Plasma Membrane Lipid Mobility Research Articles

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM1-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells). Computer-assisted interpretation of EPR spectra revealed three modes of spin probe movement reflecting the properties of three types of PM nanodomains. Domains with an intermediate order parameter (domain 2) were the most affected by the presence of the GnRH-Rs, which increased PM ordering (order parameter (S)) and rotational mobility of PM lipids (decreased rotational correlation time (τc)). Depletion of cholesterol by methyl-β-cyclodextrin (methyl-β-CD) inhibited agonist-induced GnRH-R internalization and intracellular Ca2+ activity and resulted in an overall reduction in PM order; an observation further supported by molecular dynamics (MD) simulations of model membrane systems. This study provides evidence that GnRH-R PM localization may be related to a subdomain of lipid rafts that has lower PM ordering, suggesting lateral heterogeneity within lipid raft domains.

Lateral mobility of plasma membrane lipids in bull spermatozoa: heterogeneity between surface domains and rigidification following cell death.

Compartmentalization of surface membrane antigens into discrete regions or domains is a characteristic feature of differentiated cells. In mammalian spermatozoa at least 5 surface domains are known, implying the presence of barriers or boundaries within the plasma membrane. Using the technique of fluorescence recovery after photobleaching (FRAP) to measure diffusibility of fluorescent lipid analogues 1,1'-dihexadecyl-3,3,3'3'-tetramethylindocarbocyanine (DiIC[16]) and 5-(N-octa-decanoyl) aminofluorescein (ODAF), we have investigated lipid topology and dynamics in the plasma membrane of ejaculated bull spermatozoa. Contrary to reports in the literature, we have found that DiIC(16) stains only dead or damaged spermatozoa whereas ODAF intercalates into the plasma membrane of both live and dead cells, each type showing a distinctive staining pattern. FRAP analysis with ODAF revealed that diffusion coefficients on live spermatozoa are significantly faster on the acrosome and postacrosome (29.3x10(-9) cm2/second) than on the midpiece and principal piece (11.8x10(-9) cm2/second). Recovery (R) is >90% in all domains. ODAF diffusion also shows regionalized temperature-sensitivity with a 4-fold increase over the sperm head and a 1.8-fold increase on the tail between 20 degrees C and 37 degrees C. Remarkably, dead or permeabilized spermatozoa rapidly develop a large immobile phase (R<25%) over the whole plasma membrane. This rigidification is temperature insensitive and irreversible suggesting major changes in the physical state of membrane lipids. It is concluded that lipid diffusion in the plasma membrane of live bull spermatozoa is rapid and varies significantly between surface domains. Following permeabilization or cell death, however, a large immobile phase develops indicating substantial changes in membrane lipid disposition.

Enhancement of Melphalan Toxicity by Octanol in Ovarian Adenocarcinoma Cell Lines: Effects of Altered Cell-Cell Communication, Glutathione Levels, and Plasma Membrane Fluidity

Enhancement of Melphalan Toxicity by Octanol in Ovarian Adenocarcinoma Cell Lines: Effects of Altered Cell-Cell Communication, Glutathione Levels, and Plasma Membrane Fluidity. Barhoumi, R., Bailey, R. H., Hutchinson, R. W., Bowen, J. A., and Burghardt, R. C. (1995). Fundam. Appl. Toxicol. 25, 70-79. A2780 and COLO-316 ovarian adenocarcinoma cell lines were exposed to 1.0 mM 1-octanol for 12 hr in order to evaluate the potential effects of inhibition of gap junction-mediated intercellular communication (GJIC) on cellular responses to the chemotherapeutic drug melphalan. Other cellular endpoints relevant to drug-resistance mechanisms which were monitored after treatments included cellular glutathione levels, glutathione S-transferase activity, mitochondrial membrane potential, and plasma membrane lipid mobility. In cells which were sensitive to melphalan, octanol enhanced melphalan toxicity in the GJIC-competent (A2780/S) but not GJIC-incompetent (COLO-316/S) sensitive cells. Although octanol increases plasma membrane lipid mobility in A2780/S and COLO-316/S, it appears that enhancement of A2780/S sensitivity to melphalan may be due to inhibition of GJIC. In melphalan-resistant cells (A2780/R and COLO-316/R), 1.0 mM octanol treatment for 12 hr combined with melphalan reversed the resistance of the cells to the drug. Therefore, alterations in cellular glutathione metabolism and effects on the plasma membrane in addition to uncoupling of GJIC may be involved in sensitizing communication-competent and communication incompetent resistant cells because COLO-316/R lacks gap junction-mediated intercellular communication. Further, analysis of mitochondrial membrane potential provided an index of acquired drug resistance and the efficacy of melphalan and combined octanol/melphalan toxicity.

