Spatially-resolved optical imaging of membrane potentials induced by applied electric fields.
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Animals↗
Biomedical subjects
W W Webb
Publications and source records attributed to W W Webb.
Coupling of ATP synthesis to reversal of rat liver microsomal Ca2+-ATPase.
The reversal of the rat liver microsomal Ca2+-ATPase transport cycle was studied. Microsomes were loaded with 45Ca2+ (approximately 30 nmol/mg of protein) in an ATP-dependent process, and the time dependency of the microsomal 45Ca2+ efflux was determined with various ADP and inorganic phosphate (Pi) concentrations. Pseudo-first-order rate constants (K'e) for 45Ca2+ efflux were determined. Although there was considerable 45Ca2+ efflux in the absence of added ADP or Pi, the addition of ADP or Pi alone had minimal effects upon the K'e; in contrast, a 2.5-fold increase in the K'e was observed in the presence of both ADP and Pi. The apparent Km values for ADP and Pi were 4 microM and 0.22 mM, respectively. Stimulation of 45Ca2+ efflux by ADP and Pi was associated with ATP synthesis. The calcium ionophore A23187 prevented ATP synthesis, which indicates that the Ca2+ gradient facilitates the coupling of ATP synthesis to Ca2+ efflux.
Adenosine Diphosphate↗
Electric field-induced redistribution and postfield relaxation of low density lipoprotein receptors on cultured human fibroblasts.
The lateral mobility of unliganded low density lipoprotein-receptor (LDL-R) on the surface of human fibroblasts has been investigated by studying the generation and relaxation of concentration differences induced by exposure of the cultured cells to steady electric fields. The topographic distribution of receptors was determined by fluorescence microscopy of cells labeled with the intensely fluorescent, biologically active LDL derivative dioctadecylindolcarbocyanine LDL (dil(3)-LDL), or with native LDL and anti-LDL indirect immunofluorescence. Exposure of the LDL-receptor-internalization defective J. D. cells (GM2408A) to an electric field of 10 V/cm for 1 h at 22 degrees C causes greater than 80% of the cells to have an asymmetric distribution of LDL-R; receptors accumulate at the more negative pole of the cell. In contrast, only 20% of LDL-internalization normal GM3348 cells exposed to identical conditions have asymmetrical distributions. Phase micrographs taken during electric-field exposure rule out cell movement as the responsible mechanism for the effect. In both cell types, postfield labeling with the F-actin-specific fluorescent probe nitrobenzoxadiazole-phallacidin shows that no topographic alteration of the actin cytoskeleton accompanies the redistribution of cell surface LDL-Rs, and indirect immunofluorescence labeling of the coat protein clathrin shows that coated pits do not redistribute asymmetrically. Measurements of the postfield relaxation in the percentage of GM2408A cells showing an asymmetric distribution allow an estimate of the effective postfield diffusion coefficient of the unliganded LDL-R. At 37 degrees C, D = 2.0 X 10(-9) cm2/s, decreasing to 1.1 X 10(-9) cm2/s at 22 degrees C, and D = 3.5 X 10(-10) cm2/s at 10 degrees C. These values are substantially larger than those measured by photobleaching methods for the LDL-R complexed with dil(3)-LDL on intact cells, but are comparable to those measured on membrane blebs, and are consistent with diffusion coefficients measured for other unliganded integral membrane receptor proteins by postfield-relaxation methods.
Cell Membrane↗
Wechsler Adult Intelligence Scale and Wechsler Adult Intelligence Scale-Revised in a neurologically impaired population.
Compared Verbal, Performance, and Full Scale IQ scores from two groups of neurologically impaired patients (N = 114) similar in age, years of education, occupation, race, sex, and etiology and location of cerebral dysfunction. One group had been given the WAIS and the other the WAIS-R. All three IQ scores were higher for the WAIS group, with Full Scale and Verbal scores significantly (p less than .05) higher. Changes in item content and standardization sample cohort effects are offered as partial possible explanation for the results. The IQ scores from the two tests cannot be considered as interchangeable for neurological patients.
Brain Damage, Chronic↗
Thermal fluctuations of large cylindrical phospholipid vesicles.
The time correlation function of the shape fluctuations of large (greater than 10 micron), cylindrical, hydrated, phospholipid-membrane vesicles consisting of one bimolecular layer was measured. The restoring force of the membrane was due to the excess curvature of a membrane element. A value for the curvature elastic modulus, Kc, was obtained from the mean-square amplitude of the normal modes of the fluctuations using the equipartition theorem. An expression for the correlation time was found by solving the dynamics of the membrane's relaxation against the low Reynolds number viscous drag of the surrounding fluid. The amplitudes and correlation times of the fundamental bending mode of the cylindrical vesicles both yield Kc = 1-2 X 10(-12) ergs.
