Rate Of Lateral Diffusion Research Articles

Regulation of band 3 rotational mobility by ankyrin in intact human red cells.

Ankyrin mutations and combined spectrin and ankyrin deficiency are prominent features of red blood cells (RBCs) in patients with hereditary spherocytosis (HS). Band 3 is the most abundant integral protein in the human RBC membrane. Previous studies have shown that the lateral mobility, but not the rotational mobility, of band 3 is increased in RBCs from patients with severe autosomal recessive HS and selective spectrin deficiency. These observations are consistent with the steric hindrance model of lateral mobility restriction. Here we use the fluorescence photobleaching recovery and polarized fluorescence depletion techniques to measure the lateral and rotational mobility of band 3 in intact RBCs from six patients with HS, ankyrin mutations, and combined spectrin and ankyrin deficiency. As predicted by the steric hindrance model, the lateral diffusion rate of band 3 is greater in spectrin- and ankyrin-deficient RBCs than in control cells, and the magnitude of the increase correlates with the degree of spectrin deficiency. Unlike RBCs from patients with HS and selective spectrin deficiency, however, HS RBCs with ankyrin mutations exhibit a marked increase in band 3 rotational diffusion. The magnitude of the increase correlates inversely with the ankyrin/band 3 ratio and with the fraction of band 3 retained in the membrane skeleton following detergent extraction. These data suggest that ankyrin deficiency relaxes rotational constraints on the major (slowly rotating) population of band 3 molecules. Increases in band 3 rotation could be due to release of band 3 from low-affinity binding sites on ankyrin.

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 ± 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was ~106 s-1, at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.Key words: band 3, reconstitution, electron paramagnetic resonance, lipid-protein interactions.

Chemical kinetics of mobile-proton generation and annihilation in SiO2 thin films

The chemical kinetics of mobile-proton reactions in the SiO2 film of Si/SiO2/Si structures were analyzed as a function of forming-gas anneal parameters in the 300–600 °C temperature range. Our data show that the initial buildup of mobile protons is limited by the rate of lateral hydrogen diffusion into the SiO2 films. The final density of mobile protons is determined by the cooling rate which terminates the annealing process and, in the case of subsequent anneals, by the temperature of the final anneal. To explain the observations, we propose a dynamical equilibrium model which assumes a reversible interfacial reaction with a temperature-dependent balance.

Geometry-induced inhomogeneity of distribution of cell adhesion molecules along branching processes

There is evidence that inhomogeneity of lateral distribution of cell adhesion molecules (CAM) in the plasma membrane is crucial for cell motility and growth. It is known that the cells move when the cell-substratum adhesiveness is within a certain critical range. We have carried out simulation studies of the factors governing inhomogeneous CAM distribution along uniform and branching cylinder-shaped cell processes on a flat substrate evenly covered with a ligand. Our model included the following mechanisms: intracellular transportation of CAM to an active (growing) part of the cell, installation in the plasma membrane, lateral diffusive redistribution of mobile CAM, formation and dissociation of CAM/ligand complexes, and CAM internalization by endocytosis. Since the rate of growth is two and one order of magnitude slower than the rate of trafficking and lateral diffusion, respectively, we analyzed steady distributions of CAM. We showed that interplay of the mechanisms included in the model may lead to the occurrence of CAM distributions with inhomogeneity, whose range is critical for translocation of an active part of the cell. It was shown that a difference in the diameters of asymmetric sister branches can cause different inhomogeneous distributions of CAM along these branches and thus can define conditions of their growth. Depending on the branching geometry, one or both sister branches may appear within this critical range, and a difference in the diameters may define a difference in the rate of growth and, correspondingly, in the length. This may constitute the basis for self-control of neurite outgrowth.

