Tectorial membrane changes in hypothyroid rats during postnatal development

The morphological changes of the tectorial membrane (TM) during the postnatal development (0, 3, 6, 12 and 25 day old) of the organ of Corti were studied by light microscopy in 20 control and hypothyroid rats. Hypothyroidism was induced by daily administration of propylthioruracil (PTU) until the end of lactation. The auditive receptor in the cochlea of the hypothyroid animals shows serious structural alterations compared with those of normal ones: abnormal persistence of K?lliker's organ, immaturity of sensory cells and supporting cells and a specific distortion of the TM. Differences with controls were first observed on the sixth postnatal day of the hypothyroid rats. The inner spiral sulcus was not shaped and the TM was attached to the K?lliker's organ. In older stages (12 and 25 days), K?lliker's organ was still present. The TM acquired a shap hump with an abnormal fibrillar arrangement in its middle part. It was still attached to the outer supporting cells by a remnant of the marginal net. It was suggested that the TM is secreted by the inner spiral limbus and K?lliker's organ. An abnormal persistence of these structures in the hypothyroidism results in a retardation of Corti's organ development. However, this conclusion does not explain the absence of the outer portion of the TM. Our study confirms the hypothesis that the secretion of any components of the marginal zone of TM is made by outer supporting cells which in PTU-treated animals appear very immature and with hypoplasia.     

Bioactive polyacrylamide hydrogels with gradients in mechanical stiffness

We propose a novel, single step method for the production of polyacrylamide hydrogels with a gradient in mechanical properties. In contrast to already existing techniques such as UV photo‐polymerization with photomasks (limited penetration depth) or microfluidic gradient mixers (complex microfluidic chip), this technique is not suffering such limitations. Young's modulus of the hydrogels was varied by changing the total monomer concentration of the hydrogel precursor solution. Using programmable syringe pumps, the total monomer concentration in the solution fed to the hydrogel mold was varied from 16 wt% down to 5 wt% over the feeding time to obtain a gradient in compliance ranging from 150 kPa down to 20 kPa over a length of 10 mm down to 2.5 mm. Polymerization was achieved with the dual initiation system composed of ammonium persulfate and N,N,N′,N′‐tetramethylethylenediamine, which were both fed through separate capillaries to avoid premature polymerization. Functionalized with the model ligand collagen I, the substrates were bioactive and supported the attachment of human foreskin fibroblasts (around 30% of the cells seeded attached after 1 h). A kinetic morphology study on homogeneous hydrogels of different stiffness's indicated that fibroblasts tend to spread to their final size within 2 h on stiff substrates, while the spreading time was much longer (ca. 4–5 h) on soft substrates. These trends were confirmed on hydrogels with compliance gradients, showing well spread fibroblasts on the stiff end of the hydrogel after 2 h, while the cells on the soft end still had small area and rounded morphology. Biotechnol. Bioeng. 2013; 110: 1508–1519. © 2012 Wiley Periodicals, Inc.     

Tectorial membrane material properties in Tecta(Y)(1870C/+) heterozygous mice

The solid component of the tectorial membrane (TM) is a porous matrix made up of the radial collagen fibers and the striated sheet matrix. The striated sheet matrix is believed to contribute to shear impedance in both the radial and longitudinal directions, but the molecular mechanisms involved have not been determined. A missense mutation in Tecta, a gene that encodes for the α-tectorin protein in the striated sheet matrix, causes a 60-dB threshold shift in mice with relatively little reduction in outer hair cell amplification. Here, we show that this threshold shift is coupled to changes in shear impedance, response to osmotic pressure, and concentration of fixed charge of the TM. In Tecta^Y1870C/+ mice, the tectorin content of the TM was reduced, as was the content of glycoconjugates reacting with the lectin wheat germ agglutinin. Charge measurements showed a decrease in fixed charge concentration from −6.4±1.4 mmol/L in wild-types to −2.1±0.7 mmol/L in Tecta^Y1870C/+ TMs. TMs from Tecta^Y1870C/+ mice showed little volume change in response to osmotic pressure compared to those of wild-type mice. The magnitude of both radial and longitudinal TM shear impedance was reduced by 10±1.6 dB in Tecta^Y1870C/+ mice. However, the phase of shear impedance was unchanged. These changes are consistent with an increase in the porosity of the TM and a corresponding decrease of the solid fraction. Mechanisms by which these changes can affect the coupling between outer and inner hair cells are discussed.     

