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T D Pollard

Publications and source records attributed to T D Pollard.

At least 145 records · Page 8Linked to original sources
PubMed1989

The mechanism of assembly of Acanthamoeba myosin-II minifilaments: minifilaments assemble by three successive dimerization steps.

We used 90 degrees light scattering, analytical ultracentrifugation, and electron microscopy to deduce that Acanthamoeba myosin-II minifilaments, composed of eight molecules each, assemble by a novel mechanism consisting of three successive dimerization steps rather than by the addition of monomers or parallel dimers to a nucleus. Above 200 mM KCl, Acanthamoeba myosin-II is monomeric. At low ionic strength (less than 100 mM KCl), myosin-II polymerizes into bipolar minifilaments. Between 100 and 200 mM KCl, plots of light scattering vs. myosin concentration all extrapolate to the origin but have slopes which decrease with increasing KCl. This indicates that structures intermediate in size between monomers and full length minifilaments are formed, and that the critical concentrations for assembly of these structures is very low. Analytical ultracentrifugation has confirmed that intermediate structures exist at these salt concentrations, and that they are in rapid equilibrium with each other. We believe these structures represent assembly intermediates and have used equilibrium analytical ultracentrifugation and electron microscopy to identify them. Polymerization begins with the formation of antiparallel dimers, with the two tails overlapping by approximately 15 nm. Two antiparallel dimers then associated with a 15-nm stagger to form an antiparallel tetramer. Finally, two tetramers associate with a 30-nm stagger to form the completed minifilament. At very low ionic strengths, the last step in the assembly mechanism is largely reversed and antiparallel tetramers are the predominant species. Alkaline pH, which can also induce minifilament disassembly, produces the same assembly intermediates as are found for salt induced disassembly.

Acanthamoeba↗
PubMed1988

Tropomyosin inhibits the rate of actin polymerization by stabilizing actin filaments.

Tropomyosin inhibition of the rate of spontaneous polymerization of actin is associated with binding of tropomyosin to actin filaments. Rate constants determined by using a direct electron microscopic assay of elongation showed that alpha alpha- and alpha beta-tropomyosin have a small or no effect on the rate of elongation at either end of the filaments. The most likely explanation for the inhibition of the rate of polymerization of actin in bulk samples is that tropomyosin reduces the number of filament ends by mechanical stabilization of the filaments.

Actin Cytoskeleton↗
PubMed1988

Crystallization of actophorin, an actin filament-severing protein from Acanthamoeba.

Actophorin is an actin monomer-binding and actin filament-severing protein from Acanthamoeba castellanii. It crystallizes out of polyethylene glycol in a form suitable for high resolution x-ray analysis. The crystals are orthorhombic and have the symmetry of the space group P2(1)2(1)2(1) with lattice constants a = 39.8 +/- 0.5, b = 47.3 +/- 0.6, and c = 69.9 +/- 1.6 A. They diffract to a resolution of at least 2.8 A, and the asymmetric unit contains one actophorin monomer of Mr 15,000.

Acanthamoeba↗
PubMed1988

The primary structure of the basic isoform of Acanthamoeba profilin.

Acanthamoeba profilin-II [Kaiser, D.A., Sato, M., Ebert, R. F. and Pollard, T.D. (1986) J. Cell. Biol. 102, 221-226] was digested with trypsin or cleaved by 2-(2-nitrophenylsulphenyl)-3-methyl-3-bromoindolenine. The tryptic peptides were purified by reversed-phase-high-performance liquid chromatography and completely sequenced using automated gas-phase sequence analysis. The complete profilin-II sequence was deduced by ordering the tryptic peptides using the sequence information of the tryptophan-cleavage products. Acanthamoeba profilin-II was found to be homologous to the previously determined profilin-I sequence [Ampe, C., Vandekerckhove, J., Brenner, L., Tobacman, L. and Korn, E.D. (1985) J. Biol. Chem. 260, 834-840]. Like profilin-I, profilin-II consists of 125 amino acids, has a blocked NH2 terminus and a trimethyllysine residue at position 103. Profilin-II differs in at least 21 positions from one of the profilin-I isoforms. The amino acid exchanges are mainly concentrated in the middle part of the sequence. Profilin-II contains two more basic residues than profilin-I, which explains its higher isoelectric point.

