Plasma Membrane Microviscosity Research Articles

Unraveling Microviscosity Changes Induced in Cancer Cells by Photodynamic Therapy with Targeted Genetically Encoded Photosensitizer.

Despite the fundamental importance of cell membrane microviscosity, changes in this biophysical parameter of membranes during photodynamic therapy (PDT) have not been fully understood. In this work, changes in the microviscosity of membranes of live HeLa Kyoto tumor cells were studied during PDT with KillerRed, a genetically encoded photosensitizer, in different cellular localizations. Membrane microviscosity was visualized using fluorescence lifetime imaging microscopy (FLIM) with a viscosity-sensitive BODIPY2 rotor. Depending on the localization of the phototoxic protein, different effects on membrane microviscosity were observed. With nuclear localization of KillerRed, a gradual decrease in microviscosity was detected throughout the entire observation period, while for membrane localization of KillerRed, a dramatic increase in microviscosity was observed in the first minutes after PDT, and then a significant decrease at later stages of monitoring. The obtained data on cell monolayers are in good agreement with the data obtained for 3D tumor spheroids. These results indicate the involvement of membrane microviscosity in the response of tumor cells to PDT, which strongly depends on the localization of reactive oxygen species attack via targeting of a genetically encoded photosensitizer.

Influence of Simvastatin and Pravastatin on the Biophysical Properties of Model Lipid Bilayers and Plasma Membranes of Live Cells.

Statins are among the most widely used drugs for the inhibition of cholesterol biosynthesis, prevention of cardiovascular diseases, and treatment of hypercholesterolemia. Additionally, statins also exhibit cholesterol-independent benefits in various diseases, including neuroprotective properties in Alzheimer's disease, anti-inflammatory effects in coronary artery disease, and antiproliferative activities in cancer, which likely result from the statins' interaction and alteration of lipid bilayers. However, the membrane-modulatory effects of statins and the mechanisms by which statins alter lipid bilayers remain poorly understood. In this work, we explore the membrane-modulating effects of statins on model lipid bilayers and live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM) combined with viscosity-sensitive environmental probes, we demonstrate that hydrophobic, but not hydrophilic, statins are capable of changing the microviscosity and lipid order in model and live cell membranes. Furthermore, we show that hydrophobic simvastatin is capable of forming nanoscale cholesterol-rich domains and homogenizing the cholesterol concentrations in lipid bilayers. Our results provide a mechanistic framework for understanding the bimodal effects of simvastatin on the lipid order and the lateral organization of cholesterol in lipid bilayers. Finally, we demonstrate that simvastatin temporarily decreases the microviscosity of live cell plasma membranes, making them more permeable and increasing the level of intracellular chemotherapeutic drug accumulation.

Correlation of Plasma Membrane Microviscosity and Cell Stiffness Revealed via Fluorescence-Lifetime Imaging and Atomic Force Microscopy.

The biophysical properties of cells described at the level of whole cells or their membranes have many consequences for their biological behavior. However, our understanding of the relationships between mechanical parameters at the level of cell (stiffness, viscoelasticity) and at the level of the plasma membrane (fluidity) remains quite limited, especially in the context of pathologies, such as cancer. Here, we investigated the correlations between cells' stiffness and viscoelastic parameters, mainly determined via the actin cortex, and plasma membrane microviscosity, mainly determined via its lipid profile, in cancer cells, as these are the keys to their migratory capacity. The mechanical properties of cells were assessed using atomic force microscopy (AFM). The microviscosity of membranes was visualized using fluorescence-lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY 2. Measurements were performed for five human colorectal cancer cell lines that have different migratory activity (HT29, Caco-2, HCT116, SW 837, and SW 480) and their chemoresistant counterparts. The actin cytoskeleton and the membrane lipid composition were also analyzed to verify the results. The cell stiffness (Young's modulus), measured via AFM, correlated well (Pearson r = 0.93) with membrane microviscosity, measured via FLIM, and both metrics were elevated in more motile cells. The associations between stiffness and microviscosity were preserved upon acquisition of chemoresistance to one of two chemotherapeutic drugs. These data clearly indicate that mechanical parameters, determined by two different cellular structures, are interconnected in cells and play a role in their intrinsic migratory potential.