Plasma membrane fluidity gradients of human peripheral blood leukocytes

The shape of the fluidity gradient of the outer hemileaflet of the plasma membrane of normal, living, human white blood cells was determined using a series of n-(9-anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 11, 12, and 16, to establish a baseline for future studies on the consequences of various pathological states. Fluorescence uptake and steady-state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, with the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence was, for the n-(9-anthroyloxy) series, in the order 2 greater than 3 greater than 6 greater than 7 greater than 9 greater than 11 = 12 = 16 for neutrophils, lymphocytes, and monocytes. Anisotropy values were constant from 5 to 30 min after addition of the various probes. The orders of the anisotropy magnitudes, indicative of the shapes of the fluidity gradient, were, for neutrophils, 6 greater than 7 greater than 9 greater than 2 = 3 = 11 = 12 greater than 16, for lymphocytes, 7 greater than 6 greater than 9 greater than 11 greater than 2 = 3 greater than 11 = 12 greater than 16, and for monocytes, 9 greater than 7 greater than 6 greater than 11 greater than 2 = 3 greater than 12 greater than 16. The kinetics of anisotropy from 1 to 5 min after addition of the probes differed for each of the three cell types. Probes with an n-value less than or equal to the maxima (n = 6, neutrophils; n = 7, lymphocytes; n = 9, monocytes) rapidly (1.2 min) reached equilibrium, whereas those probes with n-values greater than the maxima took progressively longer times to equilibrate as n increased. This behavior is consistent with the existence of an energy barrier just below the approximate region sensed by the probes, which would correspond to just below 6AS for neutrophils, 7AS for lymphocytes, and 9AS for monocytes.     

A combination of immobilised pH gradients improves membrane proteomics

Membrane protein analyses have been notoriously difficult due to hydrophobicity and the general low abundance of these proteins compared to their soluble cytosolic counterparts. Shotgun proteomics has become the preferred method for analyses of membrane proteins, in particular the recent development of peptide immobilized pH gradient isoelectric focusing (IPG-IEF) as the first dimension of two-dimensional shotgun proteomics. Recently, peptide IPG-IEF has been shown to be a valuable shotgun proteomics technique through the use of acidic narrow range IPG strips, which demonstrated that small acidic p I increments are rich in peptides. In this study, we assess the utility of both broad range (BR) (p I 3-10) and narrow range (NR) (p I 3.4-4.9) IPG strips for rat liver membrane protein analyses. Furthermore, the use of these IPG strips was evaluated using label-free quantitation to demonstrate that the identification of a subset of proteins can be improved using NR IPG strips. NR IPG strips provided 2603 protein assignments on average (with 826 integral membrane proteins (IMPs)) compared to BR IPG strips, which provided 2021 protein assignments on average (with 712 IMPs). Nonredundant protein analysis demonstrated that in total from all experiments, 4195 proteins (with 1301 IMPs) could be identified with 1428 of these proteins unique to NR IPG strips with only 636 from BR IPG strips. With the use of label-free quantitation methods, 1659 proteins were used for quantitative comparison of which 319 demonstrated statistically significant increases in normalized spectral abundance factors (NSAF) in NR IPG strips compared to 364 in BR IPG strips. In particular, a selection of six highly hydrophobic transmembrane proteins was observed to increase in NSAF using NR IPG strips. These results provide evidence for the use of alternative pH gradients in combination to improve the shotgun proteomic analysis of the membrane proteome.     

