Hypo-osmotic Stress Research Articles

Tolerances to hypo-osmotic and temperature stresses in native and invasive species of Gracilaria (Rhodophyta)

:Physiological responses to the hypo-osmotic and temperature stresses of an invasive species, Gracilaria vermiculophylla, were compared to those of the native Atlantic species, G. tikvahiae and G. cervicornis. For the hypo-osmotic and lower lethal temperature experiment, a Connecticut (CT) strain of the invasive G. vermiculophylla and a Rhode Island (RI) strain of the native G. tikvahiae were cultivated at combinations of five different salinities (S = 5, 15, 20, 25 and 30) and at five different temperatures (5°C, 10°C, 15°C, 20°C and 25°C) for 3 weeks. For the upper lethal temperature experiment, two strains of G. vermiculophylla from CT and Portugal, two strains of G. tikvahiae from CT and RI and one Florida strain of G. cervicornis were cultivated at temperatures ranged from 22°C to 39°C in 2°C or 3°C increments for 14 days. Gracilaria vermiculophylla showed a wide range of temperature (5°C–34°C) and salinity (5 – 30 S) tolerance; whereas, G. tikvahiae cannot withstand harsher environmental stresses, such as prolonged exposures to salinities of ≤ 20 S and temperatures of ≤ 10°C or ≥ 34°C. Gracilaria vermiculophylla also grew faster and had higher survival rates than G. tikvahiae or G. cervicornis. These results suggest that the high tolerance and growth capacity of G. vermiculophylla may be responsible for the successful invasion of this alga into Long Island Sound and elsewhere along the east coast of North America.

Mechanisms of adaption to salinity stress in marine gastropods Littorina saxatilis: a proteomic analysis

Salinity is one of the most important abiotic environmental factors for marine animals. If salinity deviates from optimum, adaptive mechanisms switch on to maintain organism’s physiological activity. In this study the proteome of the marine snails Littorina saxatilis from natural habitats (12, 23 and 32‰ and in response to experimental salinity decreasing (from 20‰ to 10‰) was analyzed. The isolation of all snails inside their shells and gradually declining mortality was observed under an acute experimental salinity decrease. Proteomic changes were evaluated in the survived experimental mollusks compared to control individuals using differential 2D gel-electrophoresis (DIGE) and subsequent LC-MS/MS-identification of proteins. Approximately 10% of analyzed proteins underwent upor down regulation during the experiment. Proteins of folding, antioxidant response, intercellular matrix, and metabolic enzymes were identified among them. Proteomic changes observed in experimental hypoosmotic stress partially reproduced in the proteomes of molluscs that live in conditions of natural freshening (estuaries). Possible mechanisms involved in the adaptation process of L. saxatilis individuals to hypoosmotic stress are discussed.

Profiling of Up-Regulated Genes Response to Acute Hypo-Osmotic Stress in Hepatopancreas and Gill of the Pacific White Shrimps (Litopenaeus vannamei)