Effects of Paclitaxel on Plasma Membrane Microviscosity and Lipid Composition in Cancer Cells.

The cell membrane is an important regulator for the cytotoxicity of chemotherapeutic agents. However, the biochemical and biophysical effects that occur in the membrane under the action of chemotherapy drugs are not fully described. In the present study, changes in the microviscosity of membranes of living HeLa-Kyoto tumor cells were studied during chemotherapy with paclitaxel, a widely used antimicrotubule agent. To visualize the microviscosity of the membranes, fluorescence lifetime imaging microscopy (FLIM) with a BODIPY 2 fluorescent molecular rotor was used. The lipid profile of the membranes was assessed using time-of-flight secondary ion mass spectrometry ToF-SIMS. A significant, steady-state decrease in the microviscosity of membranes, both in cell monolayers and in tumor spheroids, was revealed after the treatment. Mass spectrometry showed an increase in the unsaturated fatty acid content in treated cell membranes, which may explain, at least partially, their low microviscosity. These results indicate the involvement of membrane microviscosity in the response of tumor cells to paclitaxel treatment.

Imatinib modulates pro-inflammatory microenvironment with angiostatic effects in experimental lung carcinogenesis.

Lung cancer has second highest rate of incidence and mortality around the world. Smoking cigarettes is the main stream cause of lung carcinogenesis along with other factors such as spontaneous mutations, inactivation of tumor suppressor genes. The present study was aimed to identify the mechanistic role of Imatinib in the chemoprevention of experimental lung carcinogenesis in rat model. Gross morphological observations for tumor formation, histological examinations, RT-PCR, Western blotting, fluorescence spectroscopy and molecular docking studies were performed to elucidate the chemopreventive effects of Imatinib and support our hypothesis by various experiments. It is evident that immuno-compromised microenvironment inside solid tumors is responsible for tumor progression and drug resistance. Therefore, it is inevitable to modulate the pro-inflammatory signaling inside solid tumors to restrict neoangiogenesis. In the present study, we observed that Imatinib could downregulate the inflammatory signaling and also attributed angiostatic effects. Moreover, Imatinib also altered the biophysical properties of BAL cells such as plasma membrane potential, fluidity and microviscosity to restrict their infiltration and thereby accumulation to mount immuno-compromised environment inside the solid tumors during angiogenesis. Our molecular docking studies suggest that immunomodulatory and angiostatic properties of Imatinib could be either independent of each other or just a case of synergistic pleiotropy. Imatinib was observed to activate the intrinsic or mitochondrial pathway of apoptosis to achieve desired effects in cancer cell killings. Interestingly, binding of Imatinib inside the catalytic domain of PARP-1 also suggests that it has caspase-independent properties in promoting cancer cell deaths.

Insulinreceptor interactions and EPR spectroscopy in experimental diabetes mellitus

Insulin concentration in the blood and the level of its binding in tissues inversely correlate in experimental diabetes mellitus. Insulin-binding activity in cells and the microviscosity and hydrophilism of plasma membranes are in inverse correlation. ATP modifies insulin reception.

Microscopic Viscosity of Neuronal Plasma Membranes Measured Using Fluorescent Molecular Rotors: Effects of Oxidative Stress and Neuroprotection.

Molecular mobility in neuronal plasma membranes is a crucial factor in brain function. Microscopic viscosity is an important parameter that determines molecular mobility. This study presents the first direct measurement of the microviscosity of plasma membranes of live neurons. Microviscosity maps were obtained using fluorescence lifetime imaging of environment-sensing dyes termed "molecular rotors". Neurons were investigated both in the basal state and following common neurodegenerative stimuli, excitotoxicity, or oxidative stress. Both types of neurotoxic challenges induced microviscosity decrease in cultured neurons, and oxidant-induced membrane fluidification was counteracted by the wide-spectrum neuroprotectant, the H3 peptide. These results provide new insights into molecular mobility in neuronal membranes, paramount for basic brain function, and suggest that preservation of membrane stability may be an important aspect of neuroprotection in brain insults and neurodegenerative disorders.

