Decrease In Plasma Membrane Fluidity Research Articles

Cells immersed in collagen matrices show a decrease in plasma membrane fluidity as the matrix stiffness increases

Cells are constantly adapting to maintain their identity in response to the surrounding media's temporal and spatial heterogeneity. The plasma membrane, which participates in the transduction of external signals, plays a crucial role in this adaptation. Studies suggest that nano and micrometer areas with different fluidities at the plasma membrane change their distribution in response to external mechanical signals. However, investigations linking fluidity domains with mechanical stimuli, specifically matrix stiffness, are still in progress. This report tests the hypothesis that the stiffness of the extracellular matrix can modify the equilibrium of areas with different order in the plasma membrane, resulting in changes in overall membrane fluidity distribution. We studied the effect of matrix stiffness on the distribution of membrane lipid domains in NIH-3 T3 cells immersed in matrices of varying concentrations of collagen type I, for 24 or 72 h. The stiffness and viscoelastic properties of the collagen matrices were characterized by rheometry, fiber sizes were measured by Scanning Electron Microscopy (SEM) and the volume occupied by the fibers by second harmonic generation imaging (SHG). Membrane fluidity was measured using the fluorescent dye LAURDAN and spectral phasor analysis. The results demonstrate that an increase in collagen stiffness alters the distribution of membrane fluidity, leading to an increasing amount of the LAURDAN fraction with a high degree of packing. These findings suggest that changes in the equilibrium of fluidity domains could represent a versatile and refined component of the signal transduction mechanism for cells to respond to the highly heterogeneous matrix structural composition. Overall, this study sheds light on the importance of the plasma membrane's role in adapting to the extracellular matrix's mechanical cues.

Modulation of Cholesterol Pathways in Human Macrophages Infected by Clinical Isolates of Leishmania infantum.

To increase our understanding of factors contributing to therapeutic failure (TF) in leishmaniasis, we have studied some plasma membrane features of host THP-1 cells infected with clinical isolates of Leishmania infantum from patients with leishmaniasis and TF. The fluorescent probes DPH and TMA-DPH were used to measure changes in membrane fluidity at various depths of the plasma membranes. Steady-state fluorescence anisotropy of DPH embedded in the infected THP-1 membranes showed a significant increase, thereby suggesting a substantial decrease in plasma membrane fluidity relative to controls. Considering that cholesterol affects membrane fluidity and permeability, we determined the cholesterol content in plasma membrane fractions of human macrophages infected with these L. infantum lines and observed a significant increase in cholesterol content that correlates with the measured decrease in plasma membrane fluidity. In order to define the pathways that could explain the increase in cholesterol content, we studied the transcriptomics of the cholesterol-enriched pathways in host THP-1 cells infected with TF clinical isolates by RNA-seq. Specifically, we focused on four enriched Gene Ontology (GO) terms namely cholesterol efflux, cholesterol transport, cholesterol metabolic process and cholesterol storage. Additionally, we analyzed the genes involved in these pathways. Overall, this study shows that these clinical isolates are able to modulate the expression of specific genes in host cells, thereby modifying the cholesterol content in plasma membranes and inducing changes in plasma membrane fluidity that could be associated with the parasite’s ability to survive in the host macrophages, thereby possibly contributing to immune evasion and TF.

Naturally Occurring Antifungal Agents against Zygosaccharomyces bailii and Their Synergism

Polygodial was found to exhibit a fungicidal activity against a food spoilage yeast, Zygosaccharomyces bailii, with the minimum fungicidal concentration (MFC) of 50 microg/mL (0.17 mM). The time-kill curve study showed that polygodial was fungicidal at any growth stage. The primary action of polygodial comes from its ability to disrupt the native membrane-associated function of integral proteins as nonionic surface active agents (surfactants) followed by a decrease in plasma membrane fluidity. The fungicidal activity of polygodial was increased 128-fold in combination with a sublethal amount (equivalent of 1/2 MFC) of anethole and vice versa relative to the fungicidal activity of anethole. The fungicidal activity of sorbic acid was enhanced 512-fold in combination with 1/2 MFC of polygodial. Conversely, the fungicidal activity of polygodial was enhanced 128-fold in combination with 1/2 MFC of sorbic acid.

