Stress granules: RNP-containing cytoplasmic bodies springing up under stress. The structure and mechanism of organization

In this review recent data describing stress granules are summarized. Stress granules are specific RNA-containing structures in the cytoplasm of living cells which arise under stress conditions (e. g. heat shock, UV irradiation, energy depletion and oxidative stress). It became evident that stress granules accumulate non-canonical 48S initiation complexes and contain mRNA with associated proteins, small ribosomal subunits and some initiation factors. Stress granules are depleted with ternary complex and large ribosomal subunit. It's proposed that eIF2alpha phosphorylation and ternary complex decrease can be a trigger for stress granule formation. Shuttling nuclear and cytoplasmic RNA-binding protein TIA-1 plays a crucial role in this process. It's proposed that TIA-1 forms prion-like aggregates, and these aggregates are scaffolds for other components of stress granules. Cytoskeletal structures facilitate the accumulation of stress granule components in local cytoplasmic sites. Investigation of process of stress granule formation is important for understanding of cell reaction to stress and translation regulation mechanisms.     

Cellular stress leads to the formation of membraneless stress assemblies in eukaryotic cells

In cells at steady state, two forms of cell compartmentalization coexist: membrane‐bound organelles and phase‐separated membraneless organelles that are present in both the nucleus and the cytoplasm. Strikingly, cellular stress is a strong inducer of the reversible membraneless compartments referred to as stress assemblies. Stress assemblies play key roles in survival during cell stress and in thriving of cells upon stress relief. The two best studied stress assemblies are the RNA‐based processing‐bodies (P‐bodies) and stress granules that form in response to oxidative, endoplasmic reticulum (ER), osmotic and nutrient stress as well as many others. Interestingly, P‐bodies and stress granules are heterogeneous with respect to both the pathways that lead to their formation and their protein and RNA content. Furthermore, in yeast and Drosophila, nutrient stress also leads to the formation of many other types of prosurvival cytoplasmic stress assemblies, such as metabolic enzymes foci, proteasome storage granules, EIF2B bodies, U‐bodies and Sec bodies, some of which are not RNA‐based. Nutrient stress leads to a drop in cytoplasmic pH, which combined with posttranslational modifications of granule contents, induces phase separation.     

Aspergillus oryzae AoSO Is a Novel Component of Stress Granules upon Heat Stress in Filamentous Fungi

Stress granules are a type of cytoplasmic messenger ribonucleoprotein (mRNP) granule formed in response to the inhibition of translation initiation, which typically occurs when cells are exposed to stress. Stress granules are conserved in eukaryotes; however, in filamentous fungi, including Aspergillus oryzae, stress granules have not yet been defined. For this reason, here we investigated the formation and localization of stress granules in A. oryzae cells exposed to various stresses using an EGFP fusion protein of AoPab1, a homolog of Saccharomyces cerevisiae Pab1p, as a stress granule marker. Localization analysis showed that AoPab1 was evenly distributed throughout the cytoplasm under normal growth conditions, and accumulated as cytoplasmic foci mainly at the hyphal tip in response to stress. AoSO, a homolog of Neurospora crassa SO, which is necessary for hyphal fusion, colocalized with stress granules in cells exposed to heat stress. The formation of cytoplasmic foci of AoSO was blocked by treatment with cycloheximide, a known inhibitor of stress granule formation. Deletion of the Aoso gene had effects on the formation and localization of stress granules in response to heat stress. Our results suggest that AoSO is a novel component of stress granules specific to filamentous fungi.The authors would specially like to thank Hiroyuki Nakano and Kei Saeki for generously providing experimental and insightful opinions.     

Heat and salt stress in the food pathogen Bacillus cereus

AIMS: The effects of stresses imposed on bacterial contaminants during food processing and treatment of packaging material were evaluated on the food pathogen Bacillus cereus. METHODS AND RESULTS: Conditions were established which allowed the cells to adapt to heat, ethanol and hydrogen peroxide stresses, but not to osmotic shock. Cross protection between stresses indicated a clear hierarchy of resistance with salt protecting against hydrogen peroxide, which protected against ethanol, which protected against heat shock. The cultures were shown to be most sensitive to heat, ethanol and oxidative stress at mid-exponential phase and to become resistant at stationary phase. Adaptive levels of stressor were found to induce synthesis of general stress and stress-specific proteins and differential accumulation of proteins was demonstrated between heat- or salt-stressed and unstressed cells. CONCLUSIONS: Sequencing revealed that a number of glycolytic enzymes were regulated by heat and osmotic shocks and that the chaperone GroEL was induced by heat shock. SIGNIFICANCE AND IMPACT OF THE STUDY: The implications of the physiological data in designing storage and processing conditions for food are discussed. The identification of stress-regulated proteins reveals a clear role for glycolysis in adaptation to heat shock and osmotic stress.     