Lateral diffusion of the total polar lipids from Thermoplasma acidophilum in multilamellar liposomes

31 P NMR lineshapes of multilamellar liposomes composed mostly of a bilayer-spanning tetraether lipid are consistent with rapid axially symmetric motion about the bilayer normal. The residual chemical shift anisotropy of 36 ppm is comparable to that seen for diacylphosphatidylglycerol systems and suggests comparable headgroup motion. The lateral diffusion rates for Thermoplasma acidophilum total polar lipids in mutilamellar liposomes was measured by two dimensional exchange 31 P NMR as a function of temperature. At 55°C, near the growth temperature, the rate of lateral diffusion, D L, is comparable to that of diester phospholipids in the L α liquid crystalline phase, having a value of 2×10 −8 cm 2/s. D L decreases with temperature reaching a value of 8−6×10 −9 cm 2/s at 30°C. The activation energy E a for lateral diffusion is estimated to be 10 kcal/mol (∼42 kJ/mol). The lateral diffusion rates indicate that the tetraether liposomes have a membrane viscosity at 30°C which is considerably higher than that of diester phospholipids in the liquid crystalline phase.

Influence of annexin V on the structure and dynamics of phosphatidylcholine/phosphatidylserine bilayers: a fluorescence and NMR study.

The consequences of the binding of annexin V on the structure and dynamics of PC/PS bilayers were studied by means of fluorescence polarization, 31P NMR, 2H NMR, and fluorescence recovery after photobleaching (FRAP). Even at complete coverage of the lipid bilayers by the protein, annexin V showed no influence on the lipid molecular packing and the acyl chain flexibility of both PC and PS. The fluorescence polarization of the probe DPH, the 31P NMR spectra, and deuterium quadrupolar splittings of P(d31)OPS remained unchanged. However, upon binding of annexin V, two distinct populations of PC were visible in 2H NMR, which were in slow exchange on the deuterium NMR time scale (microseconds). One component in the spectrum was identical to the protein-free sample, while a second, broad, component appeared. The presence of the protein induced a decrease in the transverse relaxation times (T2e), indicative of the appearance of slow motions (milliseconds to microseconds), in the P(d31)-OPS spectrum and in the P(d31)OPC broad component. FRAP experiments were carried out with the probes C12-NBD-PC and C12-NBD-PS: at saturation, annexin V reduced the lateral diffusion rate of PC by 40% and nearly blocked the diffusion of PS. These combined experiments are consistent with a model in which annexin V enters a proteolipidic complex in the form of an extended 2D network, stabilized by specific interactions with PS. As seen from the lateral diffusion rates and the acyl chains NMR spectral parameters, two separate lipid populations appear, presumably corresponding to those interacting with annexinV (PC and PS) and protein free domains (mainly PC).

Spin-label studies of lipid-protein interactions with reconstituted band 3, the human erythrocyte chloride-bicarbonate exchanger.

Lipid-protein interactions in reconstituted band 3 preparations were investigated by using spin-labeled lipids in conjunction with electron paramagnetic resonance (EPR) spectroscopy. Purified erythrocyte band 3 was reconstituted into egg phosphatidylcholine liposomes at high protein density with preservation predominantly of the dimeric state. Lipid-protein associations were revealed by the presence of a component in the EPR spectra that, when compared to spectra obtained from protein-free bilayers, indicated that lipid chain motions are restricted by interactions with the protein. From the fraction of the motionally restricted component obtained from the phosphatidylcholine spin-label, a value of 64 +/- 14 annular lipids per band 3 dimer was obtained. This agrees with a value of 62 for the number of lipids that may be accommodated around the electron density map of a band 3 dimer. Selectivity of various spin-labeled lipids for the protein revealed that androstanol had a lower affinity for the band 3 interface, whereas a distinct preference was observed for the negatively charged lipids phosphatidylglycerol and stearic acid over phosphatidylcholine. This preference for negatively charged lipids could not be screened by 1-M salt, indicating that electrostatic lipid-protein interactions are not dominant. Estimates of annular lipid exchange rates from measured acyl chain segmental motions suggested that the rate of exchange between bilayer and boundary lipids was approximately 10(6) s(-1), at least an order of magnitude slower than the rate of lipid lateral diffusion in protein-free bilayers.