Tolerance to NaCl induces changes in plasma membrane lipid composition, fluidity and H+-ATPase activity of tomato calli.

Two tomato (Lycopersicon esculentum Mill. cv. Pera) callus lines tolerant to NaCl were obtained by successive subcultures of NaCl-sensitive calli in 50 and 100 mM NaCl-supplemented medium. Growth and ion content, as well as plasma membrane lipid composition, fluidity and H+-ATPase (EC 3.6.1.35) activity, were studied in both NaCl-sensitive and NaCl-tolerant calli. Although calli tolerant to 100 mM NaCl exhibited a reduced growth relative to calli sensitive to NaCl or tolerant to 50 mM NaCl, growth of calli tolerant to 100 mM NaCl was higher than that of NaCl-sensitive calli grown for one subculture in 100 mM NaCl. Growth in the presence of 100 mM NaCl provoked an increase of Na+ and Cl- content, but no significant changes in K+ and Ca2+. As compared with NaCl-sensitive and 50 mM NaCl-tolerant calli, plasma membrane vesicles isolated from calli tolerant to 100 mM NaCl exhibited a higher phospholipid and sterol content as well as a lower phospholipid/free sterol ratio and a lower double bond index (DBI) of phospholipid fatty acids. The changes in plasma membrane lipid composition were correlated with a decrease of plasma membrane fluidity in calli tolerant to 100 mM NaCl, as indicated by fluorimetric studies using diphenylhexatriene (DPH) as probe. Plasma membrane-enriched vesicles isolated from calli tolerant to 100 mM NaCl showed lower ATP hydrolysis and ATP-dependent H+-pumping activities, as well as a lower passive permeability to H+ than plasma membrane from NaCl-sensitive and 50 mM NaCl-tolerant calli. The involvement of the changes in plasma membrane lipid content and composition, fluidity and H+-ATPase activity in salt tolerance of tomato calli is discussed.

Influence of nifedipine on plasma membrane fluidity and oxidative burst of polymorphonuclear leucocytes.

It has been demonstrated that the calcium antagonist nifedipine inhibits the reactive oxygen species (ROS) production by polymorphonuclear leucocytes (PMNLs) activated with phorbol myristate acetate (PMA), but the mechanism underlying this effect is still unknown. In the present study we investigated the influence of nifedipine on the PMNL plasma membrane using 1-(4-trimethylaminophenyl)-6-phenyl-1,3,5,hexatriene (TMA-DPH) fluorescence polarization (P) and on PMA- and N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced ROS production, measured by luminol-dependent chemiluminescence (CL). The plasma membrane fluidity of untreated PMNLs, expressed as P, was 0.371 +/- 0.008. After preincubation of 15 min, nifedipine induced a significant change in P values only at a concentration of 10(-4) M (P = 0.00018). After preincubation of 60 min significant changes in P values were also observed at concentrations of 10(-6) M (P = 0.023) and 10(-7) M (P = 0.023). PMA-induced ROS production by PMNLs was markedly inhibited by nifedipine. Nifedipine also determined a striking change in the FMLP-induced CL response, characterized by both an overall inhibition of PMNL activity and a modification of the kinetics of the oxidative burst (rapid increase in ROS production followed by a pronounced drop in the PMNL response). Such a pattern was found at concentrations of 10(-4) M (preincubation time: 15 min), 10(-6) M and 10(-7) M (preincubation time: 60 min). These findings indicate that nifedipine directly interacts with the PMNLs by inducing a marked decrease in plasma membrane fluidity and an inhibition of the oxidative burst.

