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D L Taylor

Publications and source records attributed to D L Taylor.

At least 199 records · Page 11Linked to original sources
PubMed1981

Probing the dynamic equilibrium of actin polymerization by fluorescence energy transfer.

Fluorescence resonance energy transfer was used to follow the dynamic equilibrium of actin polymerization. We prepared fluorescent analogs of actin by labeling actin covalently with fluorescein (as the donor) and with eosin (as the acceptor). The copolymer of donor- and acceptor-labeled actin exhibits a 60%-70% efficiency of energy transfer. We followed the subunit exchange among filaments both by mixing the donor-acceptor copolymer with unlabeled F actin, and by mixing donor-labeled F actin with acceptor-labeled F actin. The extent of subunit exchange is dependent on ionic conditions. In addition, different kinetics are observed in the two approaches in the presence of excess magnesium ions. The effects of cytochalasin B as a model actin-binding factor were also investigated. One micromolar cytochalasin B reduces the rates of subunit exchange, monomer incorporation and filament depolymerization. At 10 microM cytochalasin B, we detected destabilization of filaments using a morphological assay. The results are discussed in relation to existing models of actin subunit exchange, and to proposed mechanisms of actin-cytochalasin interactions.

Actins↗
PubMed1981

Fluorescence photobleaching recovery in solutions of labeled actin.

We have demonstrated that the technique of fluorescence photobleaching recovery (FPR) can be used to examine the state of a single component in complex self-assembling macromolecular systems. Polymerization of actin, initiated by addition of salt or Mg+2 to a low-ionic-strength solution of G-actin, has been observed by sequential measurement of FPR with the aid of fluorescein-labeled actin. Solutions of actin which had been labeled using 5-iodoacetamido fluorescein (5-IAF) showed anomalous recovery of fluorescence above the initial value, which indicates a photoinduced increase in local polymerization. No such anomaly was observed with actin that had been labeled with fluorescein isothiocyanate (FITC). The FPR data are directly interpretable in terms of the fraction of labeled protein that is immobilized in the supramolecular assembly and in terms of the average diffusion coefficient of the mobile fraction. Our data are consistent with the "treadmill" model of actin polymerization, in that they show that actin is present under polymerizing conditions either as a high polymer or as monomer or low oligomer. We believe that the FPR technique can be applied to the study of many types of reconstituted motile or cytoskeletal systems in vitro or in vivo.

Actins↗
PubMed1981

Exchange of 1,N6-etheno-ATP with actin-bound nucleotides as a tool for studying the steady-state exchange of subunits in F-actin solutions.

The fluorescent analog of ATP 1-N6-ethenoadenosine 5'-triphosphate (epsilon-ATP) exchanges readily with nucleotides bound to G-actin. The exchange can be observed by measuring the fluorescence intensity, which increases significantly when epsilon-ATP binds to actin. When excess epsilon-ATP is added to a solution of F-actin, a continuous increase in fluorescence intensity is observed, indicating that the nucleotides bound to F-actin are directly or indirectly exchangeable. The kinetics of exchange consist of a fast phase and a slow phase. Both phases are stimulated by shearing and are inhibited by phalloidin treatment, suggesting that the exchange of nucleotides is coupled to the exchange of subunits. Therefore, the exchange reaction can be used as a convenient, nonperturbing tool to study the exchange of free actin subunits with subunits in actin filaments. The exchange of actin subunits was characterized by a pulse-chase experiment. The results suggest that actin subunits assemble and disassemble through the same end of the filament during the fast phase of exchange but through opposite ends of the filament during the slow phase. In addition, the slow phase of exchange is inhibited in the absence of millimolar magnesium ions, but is not significantly affected by cytochalasin B at concentrations between 0.1 and 10 microM. These observations are discussed in relation to possible mechanisms of subunit exchange in steady-state F-actin solutions.