Stress granules: RNP-containing cytoplasmic bodies arising in stress: Structure and mechanism of organization

The review considers recent data on stress granules, which are dense RNP-containing cytoplasmic bodies that arise under stress conditions, e.g., in heat shock, UV irradiation, energy depletion, and oxidative stress. There is evidence that stress granules accumulate incomplete initiation complexes containing mRNA associated with proteins, small ribosomal subunits, and some translation initiation factors, and that stress granules are formed when cells are depleted of the ternary complex (eIF2-tRNA^Met-GTP), in particular, upon eIF2A phosphorylation or a decrease in GTP. Large ribosomal subunits and the ternary complex are absent from stress granules. The structural basis of stress granules is known. It is probable, however, that RNA-binding protein TIA-1, which normally occurs in the nucleus, forms prion-like aggregates that serve as scaffolds for other components of stress granules. The cytoskeleton facilitates the accumulation of stress granule components in local cytoplasmic sites. Studies of the formation and composition of stress granules are important for a better understanding of the regulation of translation initiation in vivo and the mechanisms of the cell response to stress factors.     

Incorporating germination‐induction into decontamination strategies for bacterial spores

Oenococcus oeni is the dominant species able to cope with a hostile environment of wines, comprising cumulative effects of low pH, high ethanol and SO₂ content, nonoptimal growth temperatures and growth inhibitory compounds. Ethanol tolerance is a crucial feature for the activity of O. oeni cells in wine because ethanol acts as a disordering agent of its cell membrane and negatively affects metabolic activity; it damages the membrane integrity, decreases cell viability and, as other stress conditions, delays the start of malolactic fermentation with a consequent alteration of wine quality. The cell wall, cytoplasmic membrane and metabolic pathways are the main sites involved in physiological changes aimed to ensure an adequate adaptive response to ethanol stress and to face the oxidative damage caused by increasing production of reactive oxygen species. Improving our understanding of the cellular impact of ethanol toxicity and how the cell responds to ethanol stress can facilitate the development of strategies to enhance microbial ethanol tolerance; this allows to perform a multidisciplinary endeavour requiring not only an ecological study of the spontaneous process but also the characterization of useful technological and physiological features of the predominant strains in order to select those with the highest potential for industrial applications.     

Changes of Saccharomyces cerevisiae cell membrane components and promotion to ethanol tolerance during the bioethanol fermentation

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane might provide main protection to tolerate accumulated ethanol, and S. cerevisiae cells might also remodel their membrane compositions or structure to try to adapt to or tolerate the ethanol stress. However, the exact changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation still remains poorly understood. This study was performed to clarify changes and roles of S. cerevisiae cell membrane components during bioethanol fermentation. Both cell diameter and membrane integrity decreased as fermentation time lasting. Moreover, compared with cells at lag phase, cells at exponential and stationary phases had higher contents of ergosterol and oleic acid (C_18:1) but lower levels of hexadecanoic (C_16:0) and palmitelaidic (C_16:1) acids. Contents of most detected phospholipids presented an increase tendency during fermentation process. Increased contents of oleic acid and phospholipids containing unsaturated fatty acids might indicate enhanced cell membrane fluidity. Compared with cells at lag phase, cells at exponential and stationary phases had higher expressions of ACC1 and HFA1. However, OLE1 expression underwent an evident increase at exponential phase but a decrease at following stationary phase. These results indicated that during bioethanol fermentation process, yeast cells remodeled membrane and more changeable cell membrane contributed to acquiring higher ethanol tolerance of S. cerevisiae cells. These results highlighted our knowledge about relationship between the variation of cell membrane structure and compositions and ethanol tolerance, and would contribute to a better understanding of bioethanol fermentation process and construction of industrial ethanologenic strains with higher ethanol tolerance.     