<p class="1Body">Suppression subtractive hybridization (SSH) was applied to screen responsively up-regulation genes in hepatopancreas and gill of Litopenaeus vannamei induced by acute hypo-osmotic stress. In the hepatopancreas, 196 clones were randomly selected and sequenced. 131 non-redundant transcripts, corresponding to 41 genes, were found with elevated expressions. They were functionally clustered into eight biological processes which were protein synthesis and processing, carbohydrate metabolism and energy production, transport, cell growth, apoptosis, cytoskeletal component, cell defense and homeostasis, signal transduction, accounting for 26.2%, 14.3%, 16.7%, 4.7%, 4.7%, 2.3%, 11.9% and 7.1%, respectively. When confirmed by real time qRT-PCR, the gene expression levels of MnSOD, glutathione and glutamine synthetase showed significant increases (2.64-folds, 3.44-folds and 2.16-folds, respectively) in hepatopancreas (<em>P</em><0.05). Totally 81 expressed sequence tags (ESTs) were obtained by random sequencing from the gill SSH cDNA library, and 52 unigenes, including 15 contigs and 37 singlets, after CP3 online matching were gained by clustering and assembling. The sequence alignment results revealed that 30 of them had significant homology to previously identified Genbank database sequences, while 22 unigenes did not match any sequence in the datebase and presumably represented unidentified cDNAs. Functionally, the unigenes were classified into six clusters, including ion channels and transport proteins (1); protein synthesis, translation and transcription factor (2); stress resistance and antioxidant factors (3); energy metabolism (4); signal receptor and transduction (5); cell fibrin and cytoskeletal proteins (6) with proportion of 23.3%, 20.0%, 20.0%, 16.7%, 10.0% and 10.0% respectively. Real time qRT-PCR confirmation revealed significant elevation of Arginine kinase, Carbonic anhydrase and NaK-ATPase-α-subunit expressions (3.73-folds, 2.55-folds and 5.83-folds, respectively) in the gill after acute low-osmotic stress (P < 0.05). These results provided insight into critical physiology processes and pathways constituting the hypo-osmotic stress adaption program in hepatopancreas and gill of <em>L. vannamei</em>.</p>

LncRNA-Induced Nucleosome Repositioning Reinforces Transcriptional Repression of rRNA Genes upon Hypotonic Stress.

The activity of rRNA genes (rDNA) is regulated by pathways that target the transcription machinery or alter the epigenetic state of rDNA. Previous work has established that downregulation of rRNA synthesis in quiescent cells is accompanied by upregulation of PAPAS, a long noncoding RNA (lncRNA) that recruits the histone methyltransferase Suv4-20h2 to rDNA, thus triggering trimethylation of H4K20 (H4K20me3) and chromatin compaction. Here, we show that upregulation of PAPAS in response to hypoosmotic stress does not increase H4K20me3 because of Nedd4-dependent ubiquitinylation and proteasomal degradation of Suv4-20h2. Loss of Suv4-20h2 enables PAPAS to interact with CHD4, a subunit of the chromatin remodeling complex NuRD, which shifts the promoter-bound nucleosome into the transcriptional "off" position. Thus, PAPAS exerts a "stress-tailored" dual function in rDNA silencing, facilitating either Suv4-20h2-dependent chromatin compaction or NuRD-dependent changes in nucleosome positioning.

The TRPV4 Ion Channel Alters Intracellular Calcium Transients in Cardiomyocytes of Aged Mice

Cardiac excitation-contraction coupling (ECC) occurs via calcium-induced calcium release and is therefore critically dependent on precise regulation of intracellular calcium homeostasis. Cardiomyocytes of the aged heart exhibit alterations in calcium homeostasis which predisposes the heart to calcium overload, contractile dysfunction, and arrhythmia. In this investigation we tested the hypothesis that the transient receptor potential vanilloid member 4 (TRPV4) osmotically-sensitive cation channel is expressed in cardiomyocytes of the aged mouse heart and alters intracellular calcium transients. Western blot analysis of whole heart homogenates of Young (3-6 month) and Aged (24-26 month) C57BL/6 male mice revealed increased expression of TRPV4 protein in hearts of Aged. Enzymatically isolated left-ventricular cardiomyocytes were loaded with the calcium indicator dye fluo-4/AM and electrically stimulated at 0.5 Hz to induce intracellular calcium transients. Pharmacological activation of TRPV4 with the agonist RN1747 (100 nM) produced an increase in calcium transient amplitude in cardiomyocytes of Aged (F/F0:3.3±0.4 RN1747 versus 2.6±0.4 control, P<0.05), but not Young (F/F0:2.8±0.3 RN1747 versus 2.9±0.2 control) mice. Following hypo-osmotic stress (250 mOsm, 40 minutes), cardiomyocytes of Aged exhibited enhanced responsiveness to RN1747 (F/F0:4.3±0.6 RN1747 versus 3.5±0.4 control, P<0.05) which associated with altered transient decay kinetics and cellular calcium overload (7/8 cells); these effects were prevented by the TRPV4 antagonist HC067047 (1 uM; F/F0:2.9±0.3 RN1747+HC versus 3.0±0.2 HC, 0/5 cells). In conclusion, the TRPV4 ion channel is expressed in cardiomyocytes of the aged heart and alters calcium transients during ECC.

