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T D Pollard

Publications and source records attributed to T D Pollard.

At least 199 records · Page 11Linked to original sources
PubMed1982

Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2.

Acanthamoeba profilin strongly inhibits in a concentration-dependent fashion the rate and extent of Acanthamoeba actin polymerization in 50 mM KCl. The lag phase is prolonged indicating reduction in the rate of nucleus formation. The elongation rates at both the barbed and pointed ends of growing filaments are inhibited. At steady state, profilin increases the critical concentration for polymerization but has no effect on the reduced viscosity above the critical concentration. Addition of profilin to polymerized actin causes it to depolymerize until a new steady-state, dependent on profilin concentration, is achieved. These effects of profilin can be explained by the formation of a 1:1 complex with actin with a dissociation constant of 1 to 4 microM. MgCl2 strongly inhibits these effects of profilin, most likely by binding to the high-affinity divalent cation site on the actin. Acanthamoeba profilin has similar but weaker effects on muscle actin, requiring 5 to 10 times more profilin than with amoeba actin.

Actins↗
PubMed1982

Nucleated polymerization of actin from the membrane-associated ends of microvillar filaments in the intestinal brush border.

We examined the nucleated polymerization of actin from the two ends of filaments that comprise the microvillus (MV) core in intestinal epithelial cells by electron microscopy. Three different in vitro preparations were used to nucleate the polymerization of muscle G-actin: (a) MV core fragments containing "barbed" and "pointed" filament ends exposed by shear during isolation, (b) isolated, membrane-intact brush borders, and (c) brush borders demembranated with Triton-X 100. It has been demonstrated that MV core fragments nucleate filament growth from both ends with a strong bias for one end. Here we identify the barbed end of the core fragment as the fast growing end by decoration with myosin subfragment one. Both cytochalasin B (CB) and Acanthamoeba capping protein block filament growth from the barbed but not the pointed end of MV core fragments. To examine actin assembly from the naturally occurring, membrane-associated ends of MV core filaments, isolated membrane-intact brush borders were used to nucleate the polymerization of G-actin. Addition of salt (75 mM KCl, 1 mM MgSO4) to brush borders preincubated briefly at low ionic strength with G-actin induced the formation of 0.2-0.4 micron "growth zones" at the tips of microvilli. The dense plaque at the tip of the MV core remains associated with the membrane and the presumed growing ends of the filaments. We also observed filament growth from the pointed ends of core filaments in the terminal web. We did not observe filament growth at the membrane-associated ends of core filaments when the latter were in the presence of 2 microM CB or if the low ionic strength incubation step was omitted. Addition of G-actin to demembranated brush borders, which retain the dense plaque on their MV tips, resulted in filament growth from both ends of the MV core. Again, 2 microM CB blocked filament growth from only the barbed (tip) end of the core. The dense plaque remained associated with the tip-end of the core in the presence of CB but usually was dislodged in control preparations where nucleated polymerization from the tip-end of the core occurred. Our results support the notion that microvillar assembly and changes in microvillar length could occur by actin monomer addition/loss at the barbed, membrane-associated ends of MV core filaments.

Actins↗
PubMed1982

Structure and polymerization of Acanthamoeba myosin-II filaments.

Acanthamoeba myosin-II forms filaments of two different sizes. Thin bipolar filaments 7 nm wide and 200 nm long consist of 16 myosin-II molecules. Thick bipolar filaments of variable width (14-19 nm) consist of 40 or more myosin-II molecules. Both have a central bare zone 90 nm long and myosin heads projecting laterally at the ends. The heads are arranged in rows spaced 15 nm apart. In the case of the thin myosin-II filaments there are two molecules per row. The thick filaments are formed rapidly and reversibly in the presence of 6-10 mM MgCl2 (or any of five other different divalent cations tested) by the lateral aggregation of thin myosin-II filaments. Acid pH also favors thick filament formation. Neither the myosin-II concentration (50-1,000 micrograms/ml) nor ATP has an effect on the morphology of the filaments. The polymerization mechanism was studied quantitatively by measuring the amount of polymer formed (Cp) under various conditions as a function of total myosin-II concentration (Ct). Above a critical concentration of 15-40 micrograms/ml, Cp was proportional to Ct with a slope of 0.5-0.95 depending on conditions. In the range of 0.8-4.9 heavy chain phosphates per molecule, phosphorylation has no effect on the morphology of either the thin or thick myosin-II filaments and only a small effect on the extent of polymerization.

Amoeba↗
PubMed1981

Purification of a calcium-sensitive actin gelation protein from Acanthamoeba.

A new calcium-sensitive actin filament cross-linking protein has been purified from Acanthamoeba. By gel electrophoresis in sodium dodecyl sulfate, the apparent subunit molecular weight varies depending on the concentration of sulfhydryl reducing agent in the sample. The major band is 60,000 in 10% beta-mercaptoethanol, 85,000 in 1% mercaptoethanol, an 90,000 without beta-mercaptoethanol. By electron microscopy, the molecule is a rod about 3 nm wide and 55 nm long with a 5.5-nm globular region at one end, which accounts for its large Stokes radius of 8.5 nm. At low concentrations, the gelation protein cross-links actin filaments to form a solid gel. This gelation reaction is inhibited by micromolar concentrations of Ca2+, by cytochalasin B, and by capping protein and is promoted by ATP, MgCl2, and KCl.

