Jiří Hašek

Robust heat shock induces eIF2α-phosphorylation-independent assembly of stress granules containing eIF3 and 40S ribosomal subunits in budding yeast, Saccharomyces cerevisiae

Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2α/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Δlsm4ΔC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2α became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2α. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.

ELECTROMAGNETIC ACTIVITY OF YEAST CELLS IN THE M PHASE

Electromagnetic activity around yeast mitotic cells (Saccharomyces cerevisiae) was measured in the frequency range 8–9 MHz and special care was taken to extract reliable information from the raw signals. The characteristic of cold-sensitive tubulin mutants tub2-401 and tub2-406, which come to arrest before mitosis at a restrictive temperature (14°C) and which re-enter mitosis upon a shift back to a permissive temperature (28°C), was used to prepare synchronized mitotic cells. Immunofluorescence microscopy using an antitubulin antibody was used to analyze microtubular structures. The arrested tub2-401 mutant that had back-shifted to permissive temperature displayed no microtubules and no electromagnetic activity around the cells. In contrast, the arrested cells of the mutant tub2-406 displayed developed, but aberrant, nonfunctional microtubules and a high electromagnetic activity around the cells. The electromagnetic activity around the arrested mutant tub2-401 back-shifted to permissive temperature peaks at four time points which may coincide with (i) formation of the mitotic spindle, (ii) binding of chromatids to kinetochore microtubules, (iii) elongation of the spindle in anaphase A, and (iv) elongation of the spindle in anaphase B. The profile of the electromagnetic activity around the synchronized mutant tub2-406 at permissive temperature seems to be delayed by the time required for aberrant nonfunctional microtubules to be depolymerized. Experimental results presented in this paper support Pohls idea of existence of the electromagnetic field around yeast cells.

Microelectronic sensors for measurement of electromagnetic fields of living cells and experimental results

Microelectronic sensors are used for measurements of electromagnetic fields generated by synchronized cultures of yeast cells. Cold sensitive mutant tub2-401 of Saccharomyces cerevisiae is used. The measured electromagnetic signals in the frequency range from 8 to 9 MHz are compared with evolution of the reassembled microtubules. The detected signals peak in the time interval 25-30 min and 45-60 min after the release of the cells from the restrictive to the permissive temperature. The first maximum corresponds to the stage when the mitotic spindle is formed and binds chromatids. The second maximum is measured when the processes of anaphase A and of anaphase B take place.

Endogenous Electric Field and Organization of Living Matter

Microtubules in eucaryotic cells form electrically polar structures, which satisfy conditions for excitation, energy condensation, and generation of endogenous electromagnetic field with strong electric near zone component. Large energy supply connected with continuous rebuilding of the microtubular structure and very likely with activity of motor proteins, and interfacial slip layer at the microtubule surface protecting vibrations in microtubules from viscous damping of the cytosol are important conditions for excitation and formation of coherent state. Generated electric field can exert a driving force for directed transport. The Wiener-Lévy process with symmetry breaking is used to describe motion of molecules and charges. Motion of molecules with diameter 1 and 5 nm at distances up to 50 nm is analysed. Transport driven by the electric field with inseparable thermal component has greater probability to reach the target than transport by thermal motion itself. Transport of electrons display similar dependence. Probability of any action depending on the ratio of the random and of the deterministic component of motion should be high enough to provide small number of errors but sufficiently low to comply with requirements for evolutionary changes.

Electromagnetic Field of Microtubules: Effects on Transfer of Mass Particles and Electrons

Biological polar molecules and polymer structures with energy supply (such as microtubules in the cytoskeleton) can get excited and generate an endogenous electromagnetic field with strong electrical component in their vicinity. The endogenous electrical fields through action on charges, on dipoles and multipoles, and through polarization (causing dielectrophoretic effect) exert forces and can drive charges and particles in the cell. The transport of mass particles and electrons is analyzed as a Wiener-Lévy process with inclusion of deterministic force (validity of the Bloch theorem is assumed for transport of electrons in molecular chains too). We compare transport driven by deterministic forces (together with an inseparable thermal component) with that driven thermally and evaluate the probability to reach the target. Deterministic forces can transport particles and electrons with higher probability than forces of thermal origin only. The effect of deterministic forces on directed transport is dominant.