Cyclic AMP binding to the amphibian oocyte plasma membrane: possible interrealtionship between meiotic arrest and membrane fluidity

Cyclic AMP, which maintains the vertebrate oocyte in prophase arrest under physiological conditions, exhibits specific and saturable binding to the cytoplasmic face of the prophase-arrested Rana pipiens oocyte membrane. Scatchard type analyses of [ 3H]cAMP binding to isolated plasma membranes indicate a single class of binding sites with a K d = 19.3 ± 7.0 nM at cAMP concentrations below 10 −6 M and additional low affinity site(s) and/or non-specific binding at concentrations above 10 −6 M. Photoaffinity labeling of prophase oocyte plasma membranes with [ 32P]-8-N 3cAMP demonstrates cAMP/cGMP-displacable binding of 8-N 3[ 32P]cAMP to a 100–110 kDa peptide doublet. Plasma membrane fluidity was monitored by electron spin resonance in isolated plasma-vitelline membranes using a 5-doxyl stearic acid probe. Exogenous dibutryl cAMP (dbcAMP) produces an increase in membrane fluidity within minutes and blocks and/or reverses the progesterone-induced decrease in plasma membrane fluidity. The dbcAMP concentration that produced half-maximal fluidity increase (10 μM) corresponds to the half-maximal inhibiting dose of dbcAMP for progesterone induction of meiosis. Cholera toxin, which elevates intracellular cAMP and blocks meiosis, also increases membrane fluidity and inhibits progesterone-induced decrease in membrane fluidity. Elevated levels of intracellular cAMP thus appear to maintain meiotic arrest by binding to specific plasma membrane site(s) and maintaining the plasma membrane in a relatively fluid state. The progesterone-induced fall in intracellular cAMP first reported in Rana thus appears to be responsible for the progesterone-induced increase in membrane fluidity and further suggests that the change in membrane order is essential for the resumption of the meiotic divisions.

Changes in superoxide radical formation, lipid peroxidation, membrane fluidity and cathepsin B activity in aging and spawning male chinook salmon ( Oncorhynchus tschawytscha)

Superoxide radical (SOR) formation in the brain and the liver of male Chinook salmon ( Oncorhynchus tschawytscha) increased in mitochondrial and plasma membrane samples as they aged. In 2-year-old salmon, spawning also lead to a significant elevation in SOR formation in mitochondrial and plasma membrane samples. The rise in this free radical was associated with an increase in lipid peroxidation, a decrease in plasma membrane fluidity, and an elevation in cathepsin B activity in the brain and liver. In 2-year-old spawning salmon, the changes in these parameters was greater than in 2-year-old non-spawning salmon. These observations suggestion that free radical levels increase with aging and during spawning and indicate that these changes may be involved in cellular degeneration. In addition, these results support the suggest that cellular degeneration accelerates during the spawning process.

Modifications of ependymal cells membranes by galactocerebrosides in cell culture

In this paper we have demonstrated that treatment of ependymal cells in culture by galactocerebrosides induced a decrease in plasma membrane fluidity and an increase of EGF binding sites. We have shown in a previous work that galactocerebroside in vitro and in vivo caused an important morphological change in ependymal cells that grew into an astrocytic shape after a five day treatment. We discuss the hypothesis that the first event in morphological effect could be a modification of plasma membrane followed by important changes in molecules distribution.

Confluence-dependent resistance in human colon cancer cells: role of reduced drug accumulation and low intrinsic chemosensitivity of resting cells.