The Effect of Nanosized Silicon Molybdate Anions on the Plasma Membrane of Human Fetal Fibroblasts

We used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to study changes in the composition of the plasma membranes of human fetal fibroblasts under the action of nanosized anions of silicon molybdic acid. The dependences of the mass spectra of the main lipids of the plasma membranes on the silicon molybdate concentration were measured and interpreted; the dependences correlate with the layer-by-layer distributions and with the affinity of cholesterol for phospholipids. A new effect for cell biochemistry was discovered, that is, a significant decrease in the relative concentrations of cholesterol and sphingomyelin in plasma membranes under the effect of multiply charged heteropoly anions (HPAs). In aqueous silicon molybdate solutions with a concentration of c ≈ 10 µM/L and an exposure time of 48 h, the amount of cholesterol in plasma membranes decreased by 2–2.5 times, while the amount of sphingomyelin decreased by 20–25%. A new mechanism is proposed for the initial effect of HPA on plasma membranes, which consists of selective etching by multiply charged anions. According to the proposed mechanism, cholesterol and sphingomyelin, the main regulators of permeability and microviscosity of plasma membranes, are extracted from the plasma membrane at the first stage of the interaction of the polyoxometallate anion with the cell. As a consequence of the increased permeability of the plasma membranes in cells, acceleration of vital transmembrane and lateral processes may occur.

Effects of Hypoxia, Hyperbaria and Hyposmosis on Ecto- ATPase Activity in Scorpionfish (Scorpaena porcus L.) Erythrocytes

Effects of hypoxic, hyperbaric and hyposmotic exposures on activity of erythrocyte membrane-bound ecto-ATPase were studied in the scorpionfish Scorpaena porcus L. One-hour autogenic hypoxia evoked a drop while longer hypoxia (12, 24 h)–a rise in the enzyme activity. Hyperbaric exposure, irrespective of its duration, evoked the same stimulation of ecto-ATPase activity in vivo. In vitro, instead, hydrostatic pressure caused a significant drop in the enzyme activity. Hyposmosis stimulated ecto-ATPase activity when the medium was diluted to 50% of its basal level, however, a stronger dilution (70%) led to its inhibition. Under hypoxia, changes in ecto-ATPase activity of scorpionfish erythrocytes are, most likely, due to a shift in the hormonal background and the plasma acid–base equilibrium. The reasons behind the activation of erythrocyte ecto-ATPase in response to hyperbaric pressure are obscure. Under in vitro conditions, the direct effect of hyperbaria and hyposmosis on scorpionfish erythrocytes may be due to alterations in characteristics of plasma membrane microviscosity and in the ecto-ATPase conformational state as manifested in fluctuations of enzyme activity during experiments.

Low cholesterol, impulsivity and violence revisited.

To summarize the recent findings on the association of cholesterol levels with impulsivity and violence. Several authors have recently highlighted the importance of confounding factors that mask the association between cholesterol and impulsivity or violent behaviour. In particular, demographic factors and presence of psychiatric disorders can play a role in the discrepancies present in literature. Men seem to be more sensitive to low cholesterol levels as the association between low cholesterol levels and aggression is found mostly in men. Cholesterol may play a role as a moderator of the serotonergic function and interact with associations between relevant gene variants and impulsivity. Lowering cholesterol levels with statins brings about several changes in the serotonergic system, nerve cell membrane microviscosity and behaviour, and needs to be done with precaution in susceptible individuals. Cholesterol levels could serve as a biological risk marker for violence and suicidal tendencies in psychiatric patients with depression and schizophrenia. Future studies should consider the potential nonlinearity in the association of cholesterol levels with impulsivity, and dependence of the association on gender, psychiatric condition, and environmental factors.

Molecular rotors report on changes in live cell plasma membrane microviscosity upon interaction with beta-amyloid aggregates.

Amyloid deposits of aggregated beta-amyloid Aβ(1-42) peptides are a pathological hallmark of Alzheimer's disease. Aβ(1-42) aggregates are known to induce biophysical alterations in cells, including disruption of plasma membranes. We investigated the microviscosity of plasma membranes upon interaction with oligomeric and fibrillar forms of Aβ(1-42). Viscosity-sensing fluorophores termed molecular rotors were utilised to directly measure the microviscosities of giant plasma membrane vesicles (GPMVs) and plasma membranes of live SH-SY5Y and HeLa cells. The fluorescence lifetimes of membrane-inserting BODIPY-based molecular rotors revealed a decrease in bilayer microviscosity upon incubation with Aβ(1-42) oligomers, while fibrillar Aβ(1-42) did not significantly affect the microviscosity of the bilayer. In addition, we demonstrate that the neuroprotective peptide H3 counteracts the microviscosity change induced by Aβ(1-42) oligomers, suggesting the utility of H3 as a neuroprotective therapeutic agent in neurodegenerative disorders and indicating that ligand-induced membrane stabilisation may be a possible mechanism of neuroprotection during neurodegenerative disorders such as Alzheimer's disease.