RPG1: an essential gene of Saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase

Abstract In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G1 phase. We characterized a novel gene, RPG1, which is also involved in this phase. RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein. Elutriated or α-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature. The cells remained unbudded and neither grew nor duplicated DNA. rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G1 cells after a shift to a non-permissive temperature. Similarly, the asynchronous rpg1-1 cells accumulated in G1 at the non-permissive temperature, but mother and daughter cells did not separate. A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum. Our data show that Rpg1p is required for passage through the G1 phase and may be involved in growth control. Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3.

Measurement of Electrical Oscillations and Mechanical Vibrations of Yeast Cells Membrane Around 1 kHz

Fröhlich postulated coherent polar oscillations as a fundamental biophysical property of biological systems. Recently, Pelling et al. (2004, 2005) detected mechanical vibrations of yeast cell membrane with atomic force microscope (AFM) and analyzed by Fourier analysis in the frequency range 0.5–2 kHz with amplitudes of the order of 1 nm. This article describes the measurement of electric activity of yeast cells in the acoustic frequency range and of mechanical vibrations of cell membrane. Spectrum analyzer and electrically and electromagnetically screened box with point sensor and amplifiers fed by batteries were used for measurement of synchronized and non synchronized tubulin mutants of yeast cells. We show that the electric activity of synchronized cells in the M phase is greater that of non synchronized cells. That corresponds to the findings of Pohl et al. (1981). Obtained results of measurement of cell electric activity are in good agreement with AFM findings.

Yeast Fluorescence Microscopy

Fluorescence microscopy is the essential technique for investigation of the intracellular distribution of macromolecules and various organelles also in yeast cells. In this chapter, detailed practical procedures for fluorescence microscopic observations developed or adopted in our laboratory are described. These include labeling of the cell wall and chitin, F-actin structures, nuclear and mitochondrial DNA, and two different procedures for investigation of yeast cells by immunofluorescence. In addition, our experience with multicolor labeling experiments is introduced and discussed.

Deregulation of DSE1 Gene Expression Results in Aberrant Budding within the Birth Scar and Cell Wall Integrity Pathway Activation in Saccharomyces cerevisiae

ABSTRACT Strains of Saccharomyces cerevisiae lacking Isw2, the catalytic subunit of the Isw2 chromatin remodeling complex, show the mating type-independent activation of the cell wall integrity (CWI) signaling pathway. Since the CWI pathway activation usually reflects cell wall defects, we searched for the cell wall-related genes changed in expression. The genes DSE1, CTS1, and CHS1 were upregulated as a result of the absence of Isw2, according to previously published gene expression profiles (I. Frydlova, M. Basler, P. Vasicova, I. Malcova, and J. Hasek, Curr. Genet. 52:87-95, 2007). Western blot analyses of double deletion mutants, however, did not indicate the contribution of the chitin metabolism-related genes CTS1 and CHS1 to the CWI pathway activation. Nevertheless, the deletion of the DSE1 gene encoding a daughter cell-specific protein with unknown function suppressed CWI pathway activation in isw2Δ cells. In addition, the deletion of DSE1 also abolished the budding-within-the-birth-scar phenotype of isw2Δ cells. The plasmid-driven overexpression proved that the deregulation of Dse1 synthesis was also responsible for CWI pathway activation and manifestation of the budding-within-the-birth-scar phenotype in wild-type cells. The overproduced Dse1-green fluorescent protein localized to both sides of the septum and persisted in unbudded cells. Although the exact cellular role of this daughter cell-specific protein has to be elucidated, our data point to the involvement of Dse1 in bud site selection in haploid cells.

The absence of the Isw2p–Itc1p chromatin-remodelling complex induces mating type-specific and Flo11p-independent invasive growth of Saccharomyces cerevisiae

The Isw2p–Itc1p chromatin remodelling complex of Saccharomyces cerevisiae is a member of the ISWI class of ATPases with a nucleosome spacing activity, involved in regulation of expression of a broad spectrum of genes. Its absence causes derepression of a‐specific genes and aberrant morphology in α‐mating type cells. We report here that the deletion of the ISW2 gene in the originally non‐invasive BY strain induces mating type‐specific invasive growth strongly affected by nitrogen starvation. Although the Flo11 protein was postulated to be critical for haploid invasive growth, we showed that the invasive growth caused by the isw2 and itc1 deletions in α‐mating type cells was Flo11p‐independent. This type of invasive growth was proved to be a consequence of the activation of the pheromone response pathway. Our results suggest that Isw2 and Itc1 proteins do not have the same impact on the described phenomenon. Copyright