Molecular cloning and expression of the calreticulin gene of the Pacific white shrimp, Litopenaeus vannamei, in response to acute hypo-osmotic stress

Calreticulin is a ubiquitous and highly conserved calcium-binding protein that is located in the endoplasmic reticulum (ER). In the present study, the cDNA of the Litopenaeus vannamei calreticulin (LvCRT) gene was cloned using the rapid amplification of cDNA ends (RACE) technique. Its full-length cDNA was 1866bp in length, which included a 1221-bp open reading frame and encoded a 406-amino acid. In addition, a 222-bp 5′ untranslated region (UTR) and a 423-bp 3′ UTR with a polyadenylation [poly(A)] signal (AATAAA) located 27bp upstream of the poly(A) tail were identified. The predicted molecular weight and isoelectric point of the protein were 45.3kDa and 4.90, respectively. Protein analysis revealed that LvCRT contains a conserved calreticulin family signature sequence (KHEQNIDCGGGYLKVF), a signal peptide (MKTWVFLALFGVVLVES), and a conserved endoplasmic reticulum HDEL retention sequence. Furthermore, NCBI BLASTX and phylogenetic analyses showed that LvCRT has a high degree of sequence homology with CRT proteins from Fenneropenaeus chinensis and Penaeus monodon. In addition, qRT-PCR analysis indicated that the highest level of LvCRT expression was observed in the intestines, whereas the lowest was in the muscle. When the shrimps were transferred from salinity 30 to 2psu diluted seawater, the expression level of LvCRT in the hepatopancreas and hemocytes sharply increased at 6h and 12h (which were about 3.23-fold and 2.01-fold of that in the 0h control group), and then, significantly decreased at 96h and 24h (p<0.05). The expression of LvCRT in the intestines and gills was not affected by the low-salinity stress (p>0.05). In conclusion, the responses of L. vannamei to changes in salinity via the calreticulin protein facilitate a better understanding of its function in relation to stress.

Synergism of Water Shock and a Biocompatible Block Copolymer Potentiates the Antibacterial Activity of Graphene Oxide.

Graphene oxide (GO) is promising in the fight against pathogenic bacteria. However, the antibacterial activity of pristine GO is relatively low and concern over human cytotoxicity further limits its potential. This study demonstrates a general approach to address both issues. The developed approach synergistically combines the water shock treatment (i.e., a sudden decrease in environmental salinity) and the use of a biocompatible block copolymer (Pluronic F-127) as a synergist co-agent. Hypoosmotic stress induced by water shock makes gram-negative pathogens more susceptible to GO. Pluronic forms highly stable nanoassemblies with GO (Pluronic-GO) that can populate around bacterial envelopes favoring the interactions between GO and bacteria. The antibacterial activity of GO at a low concentration (50 μg mL(-1) ) increases from <30% to virtually complete killing (>99%) when complemented with water shock and Pluronic (5 mg mL(-1) ) at ≈2-2.5 h of exposure. Results suggest that the enhanced dispersion of GO and the osmotic pressure generated on bacterial envelopes by polymers together potentiate GO. Pluronic also significantly suppresses the toxicity of GO toward human fibroblast cells. Fundamentally, the results highlight the crucial role of physicochemical milieu in the antibacterial activity of GO. The demonstrated strategy has potentials for daily-life bacterial disinfection applications, as hypotonic Pluronic-GO mixture is both safe and effective.

Editorial: Single membrane channels formed by connexins or pannexins: focus on the nervous system

Editorial: Single membrane channels formed by connexins or pannexins: focus on the nervous system

ATP release, generation and hydrolysis in exocrine pancreatic duct cells.

Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide-inactivating and nucleotide-phosphorylating ecto-enzymes. We suggest that extracellular ATP homeostasis in pancreatic ducts may be important in pancreas physiology and potentially in pancreas pathophysiology.