Effects of native and oxidized apolipoprotein A-I on lipid bilayer microviscosity of erythrocyte plasma membrane

Using a pyrene as a fluorescent probe, we investigated the influence of native and oxidized apolipoprotein A-I (apo A-I) and their complexes with tetrahydrocortisol (THC) on the microviscosity of the erythrocyte plasma membrane. The addition of THC to isolated membranes led to a 17% increase in the membrane microviscosity. In contrast, native apo A-I reduced the microviscosity (i.e., increased the fluidity) of the membranes by 15%. A more pronounced increase (by 25%) in the membrane fluidity was found in the presence of the complex of apo A-I with THC. Unlike native apo A-I, oxidized apo A-I and its complex with THC did not change the membrane viscosity. In view of the fact that apo A-I plays an important role in the binding of membrane cholesterol we suggest that the observed increase in the membrane fluidity under the influence of the native apo A-I is associated with the cholesterol efflux from plasma membrane. Oxidative modification of apo A-I likely disturbs the mechanisms of the cholesterol efflux and prevents the decrease in the membrane microviscosity.

A Possible Interconnection of Cholesterol Overloading and Phagocytic Activityof the Monocytes in the Prone to Rheumatoid Arthritis Individuals

Background: The abnormalities of lipid metabolism were demonstrated in the individuals, who later develop rheumatoid arthritis (RA). Trivial infections are known to contribute to atherosclerosis. We showed that first degree relatives of RA patients suffer from frequent and lingering trivial infections. The interconnection between the disturbance of lipid metabolism and the increased trivial infection burden is hypothesized in this group. Mononuclear phagocytes (MP) are the important players in RA pathogenesis, antiinfectious defense and atherosclerosis. The aim was to investigate the interconnection between the involvement of MPs in cholesterol metabolism, their phagocytic activity and the burden of trivial infections in the RA patients, their relatives and healthy individuals without autoimmune diseases in family history. Methods: The following parameters were studied: intracellular cholesterol content (colorimetric), membrane cholesterol content and microviscosity (fluorescent), engulfment and digestion (radioisotope); reactive oxygen species generation (chemiluminescence). Results: In relatives MPs and their cell membranes were overloaded with cholesterol; microviscosity of cell membranes and membrane annular lipid regions was increased, cholesterol accumulation in the cells being in strong correlation with the incidence and duration of the trivial infections. In RA group only increased microviscosity of annular lipid regions was revealed. In the patient and relative groups the delayed engulfment and the slowed down time to reach the peak of reactive oxygen species generation after MP stimulation were revealed. Conclusion: We speculate that the abnormalities in monocyte – cholesterol interaction can exacerbate the insufficiency of antiinfectious defense and promote the aggravation of the infectious syndrome, which is known to be a risk factor of RA.

CALCIUM SIGNALING IN TUMOR CELLS

The objective. The objective of this study was to investigate change in calcium maintenance of Ehrlich ascite adenocarcinoma cells in the course of tumor growth. Materials and methods. Cell viability was determined by counting apoptotic and necrotic cells under the fluorescence microscope. Intracellular calcium concentration was determined with the fluorescent probe 2,5 mM Fura-2AM. Relative microviscosity of membranes was estimated by fluorescence method using Pyrene. The activity of Ca-ATPase was assessed by the amount of inorganic phosphate produced during ATP hydrolysis. Results and discussions. We revealed that dynamics of Ehrlich ascite adenocarcinoma growth in our experiments was different from the commonly observed one. We noticed one peculiarity. The quantity of cells decreased on the day 12 after the tumor transplantation. This diminution was caused by mitotic activity lowering and by increase of apoptotic cell quantity the day before. Ehrlich ascite adenocarcinoma growth was accompanied by change in intracellular calcium maintenance which was independent of Ca-ATPase and cell membrane microviscosity. Conclusions. Thus, our experiments have shown the change in calcium concentration. Apparently Ca2+ activated proliferation. However calcium maintenance was settled by ROS. Possibly ROS supression in tumour on the day 12 was able to put down proliferative processes stimulation.