In vitro sensitivity of HT29 human colon cancer cells to doxorubicin (DXR), vincristine (VCR), etoposide (VP16), cisplatin (CDDP), melphalan (L-PAM) and 5-fluorouracil (5FU) was markedly reduced when cell-culture density increased. For some drugs, confluence-dependent resistance (CDR) was partly due to decreased intracellular drug accumulation; the ratio of mean intracellular drug content of non confluent to confluent cells (NC/C) was 2.5 for DXR, 4.1 for VCR and 7.4 for VP16. Altered drug penetration with confluence could be related to decrease of plasma membrane fluidity as measured by the fluorescence polarization method. Reduction of drug intracellular accumulation was nil or weak for L-PAM (NC/C = 1.0), CDDP (NC/C = 1.2) and 5 FU (NC/C = 1.8). Even if drug concentration was adjusted in culture medium to produce similar intracellular drug content in confluent and non confluent cells, higher intrinsic resistance of confluent cells was still evidenced for DXR and VP16 but not for VCR, the only agent without direct interaction with DNA. DXR- and VP16-induced DNA breakage was also less important in confluent than in non-confluent cells. CDR appeared closely related to an increased proportion of non-cycling cells at confluence, as demonstrated by flow cytometry, expression of nuclear antigen recognized by Ki67 MAb and expression of topoisomerase II. CDR is probably a major factor in the poor sensitivity of colorectal adenocarcinomas to chemotherapy.

Alterations in membrane fluidity of diabetic polymorphonuclear leukocytes

Plasma membrane fluidity of polymorphonuclear leukocytes was investigated in 28 patients with insulin dependent diabetes mellitus and 30 healthy controls. Membrane fluidity was measured by steady-state fluorescence anisotropy of 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) incorporated into the plasma membrane. The fluorescence anisotropy values in resting (unstimulated) polymorphonuclear leukocytes from diabetic subjects were significantly higher than those of controls (0.318 +/- 0.003 vs 0.287 +/- 0.003, P less than 0.001). The addition of the respiratory burst stimulus phorbol myristate acetate induced a stable increase in fluorescence anisotropy values in both groups. Fluorescence anisotropy values of stimulated polymorphonuclear leukocytes from the diabetic and control groups were not significantly different (P greater than 0.05). These data demonstrate a decrease in plasma membrane fluidity of resting polymorphonuclear leukocytes obtained from diabetic subjects. This finding could be in part explained by an increase in their basal respiratory burst activity.

Cigarette smoke alters plasma membrane fluidity of rat alveolar macrophages.

This study examined the effect of cigarette smoking on the fluidity of the rat alveolar macrophage plasma membrane. Rats were subjected to 8 wk of an in vivo smoke exposure protocol, after which their alveolar macrophages were harvested. Fluidity was assessed by measuring steady-state anisotropy of isolated plasma membranes as well as of lipid vesicles made from total lipid extracts of these plasma membranes. The smoke-exposed animals showed a significant decrease in fluidity in both intact plasma membranes (p less than 0.0001) and in their lipid vesicle preparations (p less than 0.0001). To assess the time course of these changes, lipid vesicles were prepared from total cellular lipid extracts of macrophages from paired rats, control and smoke-exposed, at 1 through 4 wk after initiation of exposure. Significant decreases in fluidity were observed as early as 2 wk after smoking was begun (p less than 0.001). To assess the reversibility of these changes, paired rats were exposed for 8 wk, then withdrawn for 8, 12, and 18 wk, after which fluidity was evaluated in lipid vesicles prepared from total cellular lipids. Even after 18 wk of smoking cessation, significant decreases in fluidity persisted (p less than 0.01). We conclude that cigarette smoking causes a decrease in plasma membrane fluidity of rat alveolar macrophages. This is due at least in part to a change in the lipid portion of the membrane. These alterations occur after a very brief period of smoke exposure and persist long after cessation of smoking.

Plasma membrane changes during corpus luteum regression.