Effect of sulfide, osmotic, and thermal stresses on taurine transporter mRNA levels in the gills of the hydrothermal vent-specific mussel Bathymodiolus septemdierum

Hydrothermal vent environmental conditions are characterized by high sulfide concentrations, fluctuating osmolality, and irregular temperature elevations caused by vent effluents. These parameters represent potential stressors for organisms that inhabit the area around hydrothermal vents. Here, we aimed to obtain a better understanding of the adaptation mechanisms of marine species to hydrothermal vent environments. Specifically, we examined the effect of sulfide, osmolality, and thermal stress on the expression of taurine transporter (TAUT) mRNA in the gill of the deep-sea mussel Bathymodiolus septemdierum, which is a dominant species around hydrothermal vent sites. We analyzed TAUT mRNA levels by quantitative real-time polymerase chain reaction (PCR) in the gill of mussels exposed to sulfide (0.1 or 1mg/L Na2S·9H2O), hyper- (115% seawater) and hypo- (97.5%, 95.5%, and 85% seawater) osmotic conditions, and thermal stresses (12°C and 20°C) for 24 and 48h. The results showed that mussels exposed to relatively low levels of sulfide (0.1mg/L) and moderate heat stress (12°C) exhibited higher TAUT mRNA levels than the control. Although hyper- and hypo-osmotic stress did not significantly change TAUT mRNA levels, slight induction was observed in mussels exposed to low osmolality. Our results indicate that TAUT is involved in the coping mechanism of mussels to various hydrothermal vent stresses.

Basal cells express functional TRPV4 channels in the mouse nasal epithelium

Basal cells in the nasal epithelium (olfactory and airway epithelia) are stem/progenitor cells that are capable of dividing, renewing and differentiating into specialized cells. These stem cells can sense their biophysical microenvironment, but the underlying mechanism of this process remains unknown. Here, we demonstrate the prominent expression of the transient receptor potential vanilloid type 4 (TRPV4) channel, a Ca2+-permeable channel that is known to act as a sensor for hypo-osmotic and mechanical stresses, in the basal cells of the mouse nasal epithelium. TRPV4 mRNA was expressed in the basal portions of the prenatal mouse nasal epithelium, and this expression continued into adult mice. The TRPV4 protein was also detected in the basal layers of the nasal epithelium in wild-type but not in TRPV4-knockout (TRPV4-KO) mice. The TRPV4-positive immunoreactions largely overlapped with those of keratin 14 (K14), a marker of basal cells, in the airway epithelium, and they partially overlapped with those of K14 in the olfactory epithelium. Ca2+ imaging analysis revealed that hypo-osmotic stimulation and 4α-phorbol 12,13 didecanoate (4α-PDD), both of which are TRPV4 agonists, caused an increase in the cytosolic Ca2+ concentration in a subset of primary epithelial cells cultured from the upper parts of the nasal epithelium of the wild-type mice. This response was barely noticeable in cells from similar parts of the epithelium in TRPV4-KO mice. Finally, there was no significant difference in BrdU-labeled proliferation between the olfactory epithelia of wild-type and TRPV4-KO mice under normal conditions. Thus, TRPV4 channels are functionally expressed in basal cells throughout the nasal epithelium and may act as sensors for the development and injury-induced regeneration of basal stem cells.

Osmotic Stress Reduces Ca2+ Signals through Deformation of Caveolae

Caveolae are membrane invaginations that can sequester various signaling proteins. Caveolae have been shown to provide mechanical strength to cells by flattening to accommodate increased volume when cells are subjected to hypo-osmotic stress. We have previously found that caveolin, the main structural component of caveolae, specifically binds Gαq and stabilizes its activation state resulting in an enhanced Ca(2+) signal upon activation. Here, we show that osmotic stress caused by decreasing the osmolarity in half reversibly changes the configuration of caveolae without releasing a significant portion of caveolin molecules. This change in configuration due to flattening leads to a loss in Cav1-Gαq association. This loss in Gαq/Cav1 association due to osmotic stress results in a significant reduction of Gαq/phospholipase Cβ-mediated Ca(2+) signals. This reduced Ca(2+) response is also seen when caveolae are reduced by treatment with siRNA(Cav1) or by dissolving them by methyl-β-cyclodextran. No change in Ca(2+) release with osmotic swelling can be seen when growth factor pathways are activated. Taken together, these results connect the mechanical deformation of caveolae to Gαq-mediated Ca(2+) signals.