A Nonvolatile MOSFET Memory Device Based on Mobile Protons in the Gate Dielectric

AbstractEver since the introduction of the metal-oxide-silicon field-effect-transistor (MOSFET), the nature of mobile and trapped charge in the oxide layer has been studied in great detail. For example, contamination with alkali ions such as sodium, causing instability of the flat-band voltage, was a major concern in the early days of MOS fabrication. Another SiO2 impurity of particular interest is hydrogen, because of its beneficial property of passivating charge traps. In this work we show that annealing of Si/SiO2/Si structures in forming gas (Ar:H2; 95:5) above 400 °C can introduce mobile H+ ions into the SiO2 layer. These mobile protons are confined within the oxide layer, and their space-charge distribution is well controllable and easily rearrangeable by applying a gate bias, making them potentially useful for application in a reliable nonvolatile MOSFET memory device. We present speed, retention, endurance, and radiation tolerance data showing that this non-volatile memory technology can be competitive with existing Si-based non-volatile memory technologies such as Flash.The chemical kinetics of mobile-proton reactions in the SiO2 film are also analyzed in greater detail. Our data show that the initial buildup of mobile protons during hydrogen annealing is limited by the rate of lateral hydrogen diffusion into the buried SiO2 films. The final density of mobile protons is determined by the cooling rate which terminates the annealing process and, in the case of subsequent anneals, by the temperature of the final anneal. To explain the observations, we propose a dynamical equilibrium model. Based on these insights, the incorporation of the proton generation process into standard semiconductor process flows is discussed.

Fatty Acid Alteration and the Lateral Diffusion of Lipids in the Plasma Membrane of Keratinocytes

The fluorescent probe diI was used to study the lateral mobility of lipids inin vitrostrains of living adult human keratinocytes grown in four different media. One medium was essential fatty acid deficient (EFAD) and low in calcium ion, a medium known to yield cells that proliferate rapidly and contain lipid with extremely low levels of essential fatty acids. Two other media were supplemented with essential fatty acids (FAS), media that are known to result in cells that grow more slowly and have normalized fatty acid proportions. A fourth medium consisted of 1 μMall-trans-retinoic acid added to the fatty acid-supplemented medium (FAS-RA), a medium known to produce cells that are highly proliferative, with a growth rate greater than that of the FAS strains and similar to that of the EFAD strains. The keratinocytes grown in these four media were studied using the fluorescence recovery after photobleaching (FRAP) technique to determine the lateral diffusion rate of diI in the plasma membranes. Our results showed a positive correlation between growth rate and diffusion coefficient (D): the diffusion coefficient of diI was higher in the EFAD or FAS-RA cells than in the FAS cells. The measurement ofDamong the FAS cells fell into two groups. One group was similar to the single group seen in the EFAD cells, but the other group was composed of much lowerDvalues. The other FRAP parameters (mobile fraction and bleach depth) were larger in the “slow” group than in the “fast” group. This trend of negative correlation between these parameters andDwas also found within the fast group. These results are interpreted in terms of possible changes in membrane structure or morphology that might be indirectly associated with the fatty acid alterations, including the possible presence of areas in senescing keratinocytes where plasma membranes collapse to form an interacting system of lipid bilayers.

Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease

The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes. Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio IE/IM. Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold. Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the IE/IM for PPDPC from 0.16 to 0.05, respectively. An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented IE/IM should arise from partial lateral segregation of PPDPC in the vesicles. This notion is supported by the further 38% increase in IE/IM observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000. The importance of the length of the lipid-protein boundary is implicated by the reduction in IE/IM resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody. Interestingly, excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease. Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved. Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresce inthio- carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control vesicles lacking the reconstituted protein only 40% quenching was evident. The addition of 1,2-dipalmitoyl-sn-glycero-3-phospho-rac'-glycerol (DPPG) decreased IE/IM for PPDPC, revealing the driving force for the lateral segregation of this probe to become attenuated. More specifically for protein-free bilayers at XDPPG = 0.10 the rate of lateral diffusion of PPDPC in POPG is diminished, as evidenced by the 24% decrement in IE/IM, under these conditions the increase in IE/IM due to lac permease was strongly reduced, by approximately 84%. The present data are interpreted in terms of the hydrophobic mismatch theory, which predicts that integral membrane proteins will draw lipids of similar hydrophobic thickness into their vicinity. In brief, the approximate lengths of most of the predicted 12 hydrophobic, membrane-spanning alpha-helical segments of lactose permease range between 28.5 and 37.5 A and thus exceed the hydrophobic thickness of POPG of approximately 25.8 A. Therefore, to reduce the free energy of the assembly, longer lipids such as PPDPC and DPPF are accumulated in the immediate vicinity of lactose permease in fluid, liquid crystalline POPG bilayers.