Biophysical Phenomena↗
Fluorescent erythrosin B is preferable to trypan blue as a vital exclusion dye for mammalian cells in monolayer culture.
Erythrosin B and trypan blue are tested and compared for their effectiveness as vital exclusion stains for mammalian cells in monolayer culture. Both stains are supposed to mark cells that have lost membrane integrity. Fluorescein diacetate (FDA), an efficient vital inclusion stain, is used as a control, as it marks cells retaining membrane integrity. Erythrosin B and FDA are used as fluorescent dyes, whereas trypan blue colors via light absorption. The effectiveness of both vital exclusion stains is assayed by their ability to stain a high percentage of monolayer cells exposed to treatments lethal to an entire cell population. Two types of lethal treatment, severe heat and metabolic poison, are employed. Erythrosin B stains all monolayer cells immediately after complete lethal treatment. Trypan blue optimally stains only about 60% of monolayer cells. Cell staining by erythrosin B and by FDA are found to be mutually exclusive. This result demonstrates the coincidence of viability indications by erythrosin B and FDA and thus confirms the reliability of both viability stains as they probe membrane permeability via independent mechanisms. This study shows that erythrosin B is an effective, nontoxic, and convenient fluorescent vital exclusion dye for three mammalian cell lines in monolayer culture, but tends to disqualify trypan blue for this application.
Animals↗
Photodamage to intact erythrocyte membranes at high laser intensities: methods of assay and suppression.
A simple hemolytic assay of the photodamage suffered by individual intact erythrocytes upon localized exposure to high laser intensities such as those encountered in fluorescence photobleaching recovery (FPR) experiments has been characterized. At incident beam powers over 100,000 W/cm2 at 514 or 568 nm, hemoglobin absorption induces thermal-shock lysis. Below the thermal-shock threshold, other intracellular chromophores appear to catalyze membrane contraction and lysis with linear dose kinetics in anaerobic preparations. A variety of chemical agents that inhibit photodamage to red cell ghost membranes at low intensities (less than 450 W/cm2) do not benefit intact cells at realistic FPR bleaching doses of 10,000 to 1,000,000 W/cm2. However, we find that sample deoxygenation dramatically photostabilizes erythrocytes, thus we have developed a physiologically compatible in situ deoxygenation method utilizing submitochondrial particles. The extent to which this and other deoxygenation procedures modify fluorophore photolability has been determined. FPR measurements become practical on intact erythrocytes when a combination of low bleach power, thorough deoxygenation, and long wavelength excitable (approximately 650 nm) fluorescence probes is employed; the equivalent of 60,000 photobleaches per cell can then be tolerated without detectable damage.
Animals↗
F-actin aggregates may activate transformed cell surfaces.
Observations on the role of transformation-specific F-actin aggregates [Carley et al, 1981] in altering morphology, adhesion and intercellular interaction in transformed cells are reported here. The appearance and disappearance of membrane- and substrate-associated F-actin aggregates (MAG and SAG, respectively) are followed in a cell line temperature-sensitive for transformation. Since MAG structures also appear near the membrane in suspension cultures of transformed cells and in transformed cells in coculture with untransformed cells, they appear to function at cell-cell contacts. Unlike microfilament bundles in untransformed cells, MAG and SAG do not contain the F-actin regulatory protein tropomyosin. The lack of tropomyosin in these structures near the membrane is reminiscent of areas of an exceptionally active actin cytoskeleton usually associated with motile processes of the normal cell membrane. Such areas of membrane-cytoskeletal interaction may be involved in the aberrant cell-cell communication as well as the aggressive behavior often seen in transformed cells.
Actins↗
Regulation and drug insensitivity of F-actin association with adhesion areas of transformed cells.
F-actin aggregates have been found near the substrate attachments in a variety of transformed cells (Carley et al., 1981). Interference reflection microscopy shows that these aggregates are present in central close adhesion areas in Rous sarcoma virus (RSV)-transformed rat kidney cells. If these transformed cells are incubated with N6, O2-dibutyryl 3':5'-cyclic monophosphoric acid (db-cAMP), adenosine 5'-monophosphoric acid (5'-AMP) or adenosine, the F-actin aggregates and their associated close adhesion areas disappear, and the cells flatten out. Treatment of untransformed cells with db-cAMP spreads their focal adhesion plaques and thickens microfilament bundles. Furthermore, F-actin aggregates are substantially more resistant to cytochalasin B and the Ca2+ ionophore A23187 than microfilament bundles in untransformed cells. These differences between F-actin complexes in untransformed and in RSV-transformed cells, with respect to morphology and sensitivities to db-cAMP and cytoskeleton-disrupting drugs, define properties of the change in F-actin regulation and association with the plasma membrane due to transformation.