Laser cytometric analysis of FIV‐induced injury in astroglia

Glia are the predominant brain cells infected by the lentiviruses human immunodeficiency virus (HIV) and feline immunodeficiency virus (FIV). The importance of astrocytes in maintenance of central nervous system homeostasis suggests that astrocytes are likely to play a strategic role in the progression of neurological disease in lentiviral-infected patients. In consideration of this postulate, the ability of FIV to cause injury by infection of cultured feline astroglia was examined via vital fluorescence assays. Intracellular Ca2+ homeostasis, plasma membrane permeability and fluidity, and cytosolic glutathione (GSH) levels were evaluated. Although basal intracellular Ca2+ was not significantly different between groups, FIV-infected astroglia displayed both a significant delay in development of peak Ca2+ levels following ionophore application and a decrease in the amount of Ca2+ released from intracellular stores. Plasma membrane lipid mobility was increased in FIV-infected cells within 24 h of infection. Glutathione levels were affected in a dose dependent fashion. With a standard viral inoculum there was a decrease in GSH which became significant after 8 days postinfection. With a high inoculum dose there was rapid loss of cell viability with an increase in GSH in surviving cells. We have identified several cellular processes altered in FIV-infected astroglia and our findings suggest that FIV-infection of feline astroglia affects cellular membranes, both structurally and functionally.

Tumor necrosis factor and immune interferon act in concert to slow the lateral diffusion of proteins and lipids in human endothelial cell membranes.

Vascular endothelial surface-related activities may depend on the lateral mobility of specific cell surface macromolecules. Previous studies have shown that cytokines induce changes in the morphology and surface antigen composition of vascular endothelial cells in vitro and at sites of immune and inflammatory reactions in vivo. The effects of cytokines on membrane dynamic properties have not been examined. In the present study, we have used fluorescence photobleaching recovery (FPR) to quantify the effects of the cytokines tumor necrosis factor (TNF) and immune interferon (IFN-gamma) on the lateral mobilities of class I major histocompatibility complex protein, of an abundant 96,000 Mr mesenchymal cell surface glycoprotein (gp96), and of a phospholipid probe in cultured human endothelial cell (HEC) membranes. Class I protein and gp96 were directly labeled with fluorescein-conjugated monoclonal antibodies; plasma membrane lipid mobility was examined with the phospholipid analogue fluorescein phosphatidylethanolamine (Fl-PE). In untreated, confluent HEC monolayers, diffusion coefficients were 30 x 10(-10) cm2 s-1 for class I protein, 14 x 10(-10) cm2 s-1 for gp96, and 80 x 10(-10) cm2 s-1 for Fl-PE. Fractional mobilities were greater than 80% for each probe. Cultures treated at visual confluence for 3-4 d with either 100 U/ml TNF or 200 U/ml IFN-gamma did not exhibit significant changes in protein or lipid mobilities despite significant changes in cell morphology and membrane antigen composition. In HEC cultures treated concomitantly with TNF and IFN-gamma, however, diffusion coefficients decreased by 71-79% for class I protein, 29-55% for gp96, and 23-38% for Fl-PE. Fractional mobilities were unchanged. By immunoperoxidase transmission electron microscopy, plasma membranes of untreated and cytokine-treated HEC were flat and stained uniformly for class I antigen. "Line" FPR measurements on doubly treated HEC demonstrated isotropic diffusion of class I protein, gp96, and Fl-PE. Finally, although TNF and IFN-gamma retarded the growth of HEC cultures and disrupted the organization of cell monolayers, the slow diffusion rates of gp96 and Fl-PE in confluent doubly treated monolayers were not reproduced in sparse or subconfluent untreated monolayers. We conclude that the slowing of protein and lipid diffusion induced by the combination of TNF and IFN-gamma is not due to plasma membrane corrugations, to anisotropic diffusion barriers, or to decreased numbers of cell-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Lateral mobility of plasma membrane lipids in normal and transformed keratinocytes