Effects of Ultradry Storage on Fluidity of Plasma Membrane of <em>Haloxylon persicum</em> Seeds

The DPH fluorescent probe (1, 6-diphenyi-1, 3, 5,-hexatriene) was used to study the effects of ultradry seed storage on the fluidity of plasma membrane. Results indicated that the micro-viscosity of plasma membrane of ultradried seeds had no significant changes compared with the Haloxylon persicum seeds which were stored under 4°C condition. However, there was a little adverse effect on the seeds with extreme dehydration. The results were consistent with higher vigor level of ultradried seed. It indicated that ultradry seed storage could maintain the physiological function of seed, protect the integrity of the membrane and improve the storability of seeds.

Dendritic cell activation, phagocytosis and CD69 expression on cognate T cells are suppressed by n‐3 long‐chain polyunsaturated fatty acids

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are bioactive n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in fish oil that exert immunosuppressive effects. A significant amount of literature shows that n-3 LCPUFAs suppress dendritic cell (DC) function in vitro; however, few studies have determined if the effects are emulated at the animal level. In this study, we first focused on the functional consequences of 5% (weight/weight) fish oil on splenic CD11c(+) DCs. Administration of n-3 LCPUFAs, modelling human pharmacological intake (2% of total kcal from EPA,1·3% from DHA), to C57BL/6 mice for 3 weeks reduced DC surface expression of CD80 by 14% and tumour necrosis factor-α secretion by 29% upon lipopolysaccharide stimulation relative to a control diet. The n-3 LCPUFAs also significantly decreased CD11c(+) surface expression and phagocytosis by 12% compared with the control diet. Antigen presentation studies revealed a 22% decrease in CD69 surface expression on transgenic CD4(+) T lymphocytes activated by DCs from mice fed fish oil. We then determined if the functional changes were mechanistically associated with changes in lipid microdomain clustering or plasma membrane microviscosity with n-3 LCPUFAs, as reported for B and T lymphocytes. Fish oil administration to mice did not influence cholera-toxin induced lipid microdomain clustering or microviscosity, even though EPA and DHA levels were significantly elevated relative to the control diet. Overall, our data show that n-3 LCPUFAs exert immunosuppressive effects on DCs, validating in vitro studies. The results also show that DC microdomain clustering and microviscosity were not changed by the n-3 LCPUFA intervention used in this study.

Nanocapsulated curcumin: Oral chemopreventive formulation against diethylnitrosamine induced hepatocellular carcinoma in rat

Toxic outcome of chemical therapeutics as well as multidrug resistance are two serious phenomena for their inacceptance in cancer chemotherapy. Antioxidants like curcumin (Cur) have gained immense importance for their excellent anticarcinogenic activities and minimum toxic manifestations in biological system. However, Cur is lipophilic and thus following oral administration hardly appears in blood indicating its potential therapeutic challenge in cancer therapy. Nanocapsulated Cur has been used as a drug delivery vector to focus the effectiveness of these vesicles against hepatocellular carcinoma. The theme of work was to evaluate effectiveness in oral route of polylactide co-glycolide (PLGA) Nanocapsulated curcumin (Nano Cur) against diethylnitrosamine (DEN) induced hepatocellular carcinoma (HCC) in rat.Nano Cur of average diameter 14nm and encapsulation efficiency of 78% were prepared. Fourier Transform Infra Red (FTIR) analysis revealed that there is no chemical interaction between drug and the polymer. Three i.p. injections of the chemical hepatocarcinogen DEN at 15days interval causes hepatotoxicity, the generation of reactive oxygen species (ROS), lipid peroxidation, decrease in plasma membrane microviscosity and depletion of antioxidant enzyme levels in liver. Nano Cur (weekly oral treatment for 16weeks at 20mg/kg b.wt) in DEN induced HCC rats exerted significant protection against HCC and restored redox homeostasis in liver cells. Nanocapsulated Cur caused cancer cell apoptosis as visualized by ApoBrdU analysis. Histopathological analysis confirmed the pathological improvement in the liver. Nano Cur was found to be a potential formulation in oral route in combating the oxidative damage of hepatic cells and eliminating DEN induced hepatocellular cancer cells in rat whereas identical amount of free Cur treatment was found almost ineffective.

