共查询到20条相似文献,搜索用时 78 毫秒
1.
Sule Oncul Oleksandr A. Kucherak Alexander P. Demchenko Sophie Martin Monique Dontenwill Youri Arntz Pascal Didier Guy Duportail Yves Mély 《生物化学与生物物理学报:生物膜》2010,1798(7):1436-13032
Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells. 相似文献
2.
The fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene, has been used to investigate the effects of controlled and uncontrolled growth on the dynamic properties of the lipid regions of hepatocyte plasma membranes. DPH was incubated with plasma membranes derived from quiescent and regenerating liver and Morris hepatoma 7777, and the resulting systems were studied by fluorescence polarization spectroscopy. Membranes from the rapidly growing hepatoma exhibited a significantly lower fluorescence polarization than observed in quiescent liver, suggesting the presence of a more fluid membrane lipid domain. Membranes from regenerating liver exhibited a time-dependent increase in membrane fluidity, reaching a maximum 12 h after growth stimulation. A close correspondence between membrane fluidity and the cholesterol-phospholipid ratio was also observed where a decrease in this ratio resulted in a more fluid lipid matrix. These results suggest that cell cycling, as observed in regenerating liver and Morris hepatoma 7777, results in significant increases in membrane fluidity, a property which may play an important regulatory role in various cell functions. 相似文献
3.
The partitioning of fluorescence probes into intracellular organelles poses a major problem when fluorescence methods are applied to evaluate the fluidity properties of cell plasma membranes with intact cells. This work describes a method for resolution of fluidity parameters of the plasma membrane in intact cells labelled with the fluorescence polarization probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The method is based on selective quenching, by nonradiative energy transfer, of the fluorescence emitted from the plasma membrane after tagging the cell with a suitable membrane impermeable electron acceptor. Such selective quenching is obtained by chemical binding of 2,4,6-trinitrobenzene sulfonate (TNBS), or by incorporation of glucosamine (BGA) to DPH-labelled cells. The procedures for determination of lipid fluidity in plasma membranes of intact cells by this method are simple and straightforward. 相似文献
4.
Changes in biophysical parameters of plasma membranes influence cisplatin resistance of sensitive and resistant epidermal carcinoma cells 总被引:5,自引:0,他引:5
Liang XJ Yin JJ Zhou JW Wang PC Taylor B Cardarelli C Kozar M Forte R Aszalos A Gottesman MM 《Experimental cell research》2004,293(2):283-291
The mechanism of resistance of cancer cells to the anticancer drug cisplatin is not fully understood. Using cisplatin-sensitive KB-3-1 and -resistant KCP-20 cells, we found that the resistant cells have higher membrane potential, as determined by membrane potential sensing oxonol dye. Electron spin resonance and fluorescence polarization studies revealed that the resistant cells have more "fluid" plasma membranes than the sensitive cells. Because of this observed difference in membrane "fluidity," we attempted modification of the plasma membrane fluidity by the incorporation of heptadecanoic acid into KB-3-1 and KCP-20 cell membranes. We found that such treatment resulted in increased heptadecanoic acid content and increased fluidity in the plasma membranes of both cell types, and also resulted in increased cisplatin resistance in the KCP-20 cells. This finding is in accord with our results, which showed that the cisplatin-resistant KCP-20 cells have more fluid membranes than the cisplatin-sensitive KB-3-1 cells. It remains to be determined whether the observed differences in biophysical status and/or fatty acid composition alone, or the secondary effect of these differences on the structure or function of some transmembrane protein(s), is the reason for increased cisplatin resistance. 相似文献
5.
Capsaicin is the pungent ingredient present in hot peppers of the genus Capsicum. Capsaicin's effect on sensory neurons has been well studied; however, its effect on non-neuronal cells is still not fully understood. This study was undertaken to evaluate the effect of capsaicin on erythrocyte membrane enzymes: Na+/K(+)-ATPase and Ca(2+)-ATPase. Treatment with capsaicin (0.01-100 microM) caused a transient increase in the activities of both enzymes; the effect declined at lower concentrations of capsaicin, and no significant effect was observed at 0.01 microM capsaicin. The effect of capsaicin was fast with a significant (p<0.01) activation of enzyme activity observed within minutes of incubation. The findings on the effect of capsaicin on human erythrocyte membrane enzymes Na+/K(+)-ATPase and Ca(2+)-ATPase signify the importance of the non-neuronal effects of capsaicin, and the need for evaluating the physiological impact of high capsaicin (capsicum) consumption in some regions of the world. 相似文献
6.