Cholesterol depletion increases membrane stiffness of aortic endothelial cells

This study has investigated the effect of cellular cholesterol on membrane deformability of bovine aortic endothelial cells. Cellular cholesterol content was depleted by exposing the cells to methyl-beta-cyclodextrin or enriched by exposing the cells to methyl-beta-cyclodextrin saturated with cholesterol. Control cells were treated with methyl-beta-cyclodextrin-cholesterol at a molar ratio that had no effect on the level of cellular cholesterol. Mechanical properties of the cells with different cholesterol contents were compared by measuring the degree of membrane deformation in response to a step in negative pressure applied to the membrane by a micropipette. The experiments were performed on substrate-attached cells that maintained normal morphology. The data were analyzed using a standard linear elastic half-space model to calculate Young elastic modulus. Our observations show that, in contrast to the known effect of cholesterol on membrane stiffness of lipid bilayers, cholesterol depletion of bovine aortic endothelial cells resulted in a significant decrease in membrane deformability and a corresponding increase in the value of the elastic coefficient of the membrane, indicating that cholesterol-depleted cells are stiffer than control cells. Repleting the cells with cholesterol reversed the effect. An increase in cellular cholesterol to a level higher than that of normal cells, however, had no effect on the elastic properties of bovine aortic endothelial cells. We also show that although cholesterol depletion had no apparent effect on the intensity of F-actin-specific fluorescence, disrupting F-actin with latrunculin A abrogated the stiffening effect. We suggest that cholesterol depletion increases the stiffness of the membrane by altering the properties of the submembrane F-actin and/or its attachment to the membrane.     

Spectrin folding versus unfolding reactions and RBC membrane stiffness

Spectrin (Sp), a key component of the erythrocyte membrane, is routinely stretched to near its fully folded contour length during cell deformations. Such dynamic loading may induce domain unfolding as suggested by recent experiments. Herein we develop a model to describe the folding/unfolding of spectrin during equilibrium or nonequilibrium extensions. In both cases, our model indicates that there exists a critical extension beyond which unfolding occurs. We further deploy this model, together with a three-dimensional model of the junctional complex in the erythrocyte membrane, to explore the effect of Sp unfolding on the membrane's mechanical properties, and on the thermal fluctuation of membrane-attached beads. At large deformations our results show a distinctive strain-induced unstiffening behavior, manifested in the slow decrease of the shear modulus, and accompanied by an increase in bead fluctuation. Bead fluctuation is also found to be influenced by mode switching, a phenomenon predicted by our three-dimensional model. The amount of stiffness reduction, however, is modest compared with that reported in experiments. A possible explanation for the discrepancy is the occurrence of spectrin head-to-head disassociation which is also included within our modeling framework and used to analyze bead motion as observed via experiment.     

Modulation of membrane potential in algal cells by temperature gradients

The aim of the present study is to ascertain whether transmembrane temperature gradients couple with transport of electric charge in living cells ofValonia utricularis and eventually measure the thermodynamic coupling coefficient (s). Simple experimental procedures are described that allow generation of temperature gradients of predetermined sense and intensity across the cell membrane. Simultaneous measurement of the potential difference is ensured by standard electrophysiological methods. The mathematical expressions that allow quantitative treatment of experimental results are indicated in the article and are based on standard nonequilibrium thermodynamic and electrophysiological formalism. The value of the coupling coefficient between temperature gradient and flow of electric charge is indicated and concisely discussed in terms of possible mechanisms of ionic membrane transport.     

Determination of transmembrane pH gradients and membrane potentials in liposomes.

Techniques for determining large transbilayer pH gradients (delta pH) and membrane potentials (delta psi) induced in response to delta pH in large unilamellar vesicle liposomal systems by measuring the transbilayer redistribution of radiolabeled compounds have been examined. For liposomes with acidic interiors, it is shown that protocols using radiolabeled methylamine in conjunction with gel filtration procedures to remove untrapped methylamine provide accurate measures of delta pH in most situations. Exceptions include gel state lipid systems, where transbilayer equilibration processes are slow, and situations where the interior buffering capacity is limited. These problems can be circumvented by incubation at elevated temperatures and by using probes with higher specific activities, respectively. Determination of delta pH in vesicles with a basic interior using weak acid probes such as radiolabeled acetate in conjunction with gel filtration was found to be less reliable, and an alternative equilibrium centrifugation protocol is described. In the case of determinations of the membrane potentials induced in response to these pH gradients, probes such as tetraphenylphosphonium and thiocyanate provide relatively accurate measures of the delta psi induced. It is shown that the maximum transmembrane pH gradient that can be stably maintained by an egg phosphatidylcholine-cholesterol 100-nm-diam large unilamellar vesicle is approximately 3.7 units, corresponding to an induced delta psi of 220 mV or transbilayer electrical field of 5 x 10(5) V/cm.     