Actins↗
Fast diffusion along defects and corrugations in phospholipid P beta, liquid crystals.
The diffusion of a fluorescent lipid analogue in liquid crystals of the anisotropic P beta, phase of dimyristoylphosphatidylcholine (DMPC) had been found to be highly variable, suggesting structural defect pathways. Fluorescence photobleaching recovery (FPR) experiments imply two effective diffusion pathways with coefficients differing by at least 100. This is consistent with fast diffusion along submicroscopic bands of disordered material ("defects") in the bilayer corrugations characteristic of this phase. Due to strains during transformation from the L alpha phase, the axis of the corrugations is ordinarily disrupted by mosaic patches rotationally disoriented within the mean plane of the molecular bilayers, although larger oriented domains are sometimes adventitiously aligned into microscopically visible striped textures. The corrugations are also systematically aligned along positive disclinations pairs or "oily streaks." Thus, fast diffusion occurs parallel to the disclination lines and along the textured stripes. FPR results yield an upper limit on the effective diffusion in the ordered material of D less than or equal to 2 X 10(-16) cm2/s at 22 degrees C, D less than or equal to 3 X 10(-17) cm2/s at 13 degrees C. In contrast the diffusion coefficient along defect pathways where disordered ribbons are aligned is D approximately 4 X 10(-11) cm2/s at 16 degrees C.
Crystallization↗
Lipid diffusibility in the intact erythrocyte membrane.
The lateral diffusion of fluorescent lipid analogues in the plasma membrane of intact erythrocytes from man, mouse, rabbit, and frog has been measured by fluorescence photobleaching recovery (FPR). Intact cells from dystrophic, normoblastic, hemolytic, and spherocytotic mouse mutants; from hypercholesterolemic rabbits and humans; and from prenatal, neonatal, and juvenile mice have been compared with corresponding normals. The lateral diffusion coefficient (D) for 3,3'-dioctadecylindodicarbocyanine iodide (DiI[5]) in intact normal human erythrocytes is D = 8.2 +/- 1.2 X 10(-9) cm2/s at 25 degrees C and D = 2.1 +/- 0.4 X 10(-8) cm2/s at 37 degrees C, and varies approximately 50-fold between 1 degree and 42 degrees C. The diffusion constants of lipid analogue rhodamine-B phosphatidylethanolamine (RBPE) are about twice those of DiI[5]. The temperature dependence and magnitude of D vary by up to a factor of 3 between species and are only influenced by donor age in prenatals. DiI[5] diffusibility is not perturbed by the presence of calcium or local anesthetics or by spectrin depletion (via mutation). However, lipid-analogue diffusibility in erythrocyte ghosts may differ from intact cells. Dietary hypercholesterolemia in rabbits reduces the diffusion coefficient and eliminates the characteristic break in Arrhenius plots of D found in all other cells studied except frog.
Animals↗
Patch-recorded single-channel currents of the purified and reconstituted Torpedo acetylcholine receptor.
Small unilamellar vesicles containing purified and reconstituted nicotinic acetylcholine receptors from Torpedo electroplax have been fused by a simple freeze-thaw procedure to form large liposomes. Giga-seal patch-recording techniques were used to form isolated patches of liposome-membrane and to measure single-channel properties of the reconstituted receptor-ion channel complex. The observed properties are quantitatively similar to those reported for vertebrate muscle nicotinic acetylcholine receptor species recorded in situ. The results demonstrate that the pentameric complex consisting of the alpha 2 beta gamma delta subunits is fully functional. The methods used in these experiments should be useful in studying the effects of chemical alterations on the properties of acetylcholine receptor channels as well as other types of purified and reconstituted ion channels.
Animals↗
Delirium associated with lithium-induced hypothyroidism: a case report.
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Delirium↗
Lateral diffusion of gramicidin C in phospholipid multibilayers. Effects of cholesterol and high gramicidin concentration.