C60 fullerene affects elastic properties and osmoregulation reactions of human lymphocytes.

The influence of aqueous solution of pristine C60 fullerene (C60FAS) on functional activity of lymphocytes from a healthy person was studied for the first time. By means of atomic force microscopy, it was found that C60FAS in a concentration of 0.1 mg/ml increases the stiffness of the lymphocyte membrane by 41% (p < 0.05) and lowers the functional activity of the plasmalemma surface, thereby constraining the use of its membrane material in physiological reactions using a hypotonic model in vitro. However, a cell retains the ability to regulate its volume and demonstrates relative resistance to hypo-osmotic stress. The resistance of lymphocytes in hypo-osmotic medium is facilitated by activation of the nucleus by C60 fullerene particles, which regulates the implementation of two consistent phases of an increase and decrease of cell volume, thereby retaining cell viability. All these indicate the impact of C60 fullerene on the cellular nucleus.

Chemical analysis of Karenia papilionacea.

One of the most widely studied organisms responsible for Harmful Algal Blooms (HABs) is the marine dinoflagellate Karenia brevis. This organism produces neurotoxic compounds known as brevetoxins. A related dinoflagellate, Karenia papilionacea, has been reported to occasionally co-bloom with K. brevis but has received little attention as a possible toxin producing species. Therefore, our aim was to investigate the toxin profile for K.papilionacea. A toxic fraction was identified using a cell based cytotoxicity assay and the toxin was isolated and identified as the ladder frame polyether brevetoxin-2 (PbTx-2) using mass spectrometry (MS) and nuclear magnetic resonance (NMR). Toxin production in K.papilionacea increased in response to hypoosmotic stress, as previously observed in K.brevis.

The ClC-7 Chloride Channel Is Downregulated by Hypoosmotic Stress in Human Chondrocytes.

Articular chondrocytes in osteoarthritis (OA) patients are exposed to hypoosmotic stress because the osmolality of this synovial fluid is significantly decreased. Hypoosmotic stress can cause an efflux of Cl(-) and an associated decrease of cell volume. We have previously reported that a Cl(-) conductance contributes to the regulation of resting membrane potential and thus can alter intracellular Ca(2+) concentration ([Ca(2+)]i) in human chondrocytes. The molecular identity and pathologic function of these Cl(-) channels, however, remained to be determined. Here, we show that the ClC-7 Cl(-) channel is strongly expressed in a human chondrocyte cell line (OUMS-27) and that it is responsible for Cl(-) currents that are activated by extracellular acidification (pH 5.0). These acid-sensitive currents are inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; IC50 = 13 μM) and are markedly reduced by small-interfering RNA-induced knockdown of ClC-7. DIDS hyperpolarized these chondrocytes, and this was followed by an increase in [Ca(2+)]i. ClC-7 knockdown caused a similar hyperpolarization of the membrane potential. Short-term culture (48 hours) in hypoosmotic medium (270 mOsm) reduced the expression of ClC-7 and decreased the acid-sensitive currents. Interestingly, these hypoosmotic culture conditions, or ClC-7 knockdown, resulted in enhanced cell death. Taken together, our results show that the significant hyperpolarization due to ClC-7 impairment in chondrocytes can significantly increase [Ca(2+)]i and cell death. Thus, downregulation of ClC-7 channels during the hypoosmotic stress that accompanies OA progression is one important concept of the complex etiology of OA. These findings suggest novel targets for therapeutic intervention(s) and drug development for OA.

