Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance

Saccharomyces cerevisiae yeast cells serve as a model to elucidate the bases of salt tolerance and potassium homeostasis regulation in eukaryotic cells. In this study, we show that two widely used laboratory strains, BY4741 and W303-1A, differ not only in cell size and volume but also in their relative plasma-membrane potential (estimated with a potentiometric fluorescent dye diS-C3(3) and as Hygromycin B sensitivity) and tolerance to alkali-metal cations. W303-1A cells and their mutant derivatives lacking either uptake (trk1 trk2) or efflux (nha1) systems for alkali-metal cations are more tolerant to toxic sodium and lithium cations but also more sensitive to higher external concentrations of potassium than BY4741 cells and their mutants. Moreover, our results suggest that though the two strains do not differ in the total potassium content, the regulation of intracellular potassium homeostasis is probably not the same in BY4741 and W303-1A cells.     

Expression of the two pore domain potassium channel TREK-1 in human intervertebral disc cells

Potassium channels play a major role in intracellular homeostasis and regulation of cell volume. Intervertebral disc cells respond to mechanical loading in a complex manner. Mechanical loading may play a role in disc degeneration. Lumbar intervertebral disc samples from 5 patients (average age: 47 years, range: 25-64 years) were used for this study, investigating cells from the nucleus pulposus and the annulus fibrosus duplicate samples to determine RNA expression and protein expression. Analysis of mRNA expression by RT-PCR demonstrated that TREK 1 was expressed by nucleus pulposus (n=5) and annulus fibrosus (n=5) cells. Currently, TREK-1 is the only potassium channel known to be activated by intracellular acidosis, and responds to mechanical and chemical stimuli. Whilst the precise role of potassium channels in cellular homeostasis remains to be determined, TREK-1 may be important to protect disc cells against ischaemic damage, and subsequent disc degeneration, and may also play a role in effecting mechanotransduction. Further research is required to fully elucidate the role of the TREK-1 ion channel in intervertebral disc cells.     

Impact of improved potassium accumulation on pH homeostasis, membrane potential adjustment and survival of Corynebacterium glutamicum

Metal ion uptake is crucial for all living cells and an essential part of cellular bioenergetic homeostasis. In this study the uptake and the impact of the most abundant internal cation, potassium, were investigated in Actinobacteria, a group of high G+C Gram-positives with a number of prominent biotechnologically and medically important members. Genome analyses revealed a variety of different potassium uptake systems in this monophyletic group ranging from potassium channels common in virtually all Actinobacteria to different active carriers that were present predominantly in pathogenic members able to cope with various stress conditions. By applying Corynebacterium glutamicum as model system we provide experimental evidence that under optimal conditions a potassium channel is sufficient in bacteria for the maintenance of internal pH and membrane potential ensuring survival of cells under stress conditions. Under potassium limitation, however, viability of C. glutamicum was increased under acidic stress or during desiccation when a functional KtrAB potassium transporter from the pathogen Corynebacterium jeikeium was heterologously expressed. We provide experimental evidence that the KtrAB mediated enhanced potassium accumulation improved maintenance of internal pH and membrane potential. The results indicate that the occurrence of active potassium transport systems correlates with an improved potassium-dependent bioenergetic homeostasis and survival of bacterial cells under stress conditions.     

Dissecting the signaling pathways involved in the function of sperm flagellum

The mammalian flagellum is a specific type of motile cilium required for sperm motility and male fertility. Effective flagellar movement is dependent on axonemal function, which in turn relies on proper ion homeostasis within the flagellar compartment. This ion homeostasis is maintained by the concerted function of ion channels and transporters that initiate signal transduction pathways resulting in motility changes. Advances in electrophysiology and super-resolution microscopy have helped to identify and characterize new regulatory modalities of the mammalian flagellum. Here, we discuss what is currently known about the regulation of flagellar ion channels and transporters that maintain sodium, potassium, calcium, and proton homeostasis. Identification of new regulatory elements and their specific roles in sperm motility is imperative for improving diagnostics of male infertility.     