Differential role of microenvironment in microencapsulation for improved cell tolerance to stress

The effect of the microenvironment in alginate–chitosan–alginate (ACA) microcapsules with liquid core (LCM) and solid core (SCM) on the physiology and stress tolerance of Sacchromyces cerevisiae was studied. The suspended cells were used as control. Cells cultured in liquid core microcapsules showed a nearly twofold increase in the intracellular glycerol content, trehalose content, and the superoxide dismutase (SOD) activity, which are stress tolerance substances, while SCM did not cause the significant physiological variation. In accordance with the physiological modification after being challenged with osmotic stress (NaCl), oxidative stress (H₂O₂), ethanol stress, and heat shock stress, the cell survival in LCM was increased. However, SCM can only protect the cells from damaging under ethanol stress. Cells released from LCM were more resistant to hyperosmotic stress, oxidative stress, and heat shock stress than cells liberated from SCM. Based on reasonable analysis, a method was established to estimate the effect of microenvironment of LCM and SCM on the protection of cells against stress factors. It was found that the resistance of LCM to hyperosmotic stress, oxidative stress, and heat shock stress mainly depend on the domestication effect of LCM’s microenvironment. The physical barrier of LCM constituted by alginate–chitosan membrane and liquid alginate matrix separated the cells from the damage of oxidative stress and ethanol stress. The significant tolerance against ethanol stress of SCM attributed to the physical barrier consists of solid alginate–calcium matrix and alginate–chitosan membrane.     

甘蓝热胁迫叶片细胞的超微结构研究

高温胁迫引起植物体损伤是很常见的,人们已从多方面探讨高温胁迫对植物的影响,主要工作集中在生理生化、膜结构、热激蛋白及作物产量等方面［１—４］,超微结构的变化也有少量报道［５—７］。但目前要建立一个高温对植物体影响的模式还比较困难。本文探讨了甘蓝不同耐热品种叶片细胞超微结构在热胁迫后的差异,目的是建立植物耐热程度的细胞学指标,为育种工作者选育耐热性品种提供细胞学依据。材料和方法试验材料为甘蓝（Ｂｒａｓｓｉｃａｏｌｅｒａｃｅａｖａｒ．ｃａｐｉｔａｔａＬ．）耐热品种“夏光甘蓝”和感热品种“京丰甘蓝”。…     

uspB, a New ςS-Regulated Gene in Escherichia coli Which Is Required for Stationary-Phase Resistance to Ethanol

The open reading frame immediately upstream of uspA is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions. UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains. Overexpression of UspB causes cell death in stationary phase, whereas mutants of uspB are sensitive to exposure to ethanol but not heat in stationary phase. In contrast to uspA, stationary-phase induction of uspB requires the sigma factor ς^S. The expression of uspB is modulated by H-NS, consistent with the role of H-NS in altering ς^S levels. Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon’s previously known role in thermotolerance, osmotolerance, and oxidative stress resistance.     

Induction of Hsp104 synthesis in Saccharomyces cerevisiae in the stationary growth phase is inhibited by the petite mutation

The elevation of Hsp104 (heat shock protein) content under heat stress plays a key role in the development of thermotolerance in yeast (Saccharomyces cerevisiae) cells. Hsp104 synthesis is increased under heat stress and in the stationary growth phase. The loss of mitochondrial DNA (petite mutation) was shown to inhibit the induction of Hsp104 synthesis under heat stress (39°C) and during the transition to the stationary growth phase. Also, the petite mutation suppressed the increase in activity of antioxidant enzymes in the stationary phase, which accompanied by decrease in thermotolerance. At the same time, mutation inhibited production of reactive oxygen species and prevented cell death under heat shock in the logarithmic growth phase. The results of this study suggest that disruption of the mitochondrial functional state suppresses the expression of yeast nuclear genes upon upon entry into the stationary growth phase.     

The Ultrastructure of Polytomella agilis*

SYNOPSIS Motile cells and cysts of Polytomella agilis, obtained over the entire growth cycle, were examined by electron microscopy. In typical late log phase cells there is a concentric arrangement of the internal organelles around the centrally located nucleus. Lying just beneath the plasma membrane is a peripheral band of elongate mitochondria. Numerous well defined Golgi bodies are also distributed around the nucleus. Vesicles associated with the Golgi body increase in size with distance from the secretory edges of the organelle. Cytoplasmic membranes with associated ribosomes are found between the mitochondrial and Golgi regions. A layer of slender membrane-limited structures is located near the mitochondrial layer. These organelles, which resemble proplastids, become highly branched during late log and early stationary phase, reaching maximum development in late stationary and early pre-cyst stages. Large storage granules of varying density are found within the cell. The PAS-positive granules have been isolated and shown to contain starch. There is an increase in the amount of this storage material as the cells enter the stationary phase. The remainder of the cytoplasmic matrix is finely granular and contains numerous free ribosomes except in the region of the anterior papilla. Four flagella arise from basal bodies at the anterior end of the cell. The cyst is characterized by a thick multi-layered cell wall whose electron density obscures the limiting plasma membrane. Large storage granules are located close to and often in contact with the periphery of the cell, suggesting their involvement in the process of cell wall deposition. Altho mitochondria can still be seen in the mature cyst, other cytoplasmic organelles often appear atypical. The mature cyst has an irregular profile possibly due to shrinkage from dehydration.     