Acanthamoeba↗
PubMed1988

Resolution of Acanthamoeba castellanii chromosomes by pulsed field gel electrophoresis and construction of the initial linkage map.

Pulsed field gel electrophoresis has been used to resolve chromosome-sized DNA molecules in fungi and parasites but has not yet been used successfully to examine the chromosomes of other lower eukaryotes used extensively for biochemical research such as Acanthamoeba, Physarum, and Dictyostelium. Here we show an electrophoretic karyotype of the protozoan Acanthamoeba castellanii using orthogonal field alternating gel electrophoresis (OFAGE). There are about 20 small chromosomes ranging in size from 220 kb to greater than 2 Mb. We have assembled initial linkage groups assigning all of the cloned Acanthamoeba genes to chromosome-sized DNA molecules. Actin, suggested to have three or more non-allelic genes, maps to at least eight distinct chromosome bands. Two myosin II genes localize to two different chromosomal bands while myosin IB and 18S rRNA map to unresolved larger chromosomes.

Acanthamoeba↗
PubMed1988

Evaluation of the binding of Acanthamoeba profilin to pyrene-labeled actin by fluorescence enhancement.

We have used a fluorescence assay to measure the binding of Acanthamoeba profilin to monomeric Acanthamoeba and rabbit skeletal muscle actin labeled on cysteine-374 with pyrene iodoacetamide. The wavelengths of the pyrene excitation and emission maxima are constant at 346 and 386 nm, but the fluorescence is enhanced up to 50% by profilin. The higher fluorescence is largely due to higher absorbance in the presence of profilin. The fluorescence enhancement has a hyperbolic dependence on the concentration of profilin, suggesting a single class of binding sites. Linear Scatchard plots yield an estimate of the dissociation constant, Kd, of the complex of profilin with pyrenyl-actin. In low-ionic-strength buffers with 2 to 6 mM imidazole (pH 7.0) and 0.1 mM CaCl2 the Kd is 9 microM for both muscle and Acanthamoeba actin. In 50 mM KCl the Kd for the complex with Acanthamoeba actin is 16 microM, while the Kd for the complex with muscle actin is greater than 50 microM.

Acanthamoeba↗
PubMed1988

Mechanical properties of brain tubulin and microtubules.

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.

Animals↗
PubMed1988

Direct demonstration of actin filament annealing in vitro.

Direct electron microscopic examination confirms that short actin filaments rapidly anneal end-to-end in vitro, leading over time to an increase in filament length at steady state. During annealing of mixtures of native unlabeled filaments and glutaraldehyde-fixed filaments labeled with myosin subfragment-1, the structural polarity within heteropolymers is conserved absolutely. Annealing does not appear to require either ATP hydrolysis or the presence of exogenous actin monomers, suggesting that joining occurs through the direct association of filament ends. During recovery from sonication the initial rate of annealing is consistent with a second-order reaction involving the collision of two filament ends with an apparent annealing rate constant of 10(7) M-1s-1. This rapid phase lasts less than 10 s and is followed by a slow phase lasting minutes to hours. Annealing is calculated to contribute minimally to filament elongation during the initial stages of self-assembly. However, the rapid rate of annealing of sonicated fixed filaments observed in vitro suggests that it may be an efficient mechanism for repairing breaks in filaments and that annealing together with polymer-severing mechanisms may contribute significantly to the dynamics and function of actin filaments in vivo.

Actin Cytoskeleton↗
PubMed1988

Structural and immunological characterization of the myosin-like 110-kD subunit of the intestinal microvillar 110K-calmodulin complex: evidence for discrete myosin head and calmodulin-binding domains.