Zymogen granule membranes contain Cl- conductance and Cl/anion exchange activities that become important for primary fluid production after fusion with the apical plasma membrane of the acinar cell. We have used steady-state fluorescence anisotropy of diphenylhexatriene derivatives and measurements of Cl- transport in isolated secretory granules to determine the contribution of membrane fluidity to the regulation of transport across the granule membrane. Secretory granules from several unstimulated glands (rat pancreas and parotid, rabbit gastric glands) were shown to have low membrane fluidity compared to plasma membranes. In addition, Cl- transport activity in different granule preparations showed a strong correlation to the membrane fluidity when measured with 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), but not with 3-[p-(6-phenyl)-1,3,5-hexatrienyl)-phenyl]propionic acid (PA-DPH). These data suggest that TMA-DPH preferentially partitions into a specific lipid environment associated with, or which exerts an influence on, the Cl- transport proteins and that increases in the fluidity of this environment are associated with higher transport rates. Data from other types of plasma membranes indicate that TMA-DPH partitions much more than PA-DPH into the cytoplasmic leaflet, suggesting that this part of the granule membrane is involved in the observed fluidity changes. Furthermore, increasing the bulk membrane fluidity with the local anesthetics benzyl alcohol and n-alkanols increased the Cl- transport rates up to 10-fold. This increase was apparently through specific transporters as anion selectivity was maintained in spite of the higher absolute rates.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Dominique Illinger Martial Kubina Guy Duportail Philippe Poindron Jacques Bartholeyns Jean-Georges Kuhry 《Cell biochemistry and biophysics》1989,14(1):17-26
Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess
a variety of particular properties in interaction with intact living cells —specific and rapid incorporation into the plasma
membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in
membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here
for quantitative monitoring of phago-cytosis kinetics, by means of simple fluorescence intensity measurements. This method
is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed
on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process.
It has been tested on mouse bone marrow macrophages. 相似文献
8.
Steroid hormone-induced effects on membrane fluidity and their potential roles in non-genomic mechanisms 总被引:7,自引:0,他引:7
Steroid hormones are lipophilic suggesting they intercalate into the bilayer of target cell plasma membranes, potentially altering the fluidity and function of the membrane. The present study measured the effects of steroidal exposure on both phospholipid fluidity and integral protein mobility. Studies were performed on the effects of a variety of steroids on phosphatidylcholine liposomes, synaptosomal plasma membranes and sarcoplasmic reticulum membranes. Progesterone decreased the lipid fluidity, whereas testosterone had no effect on lipid movement. The estrogen, 17 beta-estradiol, an aromatised metabolite of testosterone, increased lipid mobility. In each case, the steroid action was concentration-dependent. The steroids all increased the activity of the Ca2+ ATPase of SR membrane, in keeping with their effects on this enzyme's aggregation state. The results suggest that, although lipid fluidity is a factor influencing protein activity, their mobility within the bilayer is the primary determinant of enzyme activity in the membrane for most proteins. 相似文献
9.
We investigated the effects of the interaction between flavanols and related procyanidins (dimer to hexamer) with both cell and synthetic membranes, on bilayer fluidity and susceptibility to oxidation. Cocoa derived dimers (0.05 to 1 microg/ml) protected Jurkat T cells from AMVN-mediated oxidation and increased plasma membrane fluidity. These effects occurred in a concentration- and chain length-dependent manner. In liposomes, procyanidins prevented the Fe2+ -induced permeabilization of the membrane. Together, these results support the hypothesis that procyanidins could interact with the polar headgroup of lipids, increasing membrane fluidity and also, preventing the access of molecules that could affect membrane integrity. 相似文献
10.
Myosin Is are associated with specific membranes, however, the mechanism for regulating their intracellular localization is unclear. As a first step towards understanding this mechanism, membrane rebinding assays using Dictyostelium myoB were performed. Crude, cytosolic myoB bound to intact, but not to NaOH-treated plasma membranes. In contrast, partially purified myoB binds to both intact and NaOH-treated plasma membranes. Chemical cross-linking of cytosolic myoB yielded several products, whereas none were found with the partially purified myoB. These results suggest a model where proteins regulating the specific binding of myoB to the plasma membrane may exist both in the cytosol and on the plasma membrane. 相似文献
11.
