Study of mechanisms of ammonia production in yeast colonies and liquid cultures

Yeast, although non-motile and unicellular organisms can create organized structures, colonies, in which cells communicate and cooperate and which in some ways resemble multicellular organisms. Our previous studies on yeast colony development revealed that colonies periodically change pH of their surroundings. Alkalization of an agar medium is accompanied by production of volatile ammonia that acts as the long-range signal. Microarray analysis of the expression changes in Saccharomyces cerevisiae colonies during their transition from acid to alkali phase revealed significant changes in yeast transcriptome. Among others, strong induction of expression of three homologous genes ATO1 (YNR010c, ADY2), ATO2 (YNR002c, FUN34) and ATO3 (YDR384c) at the beginning of the alkali phase was found. These genes encode membrane proteins that may function as ammonium/H+ antiporters. This work contributes to better understanding of both the ammonia signaling and the role of putative ammonium exporters - Ato proteins. It was revealed, that other volatile compounds - methylamine and propylamine - are (in addition to ammonia) able to induce entry into the alkali phase of yeast colony development. Moreover, the significant impact of the transport of monocarboxylic acids on ammonia production and yeast colony development...Kvasinky jsou jednobuněčné mikroorganismy, na pevném podkladu jsou však schopny vytvářet organizované struktury, kolonie, které mohou v jistých ohledech připomínat mnohobuněčné organismy. Při sledování vývoje kvasinkových kolonií v čase bylo zjištěno, že kolonie periodicky mění pH svého okolí z acidického na alkalické a naopak. Přepnutí z acidické do alkalické fáze je spojeno s produkcí amoniaku, který funguje jako signál mezi sousedními koloniemi. Data z analýz využívajících DNA čipy a další experimentální data nasvědčují, že za produkci amoniaku do okolí by mohly být zodpovědné tři homologní proteiny kódované geny ATO1 (YNR010c, ADY2), ATO2 (YNR002c, FUN34) a ATO3 (YDR384c). Cílem této práce je prohloubení znalostí jak o amoniakové signalizaci mezi kvasinkovými buňkami, tak o možných exportérech amonného kationtu - Ato proteinech. Práce přináší poznatky o dalších těkavých látkách - methylaminu a propylaminu - které jsou (vedle amoniaku) schopny indukovat vstup kvasinkové kolonie do alkalické fáze vývoje. Z hlediska amoniakové signalizace dále popisuje významný vliv transportu karboxylových kyselin na průběh vývoje kvasinkové kolonie, kdy omezený transport těchto slabých organických kyselin do buněk vybraných delečních kmenů patrně znemožňuje správný průběh amoniakové signalizace a diferenciace...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

ixFLIM: Interferometric Excitation Fluorescence Lifetime Imaging Microscopy

Fluorescence lifetime imaging microscopy (FLIM) is a well-established technique with numerous imaging applications. Yet, one of the limitations of FLIM is that it provides information about the emitting state only. Here, we present an extension of FLIM by interferometric measurement of fluorescence excitation spectra. Interferometric Excitation Fluorescence Lifetime Imaging Microscopy (ixFLIM) reports on the correlation of the excitation spectra and emission lifetime, providing the correlation between the ground-state absorption and excited-state emission. As such, it extends the applicability of FLIM and removes some of its limitations. We introduce ixFLIM on progressively more complex systems and apply it to quantitative resonance energy transfer imaging from a single measurement.Comment: 29 pages (19 main and 10 supporting), 18 figure

Tvorba proteinů Ato v tekutých kulturách koreluje s uvolňováním amoniaku

pH of the media changes during growth of both shaken or static liquid S. cerevisiae similarly to pH of the agar under colonies. Likewise in colonies, production of all Ato proteins in liquid cultures initiates during medium alkalizatio

Study of mechanisms of ammonia production in yeast colonies and liquid cultures

Yeast, although non-motile and unicellular organisms can create organized structures, colonies, in which cells communicate and cooperate and which in some ways resemble multicellular organisms. Our previous studies on yeast colony development revealed that colonies periodically change pH of their surroundings. Alkalization of an agar medium is accompanied by production of volatile ammonia that acts as the long-range signal. Microarray analysis of the expression changes in Saccharomyces cerevisiae colonies during their transition from acid to alkali phase revealed significant changes in yeast transcriptome. Among others, strong induction of expression of three homologous genes ATO1 (YNR010c, ADY2), ATO2 (YNR002c, FUN34) and ATO3 (YDR384c) at the beginning of the alkali phase was found. These genes encode membrane proteins that may function as ammonium/H+ antiporters. This work contributes to better understanding of both the ammonia signaling and the role of putative ammonium exporters - Ato proteins. It was revealed, that other volatile compounds - methylamine and propylamine - are (in addition to ammonia) able to induce entry into the alkali phase of yeast colony development. Moreover, the significant impact of the transport of monocarboxylic acids on ammonia production and yeast colony development..

AML-Related NPM Mutations Drive p53 Delocalization into the Cytoplasm with Possible Impact on p53-Dependent Stress Response

Nucleophosmin (NPM) interaction with tumor suppressor p53 is a part of a complex interaction network and considerably affects cellular stress response. The impact of NPM1 mutations on its interaction with p53 has not been investigated yet, although consequences of NPMmut-induced p53 export to the cytoplasm are important for understanding the oncogenic potential of these mutations. We investigated p53-NPM interaction in live HEK-293T cells by FLIM-FRET and in cell lysates by immunoprecipitation. eGFP lifetime-photoconversion was used to follow redistribution dynamics of NPMmut and p53 in Selinexor-treated cells. We confirmed the p53-NPMwt interaction in intact cells and newly documented that this interaction is not compromised by the NPM mutation causing displacement of p53 to the cytoplasm. Moreover, the interaction was not abolished for non-oligomerizing NPM variants with truncated oligomerization domain, suggesting that oligomerization is not essential for interaction of NPM forms with p53. Inhibition of the nuclear exporter XPO1 by Selinexor caused expected nuclear relocalization of both NPMmut and p53. However, significantly different return rates of these proteins indicate nontrivial mechanism of p53 and NPMmut cellular trafficking. The altered p53 regulation in cells expressing NPMmut offers improved understanding to help investigational strategies targeting these mutations

The transport of carboxylic acids and important role of the Jen1p transporter during the development of yeast colonies

On solid substrates, yeast colonies pass through distinct developmental phases characterized by the changes in pH of their surroundings from acidic to nearly alkaline and vice versa. At the beginning of the alkali phase colonies start to produce ammonia, which functions as a quorum-sensing molecule inducing the reprogramming of cell metabolism. Such reprogramming includes, among others, the activation of several plasma membrane transporters and is connected with colony differentiation. In the present study, we show that colony cells can use two transport mechanisms to import lactic acid: a 'saturable' component of the transport, which requires the presence of a functional Jen1p transporter, and a 'non-saturable' component (diffusion) that is independent of Jen1p. During colony development, the efficiency of both transport components changes similarly in central and outer colonial cells. Although the lactate uptake capacity of central cells gradually decreases during colony development, the lactate uptake capacity of outer cells peaks during the alkali phase and is also kept relatively high in the second acidic phase. This lactate uptake profile correlates with the localization of the Jen1p transporter to the plasma membrane of colony cells. Both lactic acid uptake mechanisms are diminished in sok2 colonies where JEN1 expression is decreased. The Sok2p transcription factor may therefore be involved in the regulation of non-saturable lactic acid uptake in yeast colonies.info:eu-repo/semantics/publishedVersio