The actin bundle within each microvillus of the intestinal brush border is tethered laterally to the membrane by spirally arranged bridges. These bridges are thought to be composed of a protein complex consisting of a 110-kD subunit and multiple molecules of bound calmodulin (CM). Recent studies indicate that this complex, termed 110K-CM, is myosin-like with respect to its actin binding and ATPase properties. In this study, possible structural similarity between the 110-kD subunit and myosin was examined using two sets of mAbs; one was generated against Acanthamoeba myosin II and the other against the 110-kD subunit of avian 110K-CM. The myosin II mAbs had been shown previously to be cross-reactive with skeletal muscle myosin, with the epitope(s) localized to the 50-kD tryptic fragment of the subfragment-1 (S1) domain. The 110K mAbs (CX 1-5) reacted with the 110-kD subunit as well as with the heavy chain of skeletal but not with that of smooth or brush border myosin. All five of these 110K mAbs reacted with the 25-kD, NH2-terminal tryptic fragment of chicken skeletal S1, which contains the ATP-binding site of myosin. Similar tryptic digestion of 110K-CM revealed that these five mAbs all reacted with a 36-kD fragment of 110K (as well as larger 90- and 54-kD fragments) which by photoaffinity labeling was shown to contain the ATP-binding site(s) of the 110K subunit. CM binding to these same tryptic digests of 110K-CM revealed that only the 90-kD fragment retained both ATP- and CM-binding domains. CM binding was observed to several tryptic fragments of 60, 40, 29, and 18 kD, none of which contain the myosin head epitopes. These results suggest structural similarity between the 110K and myosin S1, including those domains involved in ATP- and actin binding, and provide additional evidence that 110K-CM is a myosin. These studies also support the results of Coluccio and Bretscher (1988. J. Cell Biol. 106:367-373) that the calmodulin-binding site(s) and the myosin head region of the 110-kD subunit lie in discrete functional domains of the molecule.

Acanthamoeba↗
PubMed1987

A glow discharge unit to render electron microscope grids and other surfaces hydrophilic.

We describe the design, construction, and operation of a simple glow discharge unit that can be used to make surfaces such as carbon-coated electron microscopy grids and glass coverslips hydrophilic. The use of a vacuum leak detector (Tesla coil) in place of a conventional high-voltage power supply and a small plastic desiccator for the vacuum chamber make the unit very inexpensive. Owing to the small volume of the chamber and the simplicity of the unit, the whole glow discharge process can be carried out in only 2 to 3 min, a time considerably shorter than that required for conventional vacuum evaporators. The hydrophilic surface improves adsorption of particles by several orders of magnitude in preparation for negative staining, freeze-drying, and other procedures.

Electric Conductivity↗
PubMed1987

Molecular cloning of cDNA for CENP-B, the major human centromere autoantigen.

We have isolated a series of overlapping cDNA clones for approximately 95% of the mRNA that encodes CENP-B, the 80-kD human centromere autoantigen recognized by patients with anticentromere antibodies. The cloned sequences encode a polypeptide with an apparent molecular mass appropriate for CENP-B. This polypeptide and CENP-B share three non-overlapping epitopes. The first two are defined by monoclonal antibodies elicited by injection of cloned fusion protein. Epitope 1 corresponds to a major antigenic site recognized by the anticentromere autoantibody used to obtain the original clone. Epitope 2 is a novel one not recognized by the autoantibody. These epitopes were shown to be distinct both by competitive binding experiments and by their presence or absence on different subcloned portions of the fusion protein. The third independent epitope, recognized by a subset of anticentromere-positive patient sera, maps to a region substantially closer to the amino terminus of the fusion protein. DNA and RNA blot analyses indicate that CENP-B is unrelated to CENP-C, a 140-kD centromere antigen also recognized by these antisera. CENP-B is the product of a 2.9-kb mRNA that is encoded by a single genetic locus. This mRNA is far too short to encode a polypeptide the size of CENP-C. The carboxy terminus of CENP-B contains two long domains comprised almost entirely of glutamic and aspartic acid residues. These domains may be responsible for anomalous migration of CENP-B on SDS-polyacrylamide gels, since the true molecular mass of CENP-B is approximately 65 kD, 15 kD less than the apparent molecular mass deduced from gel electrophoresis. Quite unexpectedly, immunofluorescence analysis using antibodies specific for CENP-B reveals that the levels of antigen vary widely between chromosomes.