In this study we have examined possible differentiation-dependent modulations in plasma membrane lipid properties in normal keratinocytes, SV-40 transformed keratinocytes (SVK14) and a number of squamous carcinoma (SCC) cells. In normal keratinocytes the lateral diffusion coefficient of plasma membrane lipids (D) differs significantly for cells cultured permanently under low and normal Ca2+-conditions (5.16 x 10(-9) and 3.27 x 10(-9) cm2/s, respectively). When differentiation is induced by exposing low Ca2+-cultured cells to normal Ca2+ concentrations D increases to 7.07 x 10(-9) cm2/s during the initial hours of differentiation followed by a gradual sustained decrease to values also observed in cells cultured permanently under normal Ca2+-conditions. In SCC and SVK14 cells a similar initial transient increase in lateral lipid mobility is observed upon initiation of differentiation, but, in contrast to normal keratinocytes, no sustained decrease in D is seen upon prolonged culturing under normal Ca2+ conditions. The results indicate that the deficiency of the transformed cells to respond to Ca2+-induced differentiation might involve transformation-dependent alterations in membrane structure and function.

Formula omitted] activation and dna binding affinity of human lymphoid (CEM-C7) cytoplasmic receptors labeled with the antiglucocorticoid RU 38486

Administration of the antiprogesterone synthetic steroid RU-486 (17β-hydroxy-11β-[4-dimethylaminophenyl-1]-17α-[prop-1-ynyl]-estra-4,9-dien-3-one) in human and non-human primates induces menstruation and is promising as a new approach to fertility control. To explore the sites of action of RU-486, we investigated in this study the effects of RU-486 upon gonadotropin-stimulable adenylyl cyclase in membrane preparations obtained from human corpus luteum and upon LH and FSH release by a dispersed rat anterior pituitary cell culture. In the presence of a wide range of concentrations (10−10 to 10−6M), RU-486 failed to alter basal or hCG-stimulated adenylyl cyclase activities under conditions allowing either maximal or submaximal hormonal activation. Additionally, enzyme stimulation by GMP-P(NH)P (100 μM), NaF (10 mM) or forskolin (100 μM) was not affected by a high concentration (10−6M) of RU-486. These data indicate that RU-486 does not affect gonadotropin receptor binding nor does it interfere with cAMP generation. It is unlikely, therefore, that the compound may modulate human luteal function through changes in plasma membrane lipid mobility or modifications of reactions occurring in plasma membranes as suggested for other steroids in several membrane systems. The present observations are compatible with previously published in vivo studies suggesting that RU-486 activity does not involve a direct antigonadotropic effect at the primate corpus luteum level.We also found that RU-486 (10−12 to 10−7 M) did not alter the basal release of gonadotropins by the pituitary cells, nor did the compound impair the response of these cells to maximally or submaximally effective concentrations of LHRH. Thus, these data suggest that the anti-reproductive actions of RU-486 involve no direct effect upon pituitary function. Taken together, these findings support the concept that RU-486 exerts its effects on the luteal phase exclusively by a local action upon the endometrium.

The antiprogesterone steroid RU-486 does not impair gonadotropin-stimulated luteal adenylyl cyclase activity or gonadotropin release by pituitary cells

Administration of the antiprogesterone synthetic steroid RU-486 (17β-hydroxy-11β-[4-dimethylaminophenyl-1]-17α-[prop-1-ynyl]-estra-4,9-dien-3-one) in human and non-human primates induces menstruation and is promising as a new approach to fertility control. To explore the sites of action of RU-486, we investigated in this study the effects of RU-486 upon gonadotropin-stimulable adenylyl cyclase in membrane preparations obtained from human corpus luteum and upon LH and FSH release by a dispersed rat anterior pituitary cell culture. In the presence of a wide range of concentrations (10 −10 to 10 −6M), RU-486 failed to alter basal or hCG-stimulated adenylyl cyclase activities under conditions allowing either maximal or submaximal hormonal activation. Additionally, enzyme stimulation by GMP-P(NH)P (100 μM), NaF (10 mM) or forskolin (100 μM) was not affected by a high concentration (10 −6M) of RU-486. These data indicate that RU-486 does not affect gonadotropin receptor binding nor does it interfere with cAMP generation. It is unlikely, therefore, that the compound may modulate human luteal function through changes in plasma membrane lipid mobility or modifications of reactions occurring in plasma membranes as suggested for other steroids in several membrane systems. The present observations are compatible with previously published in vivo studies suggesting that RU-486 activity does not involve a direct antigonadotropic effect at the primate corpus luteum level. We also found that RU-486 (10 −12 to 10 −7 M) did not alter the basal release of gonadotropins by the pituitary cells, nor did the compound impair the response of these cells to maximally or submaximally effective concentrations of LHRH. Thus, these data suggest that the anti-reproductive actions of RU-486 involve no direct effect upon pituitary function. Taken together, these findings support the concept that RU-486 exerts its effects on the luteal phase exclusively by a local action upon the endometrium.