Determination of microviscosity and location of 1,3-Di(1-pyrenyl)propane in brain membranes

We determined the microviscosity of synaptosomal plasma membrane vesicles (SPMV) isolated from bovine cerebral cortex and liposomes of total lipids (SPMTL) and phospholipids (SPMPL) extracted from SPMV. Changes in the microviscosity induced by the range and rate of lateral diffusion were measured by the intramolecular excimerization of 1,3-di(1-pyrenyl)-propane (Py-3-Py). The microviscosity values of the direct probe environment in SPMV, SPMTL and SPMPL were 38.17, 31.11 and 27.64 cP, respectively, at 37 degrees C and the activation energies (E(a)) of the excimer formation of Py-3-Py in SPMV, SPMTL and SPMPL were 8.236, 7.448 amd 7.025 kcal/mol, respectively. Probe location was measured by polarity and polarizability parameters of the probe Py-3-Py and probe analogues, pyrene, 1-pyrenenonanol and 1-pyrenemethyl-3beta-hydroxy-22,23-bisnor-5-cholenate (PMC), incorporated into membranes or solubilized in reference solvents. There existed a good linear relationship between the first absorption peak of the(1)L(a) band and the polarizability parameter (n (2)-1)/(2n (2)+1). The calculated refractive index values for SPMV, SPMTL and SPMPL were close to 1.50, which is higher than that of liquid paraffin (n=1.475). The probe location was also determined by using a polarity parameter (f-1/2f(1)). Here f=(epsilon-1)/(2epsilon+1) is the dielectric constant function and f'=(n (2)-1)/(2n (2)+1) is the refractive index function. A correlation existed between the monomer fluorescence intensity ratio and the solvent polarity parameter. The probes incorporated in SPMV, SPMTL, and SPMPL report a polarity value close to that of 1-hexanol (epsilon=13.29). In conclusion, Py-3-Py is located completely inside the membrane, not in the very hydrophobic core, but displaced toward the polar head groups of phospholipid molecules, e.g., central methylene region of aliphatic chains of phospholipid molecules.

Evidence for the formation of microdomains in liquid crystalline large unilamellar vesicles caused by hydrophobic mismatch of the constituent phospholipids

The excimer-to-monomer fluorescence emission intensity ratio (IE/IM) of the fluorescent probe 1-palmitoyl-2-[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC, 1 mol%) was measured at 30 degrees C as a function of the thickness of fluid liposomal membranes composed of phosphatidylcholines (PCs) with homologous monounsaturated acyl chains of varying lengths N (= number of carbon atoms). Upon decreasing N from di-24:1 PC to di-14:1 PC, the rate of excimer formation was sigmoidally augmented from 0.02 to 0.06. This increase in IE/IM can arise either from enhanced lateral mobility or from the lateral enrichment of PPDPC into domains, or both. Direct evidence for partial lateral segregation of PPDPC being involved is provided by experiments where 1.6 mol% of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N- (5-fluoresceinthiocarbamoyl) (DPPF) was included together with PPDPC into the bilayers. Notably, because of spectral overlap DPPF can function as a resonance energy transfer acceptor for pyrene excimer. Fluorescence intensity ratio (F/Fo) measured at 480 nm for PPDPC/DPPF (yielding F) and PPDPC (yielding Fo) containing membranes as a function of N reveals a sharp maximum for di-20:1 PC, i.e., the quenching of pyrene excimer fluorescence by DPPF is least efficient in this lipid and is enhanced upon either decrease or increase in N. This is compatible with colocalization of DPPF in PPDPC enriched domains when N not equal to 20, whereas in di-20:1 PC these probes appear to be effectively dispersed. The driving force for the enrichment of PPDPC in thin (N < 20) and thick (N > 20) PC matrices is likely to be hydrophobic mismatch of the effective lengths of the matrix phospholipids and the fluorescent probes. We also measured fluorescence polarization (P) for 1,6-diphenyl-1,3,5-hexatriene (DPH) as well as the IE/IM for the intramolecular excimer forming probe 1,2-bis[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (bisPDPC) as a function of N. In brief, neither the fluorescence polarization data and nor the measurements of intramolecular chain dynamics using bisPDPC concur with enhanced lateral diffusion as the sole cause for the increase in the IE/IM for PPDPC in thin membranes. Our findings suggest hydrophobic mismatch as the cause of microdomain formation of lipids in fluid, liquid crystalline bilayers, while simultaneously allowing for a high rates of lateral diffusion. Such hydrophobic mismatch-induced compositional fluctuations would also offer one plausible explanation for the chain length diversity observed for biological membranes.