Amino Acid Sequence↗
PubMed1986

Crystallization of Acanthamoeba profilin-I.

Profilin-I, a protein that inhibits actin polymerization in Acanthamoeba castellanii, has been crystallized in a form suitable for high resolution x-ray analysis. The crystals have the symmetry of the space group C2 with lattice constants a = 110.4 +/- 0.2, b = 31.7 +/- 0.1, c = 33.5 +/- 0.1 A, beta = 112.2 degrees. They diffract to at least 2.0-A resolution. The asymmetric unit contains one 12,800-dalton monomer of profilin-I.

Amoeba↗
PubMed1986

Elongation of actin filaments is a diffusion-limited reaction at the barbed end and is accelerated by inert macromolecules.

We used a fluorescence method to measure the rate constants for the elongation of pyrene-labeled actin filaments in a number of different solvents. The absolute values of the rate constants were established by electron microscopy. Using glycerol, sucrose, or ethylene glycol to vary the solution viscosity, the association rate constant (k+) was 10(7) M-1 s-1 viscosity-1 (in centipoise). Consequently, plots of 1/k+ versus viscosity are linear and extrapolate to near the origin as expected for a diffusion-limited reaction where the rate constant approaches infinity at zero viscosity. By electron microscopy, we found that this inhibitory effect of glycerol is almost entirely at the fast growing, barbed end. For the pointed end, plots of 1/k+ versus viscosity extrapolate to a maximum rate of about 10(6) M-1 s-1 at zero viscosity, so that elongation at the pointed is not limited by diffusion. In contrast to these small molecules, polyethylene glycol, dextran, and ovalbumin all cause a concentration (and therefore viscosity)-dependent increase in k+. At any given viscosity, their effects are similar to each other. For example, at 3 centipoise, k+ = 2.2 X 10(7) M-1 s-1. We presume that this is due to an excluded volume effect that causes an increase in the thermodynamic activity of the actin. If the proteins in the cytoplasmic matrix have a similar effect, the association reactions of actin in cells may be much faster than expected from experiments done in dilute buffers.

Actins↗
PubMed1986

Acanthamoeba profilin affects the mechanical properties of nonfilamentous actin.

We investigated the mechanical properties of two abundant, cytoplasmic proteins from Acanthamoeba, profilin and actin, and found that while both profilin and nonfilamentous actin alone behaved as solids, mixtures of the two proteins were viscoelastic liquids. When allowed to equilibrate, profilin formed a viscoelastic solid with mechanical properties similar to filamentous and nonfilamentous actin. Consequently, profilin itself may contribute significantly to the elasticity and viscosity of cytoplasm. The addition of profilin to nonfilamentous actin caused a phase transition from gel (viscoelastic solid) to sol (viscoelastic liquid) when the concentration of free actin became too low to form a gel. In contrast, profilin had little effect on the rigidity and viscosity of actin filaments. We speculate that nonfilamentous actin and profilin, both of which form shear-sensitive structures, can be modeled as flocculant materials. We conclude that profilin may regulate the rigidity (elasticity) of the cytoplasm not only by inhibiting polymerization of actin, but also by modulating the mechanical properties of nonfilamentous actin.

Actins↗
PubMed1986

Purification and characterization of actophorin, a new 15,000-dalton actin-binding protein from Acanthamoeba castellanii.