Regional differences in the lateral mobility of plasma membrane lipids in a molluscan embryo

Regional and temporal differences in plasma membrane lipid mobility have been analyzed during the first three cleavage cycles of the embryo of the polar-lobe-forming mollusc Nassarius reticulatus by the fluorescence photobleaching recovery (FPR) method, using 1,1′-ditetradecyl 3,3,3′,3′-tetramethylindocarbocyanine iodide (C 14diI) as a fluorescent lipid probe. During this period of development the lateral diffusion coefficient of membrane lipids is consistently greater in the vegetal polar lobe area as compared to the animal plasma membrane area (on average 30%), demonstrating the existence of an animal-vegetal polarity in plasma membrane properties. At third cleavage, the differences between animal and vegetal plasma membrane region become even more pronounced; in the four animal micromeres the diffusion coefficient ( D) and mobile fraction (MF) are 2.9 ± 0.2 × 10 −9 cm 2/sec and 51 ± 2%, respectively, while in the four vegetal macromeres D = 5.0 ± 0.3 × 10 −9cm 2/sec and MF = 78 ± 2%. Superimposed upon the observed animal-vegetal polarity, the lateral diffusion in the polar lobe membrane area shows a cell-cycle-dependent modulation. The highest mean values for D are reached during the S phase (ranging from 7.0 to 7.8 × 10 −9 cm 2/sec in the three cycles measured), while at the end of G 2 phase and during early mitosis mean values for D have decreased significantly (ranging from 5.0 to 5.9 × 10 −9 cm 2/sec). Diffusion rates in the animal membranes of the embryo are constant during the three successive cell cycles ( D = 4.3−5.0 × 10 −9cm 2/sec), except for a peak at the S phase of the first cell cycle ( D = 6.0 × 10 −9cm 2/sec). These results are discussed in relation with previously observed ultrastructural heterogeneities in the Nassarius egg plasma membrane. It is speculated that the observed animalvegetal polarity in the organization of the egg membrane might play an important role in the process of cell diversification during early development.

Increased rate of capping of concanavalin a receptors during early Xenopus development is related to changes in protein and lipid mobility

The mobility characteristics of plasma membrane constituents were studied in dissociated cells from embryos of Xenopus laevis at various stages of development from early blastula until neurulation. An increased rate of fluorescein isothiocyanate-concanavalin A induced patching and capping of Con A-binding proteins during this period of development was correlated with a threefold increase in the lateral mobility of the receptor molecules, as determined by the fluorescent photobleaching recovery (FPR) method, the major change occurring at the onset of gastrulation. Using the same method, it was demonstrated that the lateral mobility of plasma membrane lipids increases twofold during this period of development. The major change being detectable, however, at the late blastula stage. This is in coincidence with the initiation of cell motility in dissociated Xenopus embryo cells. It is concluded that the lateral mobility of membrane proteins and lipids increases significantly during early Xenopus development, but are at least in part subject to different control mechanisms. The results suggest that the initiation of morphogenetic movements is related to changes in the dynamic properties of plasma membrane constituents.