Plasma membrane mechanical stress activates TRPC5 channels.

Mechanical forces exerted on cells impose stress on the plasma membrane. Cells sense this stress and elicit a mechanoelectric transduction cascade that initiates compensatory mechanisms. Mechanosensitive ion channels in the plasma membrane are responsible for transducing the mechanical signals to electrical signals. However, the mechanisms underlying channel activation in response to mechanical stress remain incompletely understood. Transient Receptor Potential (TRP) channels serve essential functions in several sensory modalities. These channels can also participate in mechanotransduction by either being autonomously sensitive to mechanical perturbation or by coupling to other mechanosensory components of the cell. Here, we investigated the response of a TRP family member, TRPC5, to mechanical stress. Hypoosmolarity triggers Ca2+ influx and cationic conductance through TRPC5. Importantly, for the first time we were able to record the stretch-activated TRPC5 current at single-channel level. The activation threshold for TRPC5 was found to be 240 mOsm for hypoosmotic stress and between −20 and −40 mmHg for pressure applied to membrane patch. In addition, we found that disruption of actin filaments suppresses TRPC5 response to hypoosmotic stress and patch pipette pressure, but does not prevent the activation of TRPC5 by stretch-independent mechanisms, indicating that actin cytoskeleton is an essential transduction component that confers mechanosensitivity to TRPC5. In summary, our findings establish that TRPC5 can be activated at the single-channel level when mechanical stress on the cell reaches a certain threshold.

Effects of salinity changes on coastal Antarctic phytoplankton physiology and assemblage composition

A natural marine phytoplankton assemblage from a coastal environment of Antarctica was experimentally exposed to low salinity sea water (30 vs 34 in the control) during 8days in order to study their physiological and community responses to hypoosmotic stress conditions. Hypoosmotic conditions favour water influx into the cells, which results in increased turgor pressure and increased oxidative stress. This stress is linked to a number of other cellular toxic processes, including damages to proteins, enzyme inactivation and DNA breakage. Inhibition of the instantaneous growth rate started after 48h exposure to low salinity, but at the end of experiment, growth was significantly higher in the low than in the normal (control) salinity treatment. Hypoosmotic conditions prevented phytoplankton biomass accumulation, as evidenced by reduced Chlorophyll-a concentrations as compared to the control treatment. However, in terms of cell numbers and species composition, we observed a gradual replacement of big centric by small pennate diatoms, which became dominant by the end of the experiment. In addition, the content of reactive oxygen species (ROS) and 2-thiobarbituric acid-reactive substances (TBARS), which are indicative of oxidative stress, were studied. In the low salinity treatments, ROS concentrations were significantly higher than control values on days 4 and 6, decreasing thereafter to nearly initial values. TBARS content increased during the first 48h and then decreased until around day 0 values. This coincided with significant increased values of the antioxidants α-tocopherol and β-carotene in low salinity treatments over the control. These results suggest the existence of protection mechanisms against lipid peroxidation, and lead to the conclusion that the response to stress is species-specific, so that at the community level a change in the relative abundance of phytoplankton taxa appears as a response to hypoosmotic conditions. This could have important consequences for the trophic food web dynamics in areas influenced by high fresh water inputs.

Eisosomes provide membrane reservoirs for rapid expansion of the yeast plasma membrane.

Cell surface area rapidly increases during mechanical and hypoosmotic stresses. Such expansion of the plasma membrane requires 'membrane reservoirs' that provide surface area and buffer membrane tension, but the sources of this membrane remain poorly understood. In principle, the flattening of invaginations and buds within the plasma membrane could provide this additional surface area, as recently shown for caveolae in animal cells. Here, we used microfluidics to study the rapid expansion of the yeast plasma membrane in protoplasts, which lack the rigid cell wall. To survive hypoosmotic stress, yeast cell protoplasts required eisosomes, protein-based structures that generate long invaginations at the plasma membrane. Both budding yeast and fission yeast protoplasts lacking eisosomes were unable to expand like wild-type protoplasts during hypoosmotic stress, and subsequently lysed. By performing quantitative fluorescence microscopy on single protoplasts, we also found that eisosomes disassembled as surface area increased. During this process, invaginations generated by eisosomes at the plasma membrane became flattened, as visualized by scanning electron microscopy. We propose that eisosomes serve as tension-dependent membrane reservoirs for expansion of yeast cells in an analogous manner to caveolae in animal cells.