Bilayer membrane bending stiffness by tether formation from mixed PC-PS lipid vesicles

Recently, a new approach to measure the bending stiffness (curvature elastic modulus) of lipid bilayer membrane was developed (Biophys. J., Vol. 55; pp. 509-517, 1989). The method involves the formation of cylindrical membrane strands (tethers) from bilayer vesicles. The bending stiffness (B) can be calculated from measurements of the tether radius (Rt) as a function of the axial force (f) on the tether: B = f.Rt/2 pi. In the present report, we apply this method to determine the bending stiffness of bilayer membranes composed of mixtures of SOPC (1-stearoyl-2-oleoyl phosphatidyl choline) and POPS (1-palmitoyl-2-oleoyl phosphatidyl serine). Three different mixtures were tested: pure SOPC, SOPC plus 2 percent (mol/mol) POPS, and SOPC plus 16 percent POPS. The bending stiffness determined for these three different lipid mixtures were not significantly different (1.6-1.8 x 10(-12) ergs). Because POPS carries a net negative charge, these results indicate that changes in the density of the membrane surface charge have no effect on the intrinsic rigidity of the membrane. The values we obtain are consistent with published values for the bending stiffness of other membranes determined by different methods. Measurements of the aspiration pressure, tether radius and the tether force were used to verify a theoretical relationship among these quantities at equilibrium. The ratio of the theoretical force to the measured force was 1.12 +/- 0.17.     

Instability of brain synaptosomal membrane preparations to repeated ultracentrifugation in isoosmotic density gradients

Synaptosomal membranes from rat brain were found to be remarkably unstable, from the criterion of buoyant density, to repeated density gradient ultracentrifugation. These membranes underwent progressive changes in buoyant density through at least three cycles of fractionation on density gradients generating large proportions of material of both higher and lower buoyant density than the original fraction. The observed density changes were not due to osmotic effects of the various gradient density zones, clumping due to the influence of divalent cations, age of the membrane preparation, length of centrifugation time, intermediate processing steps or a variety of other factors. In addition, the method of homogenization including duration and rate of homogenization had only minimal effects on this property of buoyant density instability.     

Transport of nucleic acid bases against their concentration gradients through quaternized chitosan membrane

A water-insoluble anion exchange membrane was prepared by crosslinking with ethyleneglycol diglycidylether, a membrane made of quaternized chitosan and poly(vinyl alcohol). The transports of nucleic acid bases such as uracil, cytosine, adenine, and guanine were investigated as one side of the membrane in a diaphragm cell was acidic and the other basic. Uracil was transported against its concentration gradient from the basic side to the acidic side regardless of the pH on the basic side. Cytosine, adenine, and guanine were also transported against their concentration gradients, but the direction of their transport depended upon the pH on the basic side. In particular, the transport directions for adenine and guanine were switched during identical transport experiments. Mechanisms for the transport of these nucleic acid bases against their concentration gradients through the quaternized chitosan membrane are discussed.     

Porosity Controls Spread of Excitation in Tectorial Membrane Traveling Waves

Cochlear frequency selectivity plays a key role in our ability to understand speech, and is widely believed to be associated with cochlear amplification. However, genetic studies targeting the tectorial membrane (TM) have demonstrated both sharper and broader tuning with no obvious changes in hair bundle or somatic motility mechanisms. For example, cochlear tuning of Tectb^–/– mice is significantly sharper than that of Tecta^Y1870C/+ mice, even though TM stiffnesses are similarly reduced relative to wild-type TMs. Here we show that differences in TM viscosity can account for these differences in tuning. In the basal cochlear turn, nanoscale pores of Tecta^Y1870C/+ TMs are significantly larger than those of Tectb^–/– TMs. The larger pore size reduces shear viscosity (by ∼70%), thereby reducing traveling wave speed and increasing spread of excitation. These results demonstrate the previously unrecognized importance of TM porosity in cochlear and neural tuning.     