Actins↗
PubMed1981

Spectrin promotes the association of F-actin with the cytoplasmic surface of the human erythrocyte membrane.

We studied the binding of actin to the erythrocyte membrane by a novel application of falling ball viscometry. Our approach is based on the notion that if membranes have multiple binding sites for F-actin they will be able to cross-link and increase the viscosity of actin. Spectrin- and actin-depleted inside-out vesicles reconstituted with purified spectrin dimer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out vesicles plus heat-denatured spectrin dimmer or tetramer induce large increases in the viscosity of actin. Comparable concentrations of spectrin alone, inside-out vesicles alone, inside-out plus heat denatured spectrin, ghosts, or ghosts plus spectrin have no effect on the viscosity of actin. Centrifugation experiments show that the amount of actin bound to the inside-out vesicles is enhanced in the presence of spectrin. The interactions detected by low-shear viscometry reflect actin interaction with membrane- bound spectrin because (a) prior removal of band 4.1 and ankyrin (band 2.1, the high- affinity membrane attachment site for spectrin) reduces both spectrin binding to the inside-out vesicles and their capacity to stimulate increase in viscosity of actin in the presence of spectrin + actin are inhibited by the addition of the water-soluble 72,000- dalton fragment of ankyrin, which is known to inhibit spectrin reassociation to the membrane. The increases in viscosity of actin induced by inside-out vesicles reconstituted with purified spectrin dimer or tetramer are not observed when samples are incubated at 0 degrees C. This temperature dependence may be related to the temperature-dependent associations we observe in solution studies with purified proteins: addition of ankyrin inhibits actin cross-linking by spectrin tetramer plus band 4.1 at 0 degrees C, and enhances it at 32 degrees C. We conclude (a) that falling ball viscometry can be used to assay actin binding to membranes and (b) that spectrin is involved in attaching actin filaments or oligomers to the cytoplasmic surface of the erythrocyte membrane.

Actins↗
PubMed1981

A membrane cytoskeleton from Dictyostelium discoideum. I. Identification and partial characterization of an actin-binding activity.

Dictyostelium discoideum plasma membranes isolated by each of three procedures bind F-actin. The interactions between these membranes and actin are examined by a novel application of falling ball viscometry. Treating the membranes as multivalent actin-binding particles analogous to divalent actin-gelation factors, we observe large increases in viscosity (actin cross-linking) when membranes of depleted actin and myosin are incubated with rabbit skeletal muscle F-actin. Pre-extraction of peripheral membrane proteins with chaotropes or the inclusion of Triton X-100 during the assay does not appreciably diminish this actin cross-linking activity. Lipid vesicles, heat-denatured membranes, proteolyzed membranes, or membranes containing endogenous actin show minimal actin cross-linking activity. Heat-denatured, but not proteolyzed, membranes regain activity when assayed in the presence of Triton X-100. Thus, integral membrane proteins appear to be responsible for some or all of the actin cross-linking activity of D. discoideum membranes. In the absence of MgATP, Triton X-100 extraction of isolated D. discoideum membranes results in a Triton-insoluble residue composed of actin, myosin, and associated membrane proteins. The inclusion of MgATP before and during Triton extraction greatly diminishes the amount of protein in the Triton-insoluble residue without appreciably altering its composition. Our results suggest the existence of a protein complex stabilized by actin and/or myosin (membrane cytoskeleton) associated with the D. discoideum plasma membrane.

Actins↗
PubMed1981

Detection of actin assembly by fluorescence energy transfer.

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor-labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor-labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.

Actins↗
PubMed1981

Catecholamines and monoamine oxidase activity in anxiety.