Effect of polyimide processing on multichip glass ceramic module fabrications

This paper discusses the yield analysis of the thin film wiring layers fabricated on the 127 mm multichip glass ceramic modules (MCM-D), currently used on the IBM Enterprise System/9000 family of computer processors. To select a suitable polyimide (PI) for the thin film wiring layer, modules were fabricated with either the 3,3',4,4'-benzophenone tetracarboxylic dianhydride-1,3-bis(3-aminophenoxy)benzene (BTDA-APB) or the pyromellitic dianhydride-oxydianiline (PMDA-ODA) polyimide. By keeping all other processing parameters and structures the same, the wiring layers fabricated with the PMDA-ODA polyimide exhibited significantly better yield than those made of the BTDA-APB PI. The yield loss in the modules fabricated with the BTDA-APB PI occurred during thermal processing, where some of the transmission lines fabricated atop the PI were found cracked. Further investigations indicate that, during lift-off processing, the presence of a small contaminant, such as a fiber, metal flake, a particle, or polishing scratches can expose the PI to the hot N-methylpyrollidinone (NMP) solvent. Depending on the polyimide used for thin film processing, the diffusion and swelling of the PI by the low molecular weight organic solvent can potentially produce significant damage to the polyimide/Cu wiring structure and the consequent yield loss of the modules due to line opens. The magnitude of the damage was found to depend on the rate of solvent diffusion, process temperature, lift-off time and the amount of PI swelling during processing. To understand these important process parameters in detail, in-plan solvent diffusion and PI swelling in the PMDA-ODA and BTDA-APB films were quantitatively measured. It was found that the rate of in-plane (X-Y plane) solvent diffusion in BTDA-APB is D=0.0617 /spl times/ex-(-9.4 Kcal/RT) cm/sup 2//s versus D=0.00155/spl times/exp(-10.24 Kcal/RT) cm/sup 2//s for the PMDA-ODA film. Both types of PI films were fully imidized at 375/spl deg/C in nitrogen for 1 h. Thus, the rate of lateral diffusion of NMP in BTDA-APB is approximately 150 times faster than that of the PMDA-ODA film at 85/spl deg/C. Furthermore, the swelling of PMDA-ODA PI in the 85/spl deg/C NMP bath is 45% versus 82% for the BTDA-APB PI. The poor elongation property of the BTDA-APB film (strain at break /spl sim/4-7% versus 90-130% for the PMDA-ODA PI) further aggravated the solvent problem, BTDA-APB was always found cracked or crazed after being exposed to the NMP solvent, making it very vulnerable to the lift-off process. Consequently, by selecting the PMDA-ODA polyimide for module manufacturing, along with an improved clean room manufacturing facility, the glass ceramic MCM-D has become an extremely reliable product with excellent manufacturing yield.

Mechanism of liposome destabilization by polycationic amino acids.