Structural and biochemical changes were examined in the plasma membrane of luteal cells during corpus luteum regression. Structural alterations as indicated by an increase in the liquid-crystalline to gel phase transition temperature and a decrease in plasma membrane fluidity were observed during luteolysis in microsomes and in plasma membranes prepared from prostaglandin F2 alpha-treated rats, when samples were examined by wide angle x-ray diffraction and fluorescence polarization. In addition, a significant increase in activity of the lipolytic enzyme phospholipase A2 appeared during incubation of plasma membrane samples and dispersed luteal cells at 40 degrees C in the presence of 1.0 mM CaCl2. Similar incubation conditions also produced a drop in human chorionic gonadotropin (hCG) binding in luteal samples from prostaglandin F2 alpha-treated rats. These results indicate that during luteolysis there are important structural changes in the plasmalemma of regressing luteal cells. These alterations appear related to an increase in phospholipase A2 activity and a decrease in hCG receptors. These modifications may account for the decrease in function during corpus luteum regression.

Serotonin transport and fluidity in plasma membrane vesicles: effect of hyperoxia.

Plasma membrane vesicles were prepared from porcine pulmonary artery endothelial cells by a dextran-polyethylene glycol two-phase system. Specific carrier-mediated transport of 5-hydroxytryptamine (5-HT) into the vesicles was examined. Transport required a Na+ gradient (out greater than in) across the membrane, and accumulated 5-HT rapidly effluxed out of the vesicles when the ionophore gramicidin was added. Transport was inhibited by the antidepressant imipramine. 5-HT transport into plasma membrane vesicles appeared saturable and exhibited Michaelis-Menten kinetics (Km 7.4 microM, maximal velocity 217 pmol.min-1.mg membrane protein-1). A 24-h exposure to 95% O2 at 1 atmosphere absolute resulted in a 21% decrease (P less than 0.05) in specific 5-HT transport by plasma membrane vesicles. Hyperoxia also caused a significant (P less than 0.01) decrease in plasma membrane fluidity, as measured with the fluorescence probe 1,6-diphenyl-1,3,5-hexatriene. These results indicate that pulmonary artery endothelial cell plasma membrane vesicles provide a good model for studying 5-HT transport activity in vitro. Hyperoxia affects plasma membrane fluidity and 5-HT transport in pulmonary artery endothelial cells, suggesting a possible cause-and-effect relationship between the two.

Changes of membrane fluidity and Na+,K+-ATPase activity during cellular differentiation in the guinea pig epidermis.

The dynamic properties of plasma membrane in epidermal cells were determined by means of electron spin resonance using two kinds of doxyl stearic acid spin labeling agents: 5-DSA and 12-DSA. 5-DSA and 12-DSA are stearic acid analogues with a nitroxide radical ring at the 5th and 12th carbon positions, and these motions reflect molecular motion of lipid bilayer surrounding the hydrophilic region and the hydrophobic region, respectively. Guinea pig epidermal cells were separated into three regions of keratinocytes by Percoll density gradient centrifugation; the upper, middle, and lower epidermal cells. The order parameter S values for 5-DSA and 12-DSA incorporated into the isolated keratinocytes increased, suggesting a decrease in the plasma membrane fluidity, as cells approached the upper epidermal cell layer. The Na+,K+-ATPase activity as a plasma membrane-bound enzyme was determined in each epidermal cell region, and was found to decrease gradually as the cells approached the upper layer. Accordingly, the differentiation of epidermal cells in the keratinization process was found to be associated with a decrease in plasma membrane fluidity and with a decline of Na+,K+-ATPase activity.

Accumulation of an alkyl lysophospholipid in tumor cell membranes affects membrane fluidity and tumor cell invasion

Tumor cells grown in the presence of 1-O-alkyl-2-O-methylglycero-3-phosphocholine (AMG-PC) accumulated this ether lipid in their membranes. Depending on the cell type and the dose of the compound, up to 17% of the total phospholipids of the purified plasma membranes consisted of authentic AMG-PC. Extensive incorporation of the agent resulted in a decrease in plasma membrane fluidity and inhibition of tumor cell invasiveness in embryonic chick heart fragments. The extent of AMG-PC incorporation and fluidity change was not strictly correlated with the degree to which tumor cell invasion was inhibited.