Ultrastructural Study in Leaf Cell of Brassica oleracea var. capitata Under Heat Stress

Ultrastructural changes of the leaf cells in response to heat stress in thermo-resistant cultivar and thermo-sensitive cultivar of Brassica oleracea var. capitata L. were investigated. No ultrastructural change was shown in mesophyll cell of the thermo-resistant cultivar. All membrane-bound structure remained intact. However, the cell ultrastructure of thermosensitive cultivar showed significant changes. One of the major effects of heat stress was the disruption of membrane structure. Chloroplasts and nucleus were extremely sensitive to heat stress. Chloroplasts rounded off in different extents and their outer membranes disappeared. Structural alteration of the thylakoid membrane were visualized. Large amount of plastoglobulis appeared within the chloroplast. Mitochondria were far more resistant to heat stress than chloroplasts. There was no distinct changes of mitochondria structure. Nucleus suffered from serious damage as heat caused disruption of nuclear envelope and condensation of nucleoplasm, showing, eventually, numerous fibrillar granulous material and irregularly shaped hollow spaces presented within the nucleus.     

Heat-shock proteins induce heavy-metal tolerance in higher plants

Cell cultures of Lycopersicon peruvianum L. stressed with CdSO₄ (10^–3M) show typical changes in the ultrastructure, starting with the plasmalemma and later on extending to the endoplasmic reticulum and the mitochondrial envelope. Part of the membrane material is extruded, with the formation of osmiophilic droplets which increase in size and number during the stress period. After 4 h, about 20 of the cells are dead. A short heat stress preceeding the heavy-metal stress induces a tolerance effect by preventing the membrane damage. The cells show a normal ultrastructure with one exception: cytoplasmic heat-shock granules are formed. This protective effect can be abolished by cycloheximide. Cadmium uptake is not markedly influenced by the heat stress. Cadmium is found together with sulfur in small deposits in the vacuoles of stressed cells. The precipitates contain an excess of sulfur, evidently due to the stress-induced formation of phytochelatins. The role in heavy-metal tolerance of heat-shock proteins in the plasmalemma (HSP70) and in cytoplasmic heat-stress granules (HSP17, HSP70) is discussed.Abbreviations EDX energy dispersive analysis of X-rays - ESI electron-spectroscopic imaging - HM heavy metal - HSG heat-stress granules - HSP heat-shock protein - MNDO modified neglect of diatomic overlap This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Sachsen-Anhalt and the Deutsche Forschungsgemeinschaft.     

Synthesis and localization of L-lactate oxidase in yeasts Yarrowia lipolytica

Conditions for L-lactate oxidase synthesis by the yeast Yarrowia lipolytica were investigated. The enzyme was found to be synthesized during growth on L-lactate in the exponential growth phase. L-lactate oxidase synthesis was also observed on glucose after adaptation to stress conditions (oxidative or thermal stress) during the stationary growth phase after glucose consumption. The cells grown on L-lactate exhibited high levels of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase), which exceeded those of glucose-grown cells. Ultrastructurally, L-lactate-grown cells and the cells grown on glucose and adapted to various stress conditions were also found to be similar, with increased mitochondria, elevated number and size of peroxisomes, and formation of lipid and polyphosphate inclusions. In order to determine the intracellular localization of L-lactate oxidase, the cells were disintegrated by the lytic enzyme complex from Helix pomatia. Centrifugation of the homogenate in Percoll gradient resulted in the isolation of purified fractions of the native mitochondria and peroxisomes. L-lactate oxidase was shown to be localized in peroxisomes.     