Actophorin is a new actin-binding protein from Acanthamoeba castellanii that consists of a single polypeptide with a molecular weight of 15,000. The isoelectric point is 6.1, and amino acid analysis shows an excess of acidic residues over basic residues. The phosphate content is less than 0.2 mol/mol. There is 0.4 +/- 0.1 mg of actophorin/g of cells, so that the molar ratio of actin to actophorin is about 10:1 in the cell. Unique two-dimensional maps of tryptic and chymotryptic peptides and complete absence of antibody cross-reactivity show that Acanthamoeba actophorin, profilin, capping protein, and actin are separate gene products with minimal homology. Actophorin has features of both an actin monomer-binding protein and an actin filament-severing protein. Actophorin reduces the extent of actin polymerization at steady state in a concentration-dependent fashion and forms a complex with pyrene-labeled actin that has spectral properties of unpolymerized actin. During ultracentrifugation a complex of actophorin and actin sediments more rapidly than either actin monomers or actophorin. Although actophorin inhibits elongation at both ends of actin filaments, it accelerates the late stage of spontaneous polymerization like mechanical shearing and theoretical predictions of polymer fragmentation. Low concentrations of actophorin decrease the length and the low shear viscosity of actin filaments. High concentrations cause preformed filaments to shorten rapidly. Ca2+ is not required for any of these effects. Muscle and amoeba actin are equally sensitive to actophorin.

Actin Depolymerizing Factors↗
PubMed1986

Characterization of alpha-actinin from Acanthamoeba.

Characterization of a protein from Acanthamoeba that was originally called gelation protein [T.D. Pollard, J. Biol. Chem. 256:7666-7670, 1981] has shown that it resembles the actin filament cross-linking protein, alpha-actinin, found in other cells. It comprises about 1.5% of the total amoeba protein and can be purified by chromatography with a yield of 13%. The native protein has a molecular weight of 180,000 and consists of two polypeptides of 90,000 Da. The Stokes' radius is 8.5 nm, the intrinsic viscosity is 0.35 dl/dm, and the extinction coefficient at 280 mm is 1.8 X 10(5)M-1 X cm-1. Electron micrographs of shadowed specimens show that the molecule is a rod 48 nm long and 7 nm wide with globular domains at both ends and in the middle of the shaft. On gel electrophoresis in sodium dodecylsulfate the pure protein can run as bands with apparent molecular weights of 60,000, 90,000, 95,000, or 134,000 depending on the method of sample preparation. Rabbit antibodies to electrophoretically purified Acanthamoeba alpha-actinin polypeptides react with all of these electrophoretic variants in samples of purified protein and cell extracts. By indirect fluorescent antibody staining of fixed amoebas, alpha-actinin is distributed throughout the cytoplasmic matrix and concentrated in the hyaline cytoplasm of the cortex. The protein cross-links actin filaments in the presence and absence of Ca++. It inhibits slightly the time course of the spontaneous polymerization of actin monomers but has no effect on the critical concentration for actin polymerization even though it increases the apparent rate of elongation to a small extent. Like some other cross-linking proteins, amoeba alpha-actinin inhibits the actin-activated ATPase of muscle myosin subfragment-1. Although Acanthamoeba alpha-actinin resembles the alpha-actinin from other cells in shape and ability to cross-link actin filaments, antibodies to amoeba and smooth muscle alpha-actinins do not cross react and there are substantial differences in the amino acid compositions and molecular dimensions.

Actinin↗
PubMed1986

Assembly and dynamics of the actin filament system in nonmuscle cells.

Kinetic analysis has provided a detailed quantitative description of the mechanism of actin polymerization as well as the methods to analyze the mechanisms of action of actin-binding proteins. In Acanthamoeba, five different proteins regulate the pool of monomers available for polymerization, cap the end of filaments, sever filaments, and cross-link filaments. Remarkably, many of these interactions involve very-low-affinity bonds between the protein molecules.

Actin Cytoskeleton↗