A novel role for a glycosylphosphatidylinositol-anchored aspartyl protease, CgYps1, in the regulation of pH homeostasis in Candida glabrata

Proteases, key virulence factors of many bacterial and fungal pathogens, are pivotally important for nutrient acquisition, invasion and adherence to host cells and evasion/escape from host immune cells. In this study, we report a novel role for CgYps1, member of a family of 11 GPI-linked aspartyl proteases, in a human opportunistic fungal pathogen, Candida glabrata, in the regulation of pH homeostasis under acidic environmental conditions. We show that CgYps1 is required to survive low-external-pH environment and the inability of Cgyps1Δ mutant to maintain pH homeostasis results in intracellular acidification and increased reactive oxygen species (ROS) production. We also provide evidence that the reduced intracellular pH in Cgyps1Δ mutant under acidic conditions is, partly, owing to the diminished activity of a plasma membrane proton pump, CgPma1, an orthologue of a key component of pH homeostasis machinery in Saccharomyces cerevisiae, Pma1. In addition, we have examined C. glabrata's response to low environmental pH via genome-wide expression analysis and several genes required for protein folding/modification and stress response pathways including seven of the CgYPS genes were found to be upregulated. Lastly, we show that C. glabrata responds to acidic environment by reducing total β-glucan levels in the cell wall in a CgYps1-dependent manner.     

Functional Implication of Dp71 in Osmoregulation and Vascular Permeability of the Retina

Functional alterations of Müller cells, the principal glia of the retina, are an early hallmark of most retina diseases and contribute to their further progression. The molecular mechanisms of these reactive Müller cell alterations, resulting in disturbed retinal homeostasis, remain largely unknown. Here we show that experimental detachment of mouse retina induces mislocation of the inwardly rectifying potassium channels (Kir4.1) and a downregulation of the water channel protein (AQP4) in Müller cells. These alterations are associated with a strong decrease of Dp71, a cytoskeleton protein responsible for the localization and the clustering of Kir4.1 and AQP4. Partial (in detached retinas) or total depletion of Dp71 in Müller cells (in Dp71-null mice) impairs the capability of volume regulation of Müller cells under osmotic stress. The abnormal swelling of Müller cells In Dp71-null mice involves the action of inflammatory mediators. Moreover, we investigated whether the alterations in Müller cells of Dp71-null mice may interfere with their regulatory effect on the blood-retina barrier. In the absence of Dp71, the retinal vascular permeability was increased as compared to the controls. Our results reveal that Dp71 is crucially implicated in the maintenance of potassium homeostasis, in transmembraneous water transport, and in the Müller cell-mediated regulation of retinal vascular permeability. Furthermore, our data provide novel insights into the mechanisms of retinal homeostasis provided by Müller cells under normal and pathological conditions.     

The halophilic fungus Hortaea werneckii and the halotolerant fungus Aureobasidium pullulans maintain low intracellular cation concentrations in hypersaline environments

Hortaea werneckii and Aureobasidium pullulans, black yeast-like fungi isolated from hypersaline waters of salterns as their natural ecological niche, have been previously defined as halophilic and halotolerant microorganisms, respectively. In the present study we assessed their growth and determined the intracellular cation concentrations of salt-adapted and non-salt-adapted cells of both species at a wide range of salinities (0 to 25% NaCl and 0 to 20% NaCl, respectively). Although 5% NaCl improved the growth of H. werneckii, even the minimal addition of NaCl to the growth medium slowed down the growth rate of A. pullulans, confirming their halophilic and halotolerant nature. Salt-adapted cells of H. werneckii and A. pullulans kept very low amounts of internal Na+ even when grown at high NaCl concentrations and can be thus considered Na+ excluders, suggesting the existence of efficient mechanisms for the regulation of ion fluxes. Based on our results, we can conclude that these organisms do not use K+ or Na+ for osmoregulation. Comparison of cation fluctuations after a hyperosmotic shock, to which nonadapted cells of both species were exposed, demonstrated better ionic homeostasis regulation of H. werneckii compared to A. pullulans. We observed small fluctuations of cation concentrations after a hyperosmotic shock in nonadapted A. pullulans similar to those in salt-adapted H. werneckii, which additionally confirmed better regulation of ionic homeostasis in the latter. These features can be expected from organisms adapted to survival within a wide range of salinities and to occasional exposure to extremely high NaCl concentrations, both characteristic for their natural environment.     