Levels of platelet monamine oxidase activity, state anxiety and trait anxiety were quantified twice in 20 drug-free subjects with generalized anxiety and an equal number of healthy drug-free controls at 4-week intervals. Fifteen subjects in each groups also had plasma epinephrine and norepinephrine measured. The index group received relaxation training during the interval between the two evaluation sessions. Post-relaxation training values for monoamine oxidase, epinephrine, norepinephrine and the anxiety levels were found to be significantly lower than the pre-treatment values for the index group. No significant changes were seen in the control group values. For the index group, catecholamine levels and monoamine oxidase activity were seen to correlate significant before and after relaxation training.

Adult↗
PubMed1980

Spectrin plus band 4.1 cross-link actin. Regulation by micromolar calcium.

A low-salt extract prepared from human erythrocyte membranes forms a solid gel when purified rabbit muscle G- or F-actin is added to it to give a concentration of approximately 1 mg/ml. This extract contains spectrin, actin, band 4.1, band 4.9, hemoglobin, and several minor components. Pellets obtained by centrifugation of the gelled material at 43,000 g for 10 min contain spectrin, actin, band 4.1, and band 4.9. Although extracts that are diluted severalfold do not gel when actin is added to them, the viscosity of the mixtures increases dramatically over that of G-actin alone, extract alone, or F-actin alone at equivalent concentrations. Heat-denatured extract is completely inactive. Under conditions of physiological ionic strength and pH, information of this supramolecular structure is inhibited by raising the free calcium ion concentration to micromolar levels. Low-salt extracts prepared by initial extraction at 37 degrees C (and stored at 0 degree C) gel after actin is added to them only when warmed, whereas extracts prepared by extraction at 0 degree C are active on ice as well as after warming. Preincubation of the 37 degrees C low-salt extract under conditions that favor conversion of spectrin dimer to tetramer greatly enhances gelation activity at 0 degree C. Conversely, preincubation of the 0 degree C low-salt extract under conditions that favor conversion of spectrin tetramer to dimer greatly diminishes gelation activity at 0 degree C. Spectrin dimers or tetramers are purified from the 37 dgrees or 0 degree C low-salt extract by gel filtration at 4 degrees C over Sepharose 4B. The addition of actin to either purified spectrin dimer (at 32 degrees C) or tetramer (at 0 degree C or 32 degrees C) results in relatively small increases in viscosity, whereas the addition of actin to a high-molecular-weight complex (HMW complex) containing spectrin, actin, band 4.1, and band 4.9 results in dramatic, calcium-sensitive increases in viscosity. These viscosities are comparable to those obtained with the 37 degrees or 0 degree C low-salt extracts. The addition of purified band 4.1 to either purified spectrin dimer (at 32 degrees C) or purified spectrin tetramer (at 0 degree C) plus actin results in large increases in viscosity similar to those observed for the HMW complex and the crude extract, which is in agreement with a recent report by E. Ungewickell, P. M. Bennett, R. Calvert, V. Ohanian, and W. B. Gratzer. 1979 Nature (Lond.) 280:811-814. We suggest that this spectrin-actin-band 4.1 gel represents a major structural component of the erythrocyte cytoskeleton.

Actins↗
PubMed1980

Contractile basis of ameboid movement. VII. The distribution of fluorescently labeled actin in living amebas.

The technique of molecular cytochemistry has been used to follow the distribution of fluorescently labeled actin in living Chaos carolinensis and Amoeba proteus during ameboid movement and various cellular processes. The distribution of 5-iodoacetamidofluorescein-labeled actin was compared with that of Lissamine rhodamine B sulfonyl chloride-labeled ovalbumin microinjected into the same cell and recorded with an image intensification microscope system. Actively motile cells demonstrated a rather uniform distribution of actin throughout most of the cytoplasm, except in the tail ectoplasm and in plasma gel sheets, where distinct actin structures were observed. In addition, actin-containing structures were induced in the cortex during wound healing, concanavalin A capping, pinocytosis, and contractions elicited by phalloidin injections. The formation of distinct fluorescent actin structures has been correlated with contractile activities.

Actins↗
PubMed1980

Contractile basis of ameboid movement. VII. Aequorin luminescence during ameboid movement, endocytosis, and capping.