Lateral mobility of plasma membrane lipids in dividing Xenopus eggs

The lateral mobility of plasma membrane lipids was analyzed during first cleavage of Xenopus laevis eggs by fluorescence photobleaching recovery (FPR) measurements, using the lipid analogs 5-( N-hexadecanoyl)aminofluorescein (“HEDAF”) and 5-( N-tetradecanoyl)aminofluorescein (“TEDAF”) as probes. The preexisting plasma membrane of the animal side showed an inhomogeneous, dotted fluorescence pattern after labeling and the lateral mobility of both probes used was below the detection limits of the FPR method ( D ⪡ 10 −10cm 2/sec). In contrast, the preexisting plasma membrane of the vegetal side exhibited homogeneous fluorescence and the lateral diffusion coefficient of both probes used was relatively high (HEDAF, D = 2.8 × 10 −8cm 2/sec; TEDAF, D = 2.4 × 10 −8cm 2/sec). In the cleaving egg visible transfer of HEDAF or TEDAF from prelabeled plasma membrane to the new membrane in the furrow did not occur, even on the vegetal side. Upon labeling during cleavage, however, the new membrane was uniformly labeled and both probes were mobile, as in the vegetal preexisting plasma membrane. These data show that the membrane of the dividing Xenopus egg comprises three macrodomains: (i) the animal preexisting plasma membrane; (ii) the vegetal preexisting plasma membrane; (iii) the new furrow membrane.

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity

Regional differences in the lateral mobility properties of plasma membrane lipids were studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein- -1abelled fatty acids HEDAF (5-(N-hexadecanoyl)- aminofluorescein) and TEDAF (5-(N-tetradecanoyl)-aminofluorescein) appear to distribute itself in the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching, indicating the existence of lipidic microdomains.

Lateral mobility of plasma membrane lipids in a molluscan egg: Evidence for an animal/vegetal polarity

The lateral diffusion of the lipid analog C₁₄-diI (3', 3'-dihexadecylindocarbocyanine iodide) was measured in the plasma membrane of early embryos of the mollusc Nassarius reticulatus using the FPR-(Fluorescence Photobleaching Recovery) method. At almost all stages measured (from fertilized egg up to 8-cell stage) the diffusion coefficient (D) of the mobile fraction (MF) of C₁₄-diI is significantly higher in the plasma membrane of the polar lobe as compared to the plasma membrane of the animal half of the embryo. These results demonstrate the presence of an animal/vegetal polarity in the plasma membrane of the embryo of Nassarius, possibly related with the polar localization of morphogenetic factors.

Lateral mobility of plasma membrane lipids in Xenopus eggs: Regional differences related to animal/vegetal polarity become extreme upon fertilization

Regional differences in the lateral mobility properties of plasma membrane lipids have been studied in unfertilized and fertilized Xenopus eggs by fluorescence photobleaching recovery (FPR) measurements. Out of a variety of commonly used lipid probes only the aminofluorescein-labeled fatty acids HEDAF (5-( N-hexadecanoyl)-aminofluorescein) and TEDAF (5-( N-tetradecanoyl)-aminofluorescein) appear to partition into the plasma membrane. Under all experimental conditions used these molecules show partial recovery upon photobleaching indicating the existence of lipidic microdomains. In the unfertilized egg the mobile fraction of plasma membrane lipids (∼50%) has a fivefold smaller lateral diffusion coefficient ( D = 1.5 × 10 −8 cm 2/sec) in the animal than in the vegetal plasma membrane ( D = 7.6 × 10 −8 cm 2/sec). This demonstrates the presence of an animal/vegetal polarity within the Xenopus egg plasma membrane. Upon fertilization this polarity is strongly (>100×) enhanced leading to the formation of two distinct macrodomains within the plasma membrane. At the animal side of the egg lipids are completely immobilized on the time scale of FPR measurements ( D ⪡ 10 −10 cm 2/sec), whereas at the vegetal side D is only slightly reduced ( D = 4.4 × 10 −8 cm 2/sec). The immobilization of animal plasma membrane lipids, which could play a role in the polyspermy block, probably arises by the fusion of cortical granules which are more numerous here. The transition between the animal and the vegetal domain is sharp and coincides with the boundary between the presumptive ecto- and endoderm. The role of regional differences in the plasma membrane is discussed in relation to cell diversification in early development.

551 Effects of high-dose medroxyprogesterone acetate (MPA) on plasma membrane lipid mobility

551 Effects of high-dose medroxyprogesterone acetate (MPA) on plasma membrane lipid mobility

321 Effects of high-dose medroxyprogesterone acetate (MPA) on plasma membrane lipid mobility

321 Effects of high-dose medroxyprogesterone acetate (MPA) on plasma membrane lipid mobility