Non‐enzymatic antioxidant accumulations in BR‐deficient and BR‐insensitive barley mutants under control and drought conditions

Drought is one of the most adverse stresses that affect plant growth and yield. Disturbances in metabolic activity resulting from drought cause overproduction of reactive oxygen species. It is postulated that brassinosteroids (BRs) regulate plant tolerance to the stress conditions, but the underlying mechanisms remain largely unknown. An involvement of endogenous BRs in regulation of the antioxidant homeostasis is not fully clarified either. Therefore, the aim of this study was to elucidate the role of endogenous BRs in regulation of non‐enzymatic antioxidants in barley (Hordeum vulgare) under control and drought conditions. The plant material included the ‘Bowman’ cultivar and a group of semi‐dwarf near‐isogenic lines (NILs), representing mutants deficient in BR biosynthesis or signaling. In general, accumulations of 11 compounds representing various types of non‐enzymatic antioxidants were analyzed under both conditions. The analyses of accumulations of reduced and oxidized forms of ascorbate indicated that the BR mutants contain significantly higher contents of dehydroascorbic acid under drought conditions when compared with the ‘Bowman’ cultivar. The analysis of glutathione accumulation indicated that under the control conditions the BR‐insensitive NILs contained significantly lower concentrations of this antioxidant when compared with the rest of genotypes. Therefore, we postulate that BR sensitivity is required for normal accumulation of glutathione. A complete accumulation profile of various tocopherols indicated that functional BR biosynthesis and signaling are required for their normal accumulation under both conditions. Results of this study provided an insight into the role of endogenous BRs in regulation of the non‐enzymatic antioxidant homeostasis.     

The epithelial potassium channel Kir7.1 is stimulated by progesterone

The choroid plexus (CP) epithelium secretes cerebrospinal fluid and plays an important role in healthy homeostasis of the brain. CP function can be influenced by sex steroid hormones; however, the precise molecular mechanism of such regulation is not well understood. Here, using whole-cell patch-clamp recordings from male and female murine CP cells, we show that application of progesterone resulted in specific and strong potentiation of the inwardly rectifying potassium channel Kir7.1, an essential protein that is expressed in CP and is required for survival. The potentiation was progesterone specific and independent of other known progesterone receptors expressed in CP. This effect was recapitulated with recombinant Kir7.1, as well as with endogenous Kir7.1 expressed in the retinal pigment epithelium. Current-clamp studies further showed a progesterone-induced hyperpolarization of CP cells. Our results provide evidence of a progesterone-driven control of tissues in which Kir7.1 is present.     

Characteristic phenotypes associated with ptsN-null mutants in Escherichia coli K-12 are absent in strains with functional ilvG

The phosphotransferase system (PTS), encompassing EI, HPr, and assorted EII proteins, uses phosphoenolpyruvate to import and phosphorylate sugars. A paralog of EIIA of the sugar PTS system known as ptsN has been purported to regulate organic nitrogen source utilization in Escherichia coli K-12. Its known biochemical function, however, relates to potassium homeostasis. The evidence for regulation of organic nitrogen source utilization by ptsN is based primarily on the defective growth of ΔptsN mutants on amino acid nitrogen sources and other nutrient combinations. These observations were made with E. coli strains MG1655 and W3110, which carry a nonfunctional version of ilvG. There are three isozymes that effectively catalyze the first committed step of branched-chain amino acid biosynthesis, but ilvG is unique for doing so effectively across a range of potassium concentrations. Here we show that all of the nutrient utilization phenotypes attributed to ptsN are manifested selectively in strains lacking functional ilvG. We conclude that the ptsN gene product does not regulate organic nitrogen source utilization as previously proposed.     

Suppression of Sensitivity to Drugs and Antibiotics by High External Cation Concentrations in Fission Yeast

Background

Potassium ion homeostasis plays an important role in regulating membrane potential and therefore resistance to cations, antibiotics and chemotherapeutic agents in Schizosaccharomyces pombe and other yeasts. However, the precise relationship between drug resistance in S. pombe and external potassium concentrations (particularly in its natural habitats) remains unclear. S. pombe can tolerate a wide range of external potassium concentrations which in turn affect plasma membrane polarization. We thus hypothesized that high external potassium concentrations suppress the sensitivity of this yeast to various drugs.

Methods

We have investigated the effect of external KCl concentrations on the sensitivity of S. pombe cells to a wide range of antibiotics, antimicrobial agents and chemotherapeutic drugs. We employed survival assays, immunoblotting and microscopy for these studies.