Fenofibrate attenuates contact-stimulated cell motility and gap junctional coupling in DU-145 human prostate cancer cell populations

In the present study, we investigated the effects of fenofibrate on the invasive potential of DU-145 human prostate cancer cells in the context of gap junctional intercellular coupling and the formation of reactive oxygen species. Time-lapse analyses of cell motility, accompanied by tests of cell viability, membrane microviscosity, reactive oxygen species accumulation and the function of gap junctional protein connexin 43 were performed in monolayer cultures of DU-145 cells following fenofibrate administration. Fenofibrate inhibited the motility of DU-145 cells and attenuated gap junctional intercellular coupling in a manner independent of its effects on cell viability, PPARα activation and cell membrane micro-viscosity. Instead, N-acetyl-L-cysteine, a scavenger of reactive oxygen species, restored cell motility and gap junctional coupling in fenofibrate-treated DU-145 cell populations. These data indicate that two parameters crucial for cancer cell metastatic potential, i.e. cell motility and gap junctional coupling, are inhibited by fenofibrate. Thus, fenofibrate affects prostate cancer cell invasion via an orchestrated action on versatile cancer cell properties determining this process. A novel mechanism of anti-invasive activity of fenofibrate, which depends on its interference with cell motility and the function of gap junctions regulated by reactive oxygen species, is suggested.

Quercetin in vesicular delivery systems: Evaluation in combating arsenic-induced acute liver toxicity associated gene expression in rat model

Arsenic, the environmental toxicant causes oxidative damage to liver and produces hepatic fibrosis. The theme of our study was to evaluate the therapeutic efficacy of liposomal and nanocapsulated herbal polyphenolic antioxidant Quercetin (QC) in combating arsenic induced hepatic oxidative stress, fibrosis associated upregulation of its gene expression and plasma TGF ß (transforming growth factor ß) in rat model. A single dose of Arsenic (sodium arsenite-NaAsO 2, 13 mg/kg b.wt) in oral route causes the generation of reactive oxygen species (ROS), arsenic accumulation in liver, hepatotoxicity and decrease in hepatic plasma membrane microviscosity and antioxidant enzyme levels in liver. Arsenic causes fibrosis associated elevation of its gene expression in liver, plasma TGF ß (from normal value 75.2 ± 8.67 ng/ml to 196.2 ± 12.07 ng/ml) and release of cytochrome c in cytoplasm. Among the two vesicular delivery systems formulated with QC, polylactide nanocapsules showed a promising result compared to liposomal delivery system in controlling arsenic induced alteration of those parameters. A single dose of 0.5 ml of nanocapsulated QC suspension (QC 2.71 mg/kg b.wt) when injected to rats 1 h after arsenic administration orally protects liver from arsenic induced deterioration of antioxidant levels as well as oxidative stress associated gene expression of liver. Histopathological examination also confirmed the pathological improvement in liver. Nanocapsulated plant origin flavonoidal compound may be a potent formulation in combating arsenic induced upregulation of gene expression of liver fibrosis through a complete protection against oxidative attack in hepatic cells of rat liver.

Stage-specific changes in membrane microviscosity in Misgurnus fossilis embryos

Natural and probe fluorescence as well as membrane microviscosity was studied in eggs and embryos of Misgurnus fossilis by fluorescence microscopy. The lateral mobility of the probe (pyrene) increased in loach embryos from early to late blastula, which indicates a decrease in plasma membrane microviscosity. At the later stage of mid-gastrula, the microviscosity remained largely invariant. Considering that the embryo exposure to different temperatures changes the quantum yield of fluorescence and the degree of pyrene excimerization, one can gain information about both the temperature-induced structural changes and changes in membrane microviscosity in the embryos. Natural and probe fluorescence of embryonic membranes is proposed as at tool to study morphogenetic mechanisms.