W.J. Van Blitterswijk P. Emmelot H.A.M. Hilkmann Els P.M. Oomenmeulemans M. Inbar 《生物化学与生物物理学报:生物膜》1977,467(3):309-320
The fluorescence polarization technique with 1,6-diphenyl 1,3,5-hexatriene as a probe was used to determine the lipid microviscosity, , of isolated plasma membranes of mouse thymus-derived ascitic leukemia (GRSL) cells and of extracellular membraneous vesicles exfoliated from these cells and occurring in the ascites fluid. For comparison, was also determined in isolated plasma cell supernatants.For isolated plasma membranes of thymocytes and GRSL cells values at 25° C amounted to 4.67 and 3.28 P, respectively, which were higher than the microviscosities of the corresponding intact cells, 3.24 and 1.73 P, respectively.Microviscosities inextracellular membranes of thymocytes and GRSL cells were 5.96 and 5.83 P, respectively. The fluidity difference between these membranes and plasma membranes was most pronounced for the leukemic cells and was thereby correlated with a large difference in cholesterol/phospholipid molar ratio (1.19 for extracellular membranes and 0.37 for plasma membranes). It is proposed that extracellular membraneous vesicles are shed from the surface of GRSL cells similar to the budding process of viruses, that is by selection of the most rigid parts of the host cell membrane.Liposomes of total lipid extracts of plasma membranes and extracellular membranes of both cell types exhibited about the same microviscosity as the corresponding intact membranes, indicating virtually no contribution of (glyco)-protein to the lipid fluidity as measured by the fluorescence polarization technique. For both cell types (25° C) values of liposomes consisting of membrane phospholipids varied between 1.5 and 1.9 P, much lower than the values for total lipids, indicating a significant rigidizing effect of cholesterol in each type of membrane. 相似文献
12.
Substance P (SP) originally found as a neuropeptide in capsaicin-sensitive sensory neurons, had more recently been identified in non-neuronal cells, especially under pathological conditions. Neuronal and non-neuronal SP may perform distinct functions. A simple technique to differentiate different SP sources is currently unavailable. Herein, we describe a two-step sequential acetic acid extraction to differentiate SP source. The efficiency of this two-step extraction in differentiating SP in capsaicin-sensitive neurons was verified by using capsaicin as a tool to deplete SP in sensory neurons. Specifically, Balb-c mice were treated with high dose capsaicin (200 mg/kg). Skin was removed two weeks after treatment. In a separate experiment, lung and skin tissues from control animals (untreated) were incubated in-vitro with capsaicin, and sequential acetic acid extraction was performed. Following capsaicin treatment, both in-vivo and in-vitro, SP recovered in first extraction decreased significantly in lung and skin. Lastly, presence of capsaicin solvent (10% methanol and 10% Tween 80) or protease inhibitor cocktail in solution altered SP EIA test, yielding false positive results. These results demonstrated that SP in capsaicin sensitive sensory neurons was extracted in initial extraction of 15 min while non-neuronal SP was present in second extraction. Because SP in non-neuronal tissues may possibly be more important in pathological conditions, this technique could be useful in determining effects of various treatments on neuronal and non-neuronal SP levels and their consequences. 相似文献
13.
Two mitochondrial contact site-enriched fractions were further characterized by freeze-fracture electron microscopy. Examination of the replicas revealed that these two fractions are in the form of vesicles with membrane sheets attached to the vesicles. The physical state of these fractions has been investigated by means of steady-state fluorescence polarization to assess the effects of alcohols on their fluidity properties and activity. Comparison between intact membranes and their extracted lipids shows that butanol and hexanol induce a differential increase of the overall membrane fluidity in the two contact site-enriched fractions. This alteration in the membrane dynamics leads to a complex modulation of cytochrome c oxidase activity in both fractions. These results bring further evidence for the existence of morphologically and functionally distinct domains in mitochondrial membranes. 相似文献
14.
Yong Zhou Kelsey N. Maxwell Erdinc Sezgin Maryia Lu Hong Liang John F. Hancock Elizabeth J. Dial Lenard M. Lichtenberger Ilya Levental 《The Journal of biological chemistry》2013,288(50):35660-35670
Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects. 相似文献
15.