Porosity Controls Spread of Excitation in Tectorial Membrane Traveling Waves

Cochlear frequency selectivity plays a key role in our ability to understand speech, and is widely believed to be associated with cochlear amplification. However, genetic studies targeting the tectorial membrane (TM) have demonstrated both sharper and broader tuning with no obvious changes in hair bundle or somatic motility mechanisms. For example, cochlear tuning of Tectb^–/– mice is significantly sharper than that of Tecta^Y1870C/+ mice, even though TM stiffnesses are similarly reduced relative to wild-type TMs. Here we show that differences in TM viscosity can account for these differences in tuning. In the basal cochlear turn, nanoscale pores of Tecta^Y1870C/+ TMs are significantly larger than those of Tectb^–/– TMs. The larger pore size reduces shear viscosity (by ∼70%), thereby reducing traveling wave speed and increasing spread of excitation. These results demonstrate the previously unrecognized importance of TM porosity in cochlear and neural tuning.     

Quantitation of hydrogen ion and potential gradients in gastric plasma membrane vesicles.

The ATP-dependent uptake of H+ by hog gastric parietal cell vesicles was quantitated by using the pH indicator dyes bromcresol green and malachite green, the weak bases, aminopyrine and 9-aminoacridine, and the pH electrode. A K+-dependent H+ uptake was found, with a significant difference between the quantity of H+ disappearing from the medium (deltaHo) and the quantity appearing inside the vesicle (deltaHi). 9-Aminoacridine gave a lower value for the deltaHi than any of the other probes. Probes of potential such as diethyloxadicarbocyanine or oxonol dyes showed that only secondary diffusion potentials occurred during H+ uptake and that the cationic dyes in the presence of protonophores could also be used to quantitate H+ uptake. The potential in the presence of protonophore indicated a deltaHi greater than that found with the other probes. Binding sites for acridine orange were generated either by ATP or an artificial pH gradient and corresponded to the deltaHi indicated by aminopyrine. SCN- (30mM) only partially inhibited the H+ gradient, and this, coupled with the failure to detect the physiological deltapH of 6.6, indicated that these vesicles may be an incomplete model of gastric acid secretion.     

Tectorial Membrane Morphological Variation: Effects upon Stimulus Frequency Otoacoustic Emissions

The tectorial membrane (TM) is widely believed to play an important role in determining the ear's ability to detect and resolve incoming acoustic information. While it is still unclear precisely what that role is, the TM has been hypothesized to help overcome viscous forces and thereby sharpen mechanical tuning of the sensory cells. Lizards present a unique opportunity to further study the role of the TM given the diverse inner-ear morphological differences across species. Furthermore, stimulus-frequency otoacoustic emissions (SFOAEs), sounds emitted by the ear in response to a tone, noninvasively probe the frequency selectivity of the ear. We report estimates of auditory tuning derived from SFOAEs for 12 different species of lizards with widely varying TM morphology. Despite gross anatomical differences across the species examined herein, low-level SFOAEs were readily measurable in all ears tested, even in non-TM species whose basilar papilla contained as few as 50-60 hair cells. Our measurements generally support theoretical predictions: longer delays/sharper tuning features are found in species with a TM relative to those without. However, SFOAEs from at least one non-TM species (Anolis) with long delays suggest there are likely additional micromechanical factors at play that can directly affect tuning. Additionally, in the one species examined with a continuous TM (Aspidoscelis) where cell-to-cell coupling is presumably relatively stronger, delays were intermediate. This observation appears consistent with recent reports that suggest the TM may play a more complex macromechanical role in the mammalian cochlea via longitudinal energy distribution (and thereby affect tuning). Although significant differences exist between reptilian and mammalian auditory biophysics, understanding lizard OAE generation mechanisms yields significant insight into fundamental principles at work in all vertebrate ears.