We have measured the lateral diffusion coefficient (D), of active dansyl-labeled gramicidin C (DGC), using the technique of fluorescence photobleaching recovery, under conditions in which the cylindrical dimer channel of DGC predominates. In pure, hydrated, dimyristoylphosphatidylcholine (DMPC) multibilayers (MBL), D decreases from 6 X 10(-8) cm2/s at 40 degrees C to 3 X 10(-8) cm2/s at 25 degrees C, and drops 100-fold at 23 degrees C, the phase transition temperature (Tm) of DMPC. Above Tm, addition of cholesterol decreases D; a threefold stepwise drop occurs between 10 and 20 mol %. Below Tm, increasing cholesterol increases D; a 10-fold increase occurs between 10 and 20 mol % at 21 degrees C, between 20 and 25 mol % at 15 degrees C, and between 25 and 30 mol % at 5 degrees C. In egg phosphatidylcholine (EPC) MBL, D decreases linearly from 5 X 10(-8) cm2/s at 35 degrees C to 2 X 10(-8) cm2/s at 5 degrees C; addition of equimolar cholesterol reduces D by a factor of 2. Thus this transmembrane polypeptide at low membrane concentrations diffuses quite like a lipid molecule. Its diffusivity in lipid mixtures appears to reflect predicted changes of lateral composition. Increasing gramicidin C (GC) in DMPC/GC MBL broadened the phase transition, and the diffusion coefficient of the lipid probe N-4-nitrobenzo-2-diazole phosphatidylethanolamine (NBD-PE) at 30 degrees C decreases from 8 X 10(-8) cm2/s below 5 mol % GC to 2 X 10(-8) cm2/s at 14 mol % GC; D for DGC similarly decreases from 4 X 10(-8) cm2/s at 2 mol % GC to 1.4 X 10(-8) cm2/s at 14 mol % GC. Hence, above Tm, high concentrations of this polypeptide restrict the lateral mobility of membrane components.
Cholesterol↗
Unconstrained lateral diffusion of concanavalin A receptors on bulbous lymphocytes.
The lateral diffusion coefficient, D, of concanavalin A receptors and receptor complexes on the surface of lymphocytes and RDM4 lymphomas is enhanced by several orders of magnitude to D greater than 5 X 10(-9)cm2/sec by induction of swelling of the cells to bulbous form. Treatments with concanavalin A or 7-nitrobenz-2-oxa-1,3-diazole-phallacidin induce blebs and the bulbous form. The resulting separation of the plasma membrane from most of the F-actin cytoskeleton is accompanied by release of constraints on lateral diffusion of the cell surface receptors, which allows the diffusivity of these glycoproteins to increase nearly to the limit allowed by membrane viscosity.
Actins↗
Isolated-patch recording from liposomes containing functionally reconstituted chloride channels from Torpedo electroplax.
Small unilamellar vesicles formed from purified phospholids by detergent/dialysis methods may be enlarged to 30-microns diameter by freezing and thawing. Very-high-resistance seals were formed by applying a glass micropipette to the surface of these large liposomes, and single bilayer "patches" of membrane were isolated from the liposome surface while remaining sealed to the micropipette. The exogenous channel-forming peptides gramicidin and alamethicin induced characteristic single-channel fluctuation behavior in these excised patches held under voltage-clamp conditions. Large liposomes were formed from the small unilamellar vesicles made from cholate extracts of Torpedo electroplax plasma membrane vesicles. Isolated patches formed from these reconstituted membranes displayed current fluctuations due to single voltage-gated Cl- channels from non-innervated-face membranes; the properties of these Cl- channels are identical to those observed in planar bilayer membranes after direct insertion from native membranes. This liposome-patch method combines the advantages of membrane protein incorporation into liposomes with high-resolution electrical recording methods and may provide a generally applicable approach to the study of integral membrane channel proteins after solubilization and reconstitution.
Animals↗
F-actin in conifer roots.
The distribution of F-actin in the complex tissues of a higher plant organ has been visualized by fluorescence labeling the roots of the conifers Chamaecyparis obtusa and Pseudotsuga menziesii with F-actin-specific fluorescent dye-conjugated phallicidin. F-actin is present in the parenchymatous cells of the vascular tissue. Some vascular parenchyma cells possess larger numbers of F-actin-containing structures (microfilament bundles) than are known to exist in any other higher plant cell. Tissue type appears to be an important determinant of the presence or absence of F-actin in a cell. For example, in contrast to vascular cells, cortical cells show no indication of fluorescence labeling of F-actin after incubation with fluorescent phallicidin. Cytoplasmic streaming is seen only in vascular cells and in a pattern that reflects the intracellular distribution of F-actin.
Journal Article↗
Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints.
Measurements of lateral molecular diffusion on blebs formed on the surfaces of isolated muscle cells and myoblasts are reported. These blebbed membranes retain integral proteins but apparently separate from the detectable cytoskeleton. On blebs, acetylcholine receptors, concanavalin A receptors, and stearoyldextran extrinsic model receptor molecules are free to diffuse with a diffusion coefficient (D) approximately 3 x 10(-9) cm2/s, which is close to the value predicted for hydrodynamic drag in the lipid membrane. In contrast, diffusion of these typical receptors in intact cell membranes is constrained to D approximately less than 10(-10) cm2/s with substantial fractions virtually nondiffusible (D less than 10(-12) cm2/s). Lipid analog diffusion is also slightly enhanced in blebs as expected of evanescent lipid protein interaction. This strong enhancement of membrane protein diffusion is attributed to release from unidentified natural constraints that is induced in some way by detachment of the bleb membrane.
Animals↗