Dataset Information

Sexual pheromone modulates the frequency of cytosolic Ca²⁺ bursts in Saccharomyces cerevisiae.

ABSTRACT: Transient and highly regulated elevations of cytosolic Ca²⁺ control a variety of cellular processes. Bulk measurements using radioactive Ca²⁺ and the luminescent sensor aequorin have shown that in response to pheromone, budding yeast cells undergo a rise of cytosolic Ca²⁺ that is mediated by two import systems composed of the Mid1-Cch1-Ecm7 protein complex and the Fig1 protein. Although this response has been widely studied, there is no treatment of Ca²⁺ dynamics at the single-cell level. Here, using protein calcium indicators, we show that both vegetative and pheromone-treated yeast cells exhibit discrete and asynchronous Ca²⁺ bursts. Most bursts reach maximal amplitude in 1-10 s, range between 7 and 30 s, and decay in a way that fits a single-exponential model. In vegetative cells, bursts are scarce but preferentially occur when cells are transitioning G1 and S phases. On pheromone presence, Ca²⁺ burst occurrence increases dramatically, persisting during cell growth polarization. Pheromone concentration modulates burst frequency in a mechanism that depends on Mid1, Fig1, and a third, unidentified, import system. We also show that the calcineurin-responsive transcription factor Crz1 undergoes nuclear localization bursts during the pheromone response.

SUBMITTER: Carbo N

PROVIDER: S-EPMC5305257 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Sexual pheromone modulates the frequency of cytosolic Ca2+ bursts in Saccharomyces cerevisiae.

Carbó Natalia N Tarkowski Nahuel N Ipiña Emiliano Perez EP Dawson Silvina Ponce SP Aguilar Pablo S PS

Molecular biology of the cell 20161228 4

Transient and highly regulated elevations of cytosolic Ca2+ control a variety of cellular processes. Bulk measurements using radioactive Ca2+ and the luminescent sensor aequorin have shown that in response to pheromone, budding yeast cells undergo a rise of cytosolic Ca2+ that is mediated by two import systems composed of the Mid1-Cch1-Ecm7 protein complex and the Fig1 protein. Although this response has been widely studied, there is no treatment of Ca2+</su ...[more]

PMID: 28031257

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Project description:How microbial cells leverage their phenotypic potential to survive in a changing environment is a complex biological problem, with important implications for pathogenesis and species evolution. Stochastic phenotype switching, a particularly fascinating adaptive approach observed in numerous species across the tree of life, introduces phenotypic diversity into a population through mechanisms which have remained difficult to define. Here we describe our investigations into the mechanistic basis of colony morphology phenotype switching which occurs in populations of a pathogenic isolate of Saccharomyces cerevisiae, YJM311. We observed that clonal populations of YJM311 cells produce variant colonies that display altered morphologies and, using whole genome sequence analysis, discovered that these variant clones harbored an exceptional collection of karyotypes newly altered by de novo structural genomic variations (SVs). Overall, our analyses indicate that copy number alterations, more often than changes in allelic identity, provide the causative basis of this phenotypic variation. Individual variants carried between 1 and 16 de novo copy number variations, most of which were whole chromosomal aneuploidies. Notably, we found that the inherent stability of the diploid YJM311 genome is comparable to that of domesticated laboratory strains, indicating that the collections of SVs harbored by variant clones did not arise by a chronic chromosomal instability (CIN) mechanism. Rather, our data indicate that these variant clones acquired such complex karyotypic configurations simultaneously, during stochastic and transient episodes of punctuated systemic genomic instability (PSGI). Surprisingly, we found that the majority of these highly altered variant karyotypes were propagated with perfect fidelity in long-term passaging experiments, demonstrating that high aneuploidy burdens can often be conducive with prolonged genomic integrity. Together, our results demonstrate that colony morphology switching in YJM311 is driven by a stochastic process in which genome stability and plasticity are integrally coupled to phenotypic heterogeneity. Consequently, this system simultaneously introduces both phenotypic and genomic variation into a population of cells, which can, in turn perpetuate population diversity for many generations thereafter.

Project description:Mitochondrial Ca2+ handling is accomplished by balancing Ca2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca2+ buffering in the matrix and Ca2+ efflux mainly via Ca2+ ion exchangers, such as the Na+/Ca2+ exchanger (NCLX) and the Ca2+/H+ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration.

Dataset Information

Sexual pheromone modulates the frequency of cytosolic Ca²⁺ bursts in Saccharomyces cerevisiae.

Publications

Sexual pheromone modulates the frequency of cytosolic Ca<sup>2+</sup> bursts in <i>Saccharomyces cerevisiae</i>.

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