Bhathena SJ Berlin E McClure D Peters RC 《The Journal of nutritional biochemistry》2001,12(9):529-535
It has been demonstrated that the type of dietary fat affects insulin receptors in various tissues in normal humans and animals by altering membrane fluidity. This study compares the effects of n-3 fatty acids from fish oil and n-6 fatty acids from corn oil on red blood cell membrane insulin receptors in normal and hypercholesterolemic minipigs. A group of minipigs were made hypercholesterolemic by feeding cholesterol and lard for 2 months; the other group served as controls and was fed stock diet. Both groups were then fed experimental diets containing either corn oil or menhaden oil or a mixture of the two for 23 additional weeks. Blood was collected at 0, 2, 12 and 23 weeks after the start of the experimental diets and membranes were prepared from the red blood cells. Insulin binding to red blood cell membranes was measured by radioreceptor assay. Plasma insulin was measured by radioimmunoassay. Insulin binding to red blood cell membrane was compared with the fluidity of the membrane measured and reported earlier. There was no significant effect of cholesterol feeding on plasma insulin concentrations. After 23 weeks on experimental diet plasma insulin was significantly higher in minipigs fed menhaden oil compared to those fed corn oil. No such effect was observed in hypercholesterolemic minipigs. No significant effect of either hypercholesterolemia or fish oil was observed on red blood cell insulin binding. A significant negative relationship was observed between insulin binding and anisotropy at 4°C for all probes but at 37°C significant negative relationship was observed only with polar probes. The data suggest that n-3 fatty acids from fish oil significantly increases plasma insulin in minipigs compared to n-6 fatty acids from corn oil. However, the unsaturation has no significant effect on insulin receptors on erythrocytes. Similarly, prior hypercholesterolemic state also has no effect on plasma insulin levels or the insulin binding to red blood cell membranes. 相似文献
16.
Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics 总被引:1,自引:0,他引:1
Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species. 相似文献
17.
In vitro treatment of crude particulate fractions of male rat ventral prostate and female rat liver with membrane fluidizers (aliphatic alcohols) has been previously reported by us to increase prolactin (PRL) receptor levels, presumably by unmasking cryptic prolactin receptors. The objective of this study was to determine if similar in vitro treatment of purified plasma membrane- and Golgi-rich fractions of male rat prostate and female rat liver with ethanol produced differential effects on prolactin binding in these two subcellular fractions. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. 125I-PRL specific binding to Golgi-rich fractions of male ventral prostate and female liver was approximately 4-fold higher than that observed in plasma membrane-rich fractions. The microviscosity parameter, inversely related to lipid fluidity, was consistently lower in Golgi-rich fractions than that in plasma membrane-rich fractions in both prostate and liver. In vitro ethanol treatment of prostatic and hepatic plasma membrane fractions produced a dose-related increase and then decline in prolactin binding and a maximal (60-75%) increase in prolactin binding was observed at 4.8% and 2.0% ethanol in prostatic and hepatic membranes, respectively. This in vitro treatment also produced a significant increase in apparent lipid fluidity of plasma membrane-rich fractions of prostate gland and liver. However, similar in vitro ethanol treatment of Golgi fractions of both prostate gland and liver exhibited little increase in prolactin binding without changing microviscosity. Our observations are consistent with the direct relationship between membrane fluidity and prolactin receptor levels. The changes in prostatic and hepatic plasma membrane fractions following in vitro ethanol treatment suggest that prolactin receptors located on the plasma membranes may be modulated (via membrane lipid microviscosity changes) in vivo to a greater extent by various physiological agents than those located within the Golgi fraction. 相似文献
18.
A D Whetton L Needham G P Margison N J Dodd M D Houslay 《Biochimica et biophysica acta》1984,773(1):106-112
The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation. 相似文献
19.
D Illinger M Kubina G Duportail P Poindron J Bartholeyns J G Kuhry 《Cell biophysics》1989,14(1):17-26
Trimethylammonium-diphenylhexatriene (TMA-DPH), a hydrophobic fluorescent probe, has been shown in earlier studies to possess a variety of particular properties in interaction with intact living cells--specific and rapid incorporation into the plasma membrane and partition equilibrium between the membranes and the buffer. These properties offer promising applications in membrane fluidity studies and in monitoring exocytosis kinetics. Furthermore, these properties offer a method described here for quantitative monitoring of phagocytosis kinetics, by means of simple fluorescence intensity measurements. This method is original in that it evaluates only the particles which have actually been internalized by phagocytosis, and not those adsorbed on the cell surface, and that it gives quantitative information on the amount of plasma membrane involved in the process. It has been tested on mouse bone marrow macrophages. 相似文献
20.
Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies 总被引:3,自引:0,他引:3
The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol. 相似文献