these events, the cell cycle ishalted, and polarized growth toward the pheromone-emitting cell begins, ultimately leading to cellfusion and the formation of a zygote. Previous research had confirmed the importance of calciumincorporation during the response to the pheromone, as it allows for the coordination of genes involvedin signal transduction and cell survival. Here, we have addressed the study of calcium dynamics duringthe pheromone response by observing individual cells. To achieve this, we introduced, developed, andvalidated the fluorescent calcium sensor GCaMP6f in S. cerevisiae. Through in vivo fluorescencemicroscopy experiments, we identified and tracked individual cells, observing transient increases in theform of short bursts. Moreover, the presence of the pheromone appears to increase the frequency ofthese calcium bursts, suggesting that the information transmitted by calcium is encoded in thetemporal distribution of these bursts. With the aim of understanding how different calcium flowpathways affect the dynamics of cytosolic calcium during the pheromone response, we constructedand analyzed a set of mutant strains lacking all known calcium transporters in S. cerevisiae. Our resultssuggest that the Fig1 protein, which is involved in calcium uptake from the extracellular medium, plays acentral role in calcium dynamics during the pheromone response, and surprisingly, we observed thatYvc1, the vacuolar calcium exporter, also plays a significant role in this process. We also observed thateven in the mutant strain lacking all cytosolic calcium elevation pathways (fig1Δ mid1Δ yvc1Δ) andtreated with pheromone, there is still an increase in the frequency of calcium bursts compared to thewild-type strain in a low-calcium environment. These results suggest the existence of a third calciumimport mechanism.By utilizing models and simulations of cytosolic calcium and vacuolar dynamics, wewere able to assign specific characteristics to the different calcium pathways, contributing to theexplanation of some of the results obtained in our study.