Aequorin luminescence has been utilized to determine the spatial and temporal fluctuations of the free calcium ion concentration [Ca++] in Chaos carolinensis during ameboid movement, pinocytosis, and capping. The [Ca++] increases above approximately 10(-7) M during normal ameboid movement. Three types of luminescent signals are detected in cells: continuous luminescence, spontaneous pulses, and stimulated pulses. Continuous luminescence is localized in the tails of actively motile cells, and spontaneous pulses occur primarily over the anterior regions of cells. We are sometimes able to correlate the spontaneous pulses with extending pseudopods, whereas stimulated pulses are induced by mechanical damage, electrical stimulation, concanavalin A-induced capping, and pinocytosis. The localization of both distinct actin structures and sites where [Ca++] increases suggests cellular sites of contractile activity. The independent evidence from localizing actin structures and the distribution of [Ca++] can also be viewed in relation to the solation-contraction coupling hypothesis defined in vitro.

Actins↗
PubMed1980

Intracellular pH in single motile cells.

Cytoplasmic pH in single living specimens of Chaos carolinensis is determined microfluorometrically by measuring the ratio of fluorescence intensity of microinjected fluorescein-thiocarbamyl (FTC)-ovalbumin at two different excitation wavelengths. The probe is evenly distributed throughout, and confined to, the cytoplasm, and the fluorescence intensity ratio depends only upon pH. It is independent of pathlength, concentration of probe, divalent cations, and ionic strength. Ratios are calibrated with a standard curve generated in situ by adjusting internal pH of FTC-ovalbumin-containing amebae with weak acid and weak base or by injection of strong buffers. With this technique, the average cytoplasmic pH of freely moving ameba is found to be 6.75 (SD +/- 0.3). The pH of a given spot relative to the morphology of a moving ameba remains fairly constant (+/- 0.05 U), whereas the pH of two different spots in the same cell may differ by as much as 0.4 U, and average pH in different amebae ranges from 6.3 to 7.4, with a suggestion of clustering about pH 6.5 and 6.8. During wound healing, there is a local, transient drop in pH (as great as 0.35 U) at the wound site upon puncture, proportional in extent to the degree of damage. Comparison of tails and advancing pseudopod tips reveals no significant difference in cytoplasmic pH at this level of spatial (50 microns diameter spot) and temporal (1.3 s) resolution. Fluctuations in intracellular pH and/or intracellular free Ca++ may be involved in regulation of cytoplasmic structure and contractility.

Amoeba↗
PubMed1980

Preparation and characterization of a new molecular cytochemical probe: 5-iodoacetamidofluorescein-labeled actin.

The new technique of molecular cytochemitry (Taylor DL, Wang YL (1978): Proc Natl Acad Sci USA 75:857) requires the use of functional fluorescent analogs of cellular components with optimal fluorescence characteristics. An analog of actin suitable for this technique is prepared by reacting purified rabbit striated muscle actin with 5-iodoacetamidofluorescein (5-IAF). The conjugate is purified by DEAE-cellulose ion exchange chromatography and cycles of polymerization-depolymerization, yielding a relatively homogeneous product with the fluorescein group covalently attached to cystein 373. The fluorescently labeled actin maintains normal polymerizability and activates heavy meromyosin Mg2+ adenosine triphosphatase to the same extent as unlabeled actin. Furthermore, fluoresecent paracrystals are readily detectable in fluroescence microscope upon adding excess Mg2+ or Ni2+ ions. Spectrofluorimetric studies of the bound fluorescein indicate that the peak excitation and emission wavelengths, the shapes of the spectra, and the peak fluorescence intensities are somewhat sensitive to polymerization and heavy meromyosin binding. Possible causes of these spectral changes are analyzed and future applications of this fluorescently labeled actin in vitro as well as in vivo are discussed.

Actins↗