Polycationic amino acids induce the leakage and fusion of liposomes containing anionic lipids. We have investigated the nature and extent of the changes in membrane physical properties caused by these polypeptides which could result in the observed membrane destabilization. We found that in the range of pH 5 to pH 7 both poly-L-histidine and poly-L-lysine were ineffective in shifting the bilayer to hexagonal phase transition temperature of dielaidoylphosphatidylethanolamine, either in the presence of absence of 1-palmitoyl-2-oleoylphosphatidylserine. We also studied the gel to liquid crystalline phase transition properties of 1:1 mixtures of phosphatidylserine and phosphatidylethanolamine, both in dimyristoyl forms as well as the 1-palmitoyl-2-oleoyl forms, as a function of pH and in the presence and absence of polycationic amino acids. We observed that these two lipids were largely miscible at all pH values and in the presence and absence of the polypeptides. However, there was some increased tendency for phase separation at higher pH and in the absence of polypeptide. Thus neither changes in curvature strain nor lateral phase separation induced by the polycationic amino acids could account for their marked ability to induce leakage and fusion. Phosphatidylethanolamine labelled with pyrene on one of the acyl chains gives rise to fluorescent emission from both monomer and excimer forms. The ratio of emission intensity from these two forms is indicative of lateral phase separation and the degree of lateral mobility of this probe. In equimolar mixtures of the 1-palmitoyl-2-oleoyl forms of phosphatidylserine and phosphatidylethanolamine in the liquid crystalline phase at 30 degrees C we find little effect of pH on the ratio of excimer to monomer emission intensity. However poly-L-lysine markedly lowers the fraction of excimer emission from these liposomes through the pH range from 5 to 7. Poly-L-histidine lowers the excimer to monomer emission ratio at pH 5 but not at pH 7. This is opposite to what one would expect for lateral phase separation and is interpreted at being the consequence of the polypeptide lowering the rate of lateral diffusion of the lipids. This effect of poly-L-histidine is observed over a range of temperatures from 0 to 40 degrees C in both gel and liquid crystalline phases. There is no evidence from the behaviour of the pyrene fluorescent probe for lipid interdigitation.(ABSTRACT TRUNCATED AT 400 WORDS)

(Galacto) lipid export from envelope to thylakoid membranes in intact chloroplasts. II. A general process with a key role for the envelope in the establishment of lipid asymmetry in thylakoid membranes

The transfer in organelle of newly synthesized lipid molecules from inner envelope to thylakoid membranes, as well as their subsequent transbilayer distribution in these membranes, have been studied in intact chloroplasts isolated from young and mature spinach, young pea and mature lettuce leaves, using a recently developed methodology (Rawyler, A., Meylan, M. and Siegenthaler, P.A. (1992) Biochim. Biophys. Acta 1104, 331–341). Three radiolabelled precursors were used. UDP-[ 14C]galactose allowed to follow the fate of mono- and digalactosyldiacylglycerol (MGDG and DGDG) made from polyunsaturated, preexisting diacylglycerol (DAG), whereas [ 14C]acetate and [ 14C]glycerol 3-phosphate were used to follow the fate of MGDG and phosphatidylglycerol (PG), respectively, after de novo synthesis. MGDG, DGDG and PG molecules assembled at the envelope level were found to be exportable to thylakoids in amounts strictly proportional to the amounts synthesized, provided that the necessary substrates were not limiting. Lipid export was class-selective; under our conditions, as much as 50–80% of the MGDG, 87% of the PG and 20–30% of the DGDG synthesized were exported to thylakoids. However, within the MGDG class labelled from [ 14C]acetate, there was hardly any selectivity in the export of its various molecular species. For MGDG, the proportionality coefficient, which reflects the efficiency of the export process, was higher in chloroplasts from young than from mature leaves, and higher in spinach than in pea and lettuce. Temperature affected the efficiency of galactolipid export in a class-dependent way. MGDG synthesis and export had similar Q 10 values of about 4 in young and 3 in mature spinach leaves, while the Q 10 of DGDG export was higher than that of its synthesis. In most cases, the transmembrane distribution of labelled lipids in thylakoids was found to match closely the corresponding distribution of mass, regardless of plant age and species and of incubation time and temperature. In some cases however, small but significant differences occurred between the label and the mass transbilayer distributions of MGDG (labelled molecules more inwardly oriented), DGDG and PG (more outwardly oriented). We propose a general model in which the thylakoid lipid asymmetry is primarily preestablished in the chloroplast envelope by the topography of its lipid-synthesizing enzymes, together with the occurrence of relatively fast lateral diffusion and translocation rates of the newly synthesized lipids. Transient fusions between inner envelope and thylakoid membranes would allow lipid export by lateral diffusion and build the observed lipid asymmetry in the latter.

Lateral diffusion of molecules partitioned into C-18 ligands on silica surfaces.

Lateral diffusion rates of a fluorescent probe molecule partitioned into chromatographic C-18 ligands on a planar silica substrate were measured as a function of overlaying solvent composition. Diffusion coefficients of the probe were measured with a fluorescence recovery after patterned photobleaching experiment. A total internal reflection fluorescence flow cell held the derivatized planar substrate in contact with various aqueous solutions and allowed the fluorescence signal from two interfering, total internally reflected laser beams to be imaged onto a detector. Diffusion coefficients for the partitioned probe were approximately 3 orders of magnitude smaller than diffusion in solution and were found to increase with increasing methanol concentration in the overlaying aqueous solvent mixture. Inability of a solution phase ionic quencher to quench the fluorescence of sorbed rubrene confirmed that the probe is fully partitioned into the C-18 chains.