Results

We demonstrate that KCl, and to a lesser extent NaCl and RbCl can suppress the sensitivity of S. pombe to a wide range of antibiotics. Ammonium chloride and potassium hydrogen sulphate also suppressed drug sensitivity. This effect appears to depend in part on changes to membrane polarization and membrane transport proteins. Interestingly, we have found little relationship between the suppressive effect of KCl on sensitivity and the structure, polarity or solubility of the various compounds investigated.

Conclusions

High concentrations of external potassium and other cations suppress sensitivity to a wide range of drugs in S. pombe. Potassium-rich environments may thus provide S. pombe a competitive advantage in nature. Modulating potassium ion homeostasis may sensitize pathogenic fungi to antifungal agents.     

Molecular mechanisms of apoptosis

Apoptosis (Programmed Cell Death) is a genetically regulated, morphologically distinct form of cell death that can be initiated by many different physiological and pathological stimuli. Such strategic intracellular programming is initiated in many instances during normal life cycle and development in order to maintain the homeostasis of a multicellular organism, to eliminate unwanted cells. However, apoptosis is also involved in a wide range of pathologic conditions, including neurodegenerative and cardiovascular diseases, cancer and autoimmune diseases. Therefore, the ability to understand and manipulate the cell death machinery is an obvious goal of medical research. Here we review the basic components of the death machinery, discuss their interaction in regulation of apoptosis, and describe the main pathways that are used to activate apoptosis.     

A screen for nigericin-resistant yeast mutants revealed genes controlling mitochondrial volume and mitochondrial cation homeostasis

Little is known about the regulation of ion transport across the inner mitochondrial membrane in Saccharomyces cerevisiae. To approach this problem, we devised a screening procedure for facilitating the identification of proteins involved in mitochondrial ion homeostasis. Taking advantage of the growth inhibition of yeast cells by electroneutral K(+)/H(+) ionophore nigericin, we screened for genetic mutations that would render cells tolerant to this drug when grown on a nonfermentable carbon source and identified several candidate genes including MDM31, MDM32, NDI1, YMR088C (VBA1), CSR2, RSA1, YLR024C, and YNL136W (EAF7). Direct examination of intact cells by electron microscopy indicated that mutants lacking MDM31 and/or MDM32 genes contain dramatically enlarged, spherical mitochondria and that these morphological abnormalities can be alleviated by nigericin. Mitochondria isolated from the Deltamdm31 and Deltamdm32 mutants exhibited limited swelling in an isotonic solution of potassium acetate even in the presence of an exogenous K(+)/H(+) antiport. In addition, growth of the mutants was inhibited on ethanol-containing media in the presence of high concentrations of salts (KCl, NaCl, or MgSO(4)) and their mitochondria exhibited two- (Deltamdm31 and Deltamdm32) to threefold (Deltamdm31Deltamdm32) elevation in magnesium content. Taken together, these data indicate that Mdm31p and Mdm32p control mitochondrial morphology through regulation of mitochondrial cation homeostasis and the maintenance of proper matrix osmolarity.     

Electromechanics and Volume Dynamics in Nonexcitable Tissue Cells

Cell volume regulation is fundamentally important in phenomena such as cell growth, proliferation, tissue homeostasis, and embryogenesis. How the cell size is set, maintained, and changed over a cell’s lifetime is not well understood. In this work we focus on how the volume of nonexcitable tissue cells is coupled to the cell membrane electrical potential and the concentrations of membrane-permeable ions in the cell environment. Specifically, we demonstrate that a sudden cell depolarization using the whole-cell patch clamp results in a 50% increase in cell volume, whereas hyperpolarization results in a slight volume decrease. We find that cell volume can be partially controlled by changing the chloride or the sodium/potassium concentrations in the extracellular environment while maintaining a constant external osmotic pressure. Depletion of external chloride leads to a volume decrease in suspended HN31 cells. Introducing cells to a high-potassium solution causes volume increase up to 50%. Cell volume is also influenced by cortical tension: actin depolymerization leads to cell volume increase. We present an electrophysiology model of water dynamics driven by changes in membrane potential and the concentrations of permeable ions in the cells surrounding. The model quantitatively predicts that the cell volume is directly proportional to the intracellular protein content.