Altered Astrocytic Swelling in the Cortex of α-Syntrophin-Negative GFAP/EGFP Mice

Brain edema accompanying ischemic or traumatic brain injuries, originates from a disruption of ionic/neurotransmitter homeostasis that leads to accumulation of K+ and glutamate in the extracellular space. Their increased uptake, predominantly provided by astrocytes, is associated with water influx via aquaporin-4 (AQP4). As the removal of perivascular AQP4 via the deletion of α-syntrophin was shown to delay edema formation and K+ clearance, we aimed to elucidate the impact of α-syntrophin knockout on volume changes in individual astrocytes in situ evoked by pathological stimuli using three dimensional confocal morphometry and changes in the extracellular space volume fraction (α) in situ and in vivo in the mouse cortex employing the real-time iontophoretic method. RT-qPCR profiling was used to reveal possible differences in the expression of ion channels/transporters that participate in maintaining ionic/neurotransmitter homeostasis. To visualize individual astrocytes in mice lacking α-syntrophin we crossbred GFAP/EGFP mice, in which the astrocytes are labeled by the enhanced green fluorescent protein under the human glial fibrillary acidic protein promoter, with α-syntrophin knockout mice. Three-dimensional confocal morphometry revealed that α-syntrophin deletion results in significantly smaller astrocyte swelling when induced by severe hypoosmotic stress, oxygen glucose deprivation (OGD) or 50 mM K+. As for the mild stimuli, such as mild hypoosmotic or hyperosmotic stress or 10 mM K+, α-syntrophin deletion had no effect on astrocyte swelling. Similarly, evaluation of relative α changes showed a significantly smaller decrease in α-syntrophin knockout mice only during severe pathological conditions, but not during mild stimuli. In summary, the deletion of α-syntrophin markedly alters astrocyte swelling during severe hypoosmotic stress, OGD or high K+.

The rate of hypo-osmotic challenge influences regulatory volume decrease (RVD) and mechanical properties of articular chondrocytes

Osteoarthritis (OA) is associated with a gradual reduction in the interstitial osmotic pressure within articular cartilage. The aim of this study was to compare the effects of sudden and gradual hypo-osmotic challenge on chondrocyte morphology and biomechanics. Bovine articular chondrocytes were exposed to a reduction in extracellular osmolality from 327 to 153 mOsmol/kg applied either suddenly (<5 s) or gradually (over 180 min). Temporal changes in cell diameter and the existence of regulatory volume decrease (RVD) were quantified along with changes in cortical actin and chromatin condensation. The cellular viscoelastic mechanical properties were determined by micropipette aspiration. In response to a sudden hypo-osmotic stress, 66% of chondrocytes exhibited an increase in diameter followed by RVD, whilst 25% showed no RVD. By contrast, cells exposed to gradual hypo-osmotic stress exhibited reduced cell swelling without subsequent RVD. There was an increase in the equilibrium modulus for cells exposed to sudden hypo-osmotic stress. However, gradual hypo-osmotic challenge had no effect on cell mechanical properties. This cell stiffening response to sudden hypo-osmotic challenge was abolished when actin organization was disrupted with cytochalasin D or RVD inhibited with REV5901. Both sudden and gradual hypo-osmotic challenge reduced cortical F-actin distribution and caused chromatin decondensation. Sudden hypo-osmotic challenge increases chondrocyte mechanics by activation of RVD and interaction with the actin cytoskeleton. Moreover, the rate of hypo-osmotic challenge is shown to have a profound effect on chondrocyte morphology and biomechanics. This important phenomenon needs to be considered when studying the response of chondrocytes to pathological hypo-osmotic stress.