Determination of ion-exchanged channel waveguide profile parameters by mode-index measurements

We report a method for characterizing ion-exchanged channel waveguides in terms of the diffusion depth, width, and surface-index change from mode-index measurements. The method is then used to study the effect of the diffusion time and the mask width on these parameters in K(+)-Na(+)-exchanged guides in BK7 glass. It is observed that the mask width affects the waveguide dimensions in ways not reported in earlier studies. Our results reveal that the diffusion depth depends on the mask opening and saturates with time to a maximum value for a given mask width. The lateral-diffusion rate and the side diffusion beneath the mask are independent of the mask width, which indicates that the lateral electric field associated with the metallic mask plays an important role in such diffusion. We discuss the implications of this behavior to modeling and fabricating integrated optical devices.

Lateral mobility of plasma membrane proteins in dividing eggs of the loach (Misgurnus fossilis): Regional differences and changes during the cell cycle

Regional differences in lateral diffusion rates of fluorescence-labeled proteins have been studied in the plasma membrane of dividing eggs of the loach (Misgurnus fossilis) by fluorescence recovery after photobleaching (FRAP). Apparent animal-vegetal differences in fluorescence intensity, lateral diffusion coefficients, and fractions of mobile proteins have been found, with all these quantities being higher in the animal pole region than in the yolk region. Cyclic changes in protein diffusion coefficients and mobile fractions during the first few cell cycles have also been recorded. Soon after the end of a cleavage, the diffusion coefficient reaches its minimal value and increases rapidly before the next cleavage.

Lateral mobility of Na,K-ATPase and membrane lipids in renal cells. Importance of cytoskeletal integrity.

Because membrane fluidity is an important determinant of membrane function, the lateral diffusion rate (DL) of the membrane protein Na,K-ATPase was determined in intact renal proximal tubule epithelial cells by the technique of fluorescence redistribution after photobleaching (FRAP). In normal cells the DL of Na,K-ATPase in the basal membrane was 3.31 x 10(-10) cm2/sec. Treatment with cytochalasin D to promote actin filament depolymerization caused a sevenfold increase in DL. Exposure of cells to a Ca(2+)-free medium or to hypoxia and reoxygenation, which have similar disruptive effects on the cytoskeleton, also caused increases in DL. Disruption of actin microfilament structure also increased the mobile fraction of Na,K-ATPase. Using a confocal laser microscopic technique only 14.9% of total Na,K-ATPase was observed to reside in the apical membrane domain of normal cells. Microfilament depolymerization caused this fraction to increase to 47.7%. Thus, the translocation of Na,K-ATPase from the basolateral to the apical domain induced by cytoskeletal protein dysfunction was enabled by an increased rate of lateral diffusion of Na,K-ATPase. The behavior of a variety of membrane lipids following actin depolymerization was more heterogeneous. Some lipids showed a similar increase in DL, whereas others showed very little dependence upon the cytoskeleton for lateral restraint.

Lipid bilayer heterogeneities and modulation of phospholipase A 2 activity

The regulation of phospholipase A 2 (PLA 2) activity toward synthetic vesicular substrates is a model for the modulation of enzyme function by biological membranes. PLA 2's catalytic rate toward membrane phospholipids can be modified by several orders of magnitude by altering the membrane's composition and structure. The physical basis of this sensitivity is the subject of this report. The results described here imply that the salient features of membrane-structure which modulate PLA 2 activity include: compositional phase separation; membrane curvature and, possibly, curvature-associated defects; and dynamic product inhibition due to limitations imposed by the rate of lateral diffusion of lipid in the membrane. Furthermore, it is shown that the effects of membrane structure on the catalytic rate are not exerted merely by enhancing association of PLA 2 with the membrane surface: a membrane-bound inactive state is spectroscopically identified. Finally, these results are discussed in the context of some published models for the role of membrane structure in the regulation of membrane-bound enzymes.