Effects of heat shock and ethanol stress on the viability of aSaccharomyces uvarum (carlsbergensis) brewing yeast strain during fermentation of high gravity wort

Summary The effects of heat shock and ethanol stress on the viability of a lager brewing yeast strain during fermentation of high gravity wort were studied. These stress effects resulted in reduced cell viability and inhibition of cell growth during fermentation. Cells were observed to be less tolerant to heat shock during the fermentation of 25°P (degree Plato) wort than cells fermenting 16°P wort. Degree Plato (^oP) is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C. Relieving the stress effects of ethanol by washing the cells free of culture medium, improved their tolerance to heat shock. Cellular changes in yeast protein composition were observed after 24 h of fermentation at which time more than 2% (v/v) ethanol was present in the growth medium. The synthesis of these proteins was either induced by ethanol or was the result of the transition of cells from exponential phase to stationary phase of growth. No differences were observed in the protein composition of cells fermenting 16°P wort compared to those fermenting 25°P wort. Thus, the differences in the tolerance of these cells to heat shock may be due to the higher ethanol concentration produced in 25°P wort which enhanced their sensitivity to heat shock.     

Nature of the sulfur-containing component and its function in Thiobacillus ferrooxidans

As was shown using various reagents (Ag+, Cd2+) and solvents (ethanol, methanol), Thiobacillus ferrooxidans cells accumulate colloidal sulfur when they grow in the medium 9K containing elemental sulfur. Colloidal sulfur is accumulated in the periplasmic space, in large, bipolarly arranged spherical structures and in simple invaginates of the cytoplasmic membrane. T. ferrooxidans cells accumulate the sulfur at a highest rate during the stationary phase of growth and can use it as a source of energy under the conditions of starvation. The factors causing sulfur accumulation in T. ferrooxidans cells are discussed.     

The role of periplasmic antioxidant enzymes (superoxide dismutase and thiol peroxidase) of the Shiga toxin-producing Escherichia coli O157:H7 in the formation of biofilms

This study examined the role of the periplasmic oxidative defense proteins, copper, zinc superoxide dismutase (SodC), and thiol peroxidase (Tpx), from the Shiga toxin-producing Escherichia coli O157:H7 (STEC) in the formation of biofilms. Proteomic analyses have shown significantly higher expression levels of both periplasmic antioxidant systems (SodC and Tpx) in STEC cells grown under biofilm conditions than under planktonic conditions. An analysis of their growth phase-dependent gene expression indicated that a high level of the sodC expression occurred during the stationary phase and that the expression of the tpx gene was strongly induced only during the exponential growth phase. Exogenous hydrogen peroxide reduced the aerobic growth of the STEC sodC and tpx mutants by more than that of their parental strain. The two mutants also displayed significant reductions in their attachment to both biotic (HT-29 epithelial cell) and abiotic surfaces (polystyrene and polyvinyl chloride microplates) during static aerobic growth. However, the growth rates of both wild-type and mutants were similar under aerobic growth conditions. The formation of an STEC biofilm was only observed with the wild-type STEC cells in glass capillary tubes under continuous flow-culture conditions compared with the STEC sodC and tpx mutants. To the best of our knowledge, this is the first mutational study to show the contribution of sodC and tpx gene products to the formation of an E. coli O157:H7 biofilm. These results also suggest that these biofilms are physiologically heterogeneous and that oxidative stress defenses in both the exponential and stationary growth stages play important roles in the formation of STEC biofilms.     

Ultrastructural study of the life cycle of <Emphasis Type="Italic">Rickettsia slovaca</Emphasis>, wild and standard type,cultivated in L929 and vero cell lines

Ultrastructural changes induced by Rickettsia slovaca standard type (ST) and wild type (WT) were examined during their life cycle in L929 and Vero cells. R. slovaca invaded the cytoplasm of the host cell by phagocytosis on the 1st d p.i. Rickettsiae adhering to the cytoplasmic membrane were engulfed by cellular extensions and occurred in phagocytic vacuoles. Binary fission of rickettsia was observed. The nuclear chromatin of eukaryotic cells was rearranged and condensed during 3rd and 6th d p.i. Finally, loss of the plasma membrane integrity, destruction of cytoplasm and nucleus resulted in cell lysis. Degeneration of the host cell caused by WT and ST was observed after 4 and 5 d p.i. in L929 cells and after 3 and 6 d p.i. in Vero cells, respectively. WT type was able to penetrate into the nucleus of the host cell and was responsible for dilatation of the perinuclear space and endoplasmic reticulum, causing more pronounced and different cytopathological changes than the ST.