Actins↗
PubMed1981

Electron microscopic localization of cytoplasmic myosin with ferritin-labeled antibodies.

We localized myosin in vertebrate nonmuscle cells by electron microscopy using purified antibodies coupled with ferritin. Native and formaldehyde-fixed filaments of purified platelet myosin filaments each consisting of approximately 30 myosin molecules bound an equivalent number of ferritin-antimyosin conjugates. In preparations of crude platelet actomyosin, the ferritin-antimyosin bound exclusively to similar short, 10-15 nm wide filaments. In both cases, binding of the ferritin-antimyosin to the myosin filaments was blocked by preincubation with unlabeled antimyosin. With indirect fluorescent antibody staining at the light microscope level, we found that the ferritin-antimyosin and unlabeled antimyosin stained HeLa cells identically, with the antibodies concentrated in 0.5-microns spots along stress fibers. By electron microscopy, we found that the concentration of ferritin-antimyosin in the dense regions of stress fibers was five to six times that in the intervening less dense regions, 20 times that in the cytoplasmic matrix, and 100 times that in the nucleus. These concentration differences may account for the light microscope antibody staining pattern of spread interphase cells. Some, but certainly not all, of the ferritin-antimyosin was associated with 10-15-nm filaments. In mouse intestinal epithelial cells, ferritin-antimyosin was located almost exclusively in the terminal web. In isolated brush borders exposed to 5 mM MgCl2, ferritin-antimyosin was also concentrated in the terminal web associated with 10-15-nm filaments.

Actomyosin↗
PubMed1981

Direct measurement of actin polymerization rate constants by electron microscopy of actin filaments nucleated by isolated microvillus cores.

We used actin filament bundles isolated from intestinal brush-border microvilli to nucleate the polymerization of pure muscle actin monomers into filaments. Growth rates were determined by electron microscopy by measuring the change in the length of the filaments as a function of time. The linear dependence of the growth rates on the actin monomer concentration provided the rate constants for monomer association and dissociation at the two ends of the growing filament. The rapidly growing ("barbed") end has higher association and dissociation rate constants than the slowly growing ("pointed") end. The values of these rate constants differ in 20 mM KCl compared with 75 mM KCl, 5 mM MgSO4. 2 microM cytochalasin B blocks growth entirely at the barbed end, apparently by reducing both association and dissociation rate constants to near zero, but inhibits growth at the pointed end to only a small extent.

Actins↗
PubMed1981

Relation between cell activity and the distribution of cytoplasmic actin and myosin.

We documented the activity of cultured cells on time-lapse videotapes and then stained these identified cells with antibodies to actin and myosin. This experimental approach enabled us to directly correlate cellular activity with the distribution of cytoplasmic actin and myosin. When trypsinized HeLa cells spread onto a glass surface, the cortical cytoplasm was the most actively motile and random, bleb-like extensions (0.5-4.0 micrometer wide, 2-5 micrometer long) occurred over the entire surface until the cells started to spread. During spreading, ruffling membranes were found at the cell perimeter. The actin staining was found alone in the surface blebs and ruffles and together with myosin staining in the cortical cytoplasm at the bases of the blebs and ruffles. In well-spread, stationary HeLa cells most of the actin and myosin was found in stress fibers but there was also diffuse antiactin fluorescence in areas of motile cytoplasm such as leading lamellae and ruffling membranes. Similarly, all 22 of the rapidly translocating embryonic chick cells had only diffuse actin staining. Between these extremes were slow-moving HeLa cells, which had combinations of diffuse and fibrous antiactin and antimyosin staining. These results suggest that large actomyosin filament bundles are associated with nonmotile cytoplasm and that actively motile cytoplasm has a more diffuse distribution of these proteins.

Actins↗
PubMed1981

Crystalline actin sheets: their structure and polymorphism.

Crystalline sheets of Acanthamoeba actin induced by the trivalent lanthanide gadolinium exist in three different polymorphic forms, which show different striation patterns and surface topographies. We have called these different forms "rectangular" and "square" sheets, and "cylinders" and have shown that each of the three forms is constructed from common "basic" lattices associated in different ways. We have used image processing of electron micrographs to obtain a model for the actin molecule in projection to a resolution of 1.5 nm. The overall dimensions observed in these images are 5.6 x 3.3 x 4.5 nm, and the molecule itself appears distinctly bilobed with the two lobes separated by a cleft. actin monomers in the sheets are arranged with P2 symmetry and are therefore packed in a manner different from that of the molecules in actin filaments. Because approximately 35% of the surface area of the actin molecule is exposed on the surface of these sheets, the sheets should be useful to study the stoichiometric binding of actin-binding proteins to the actin molecule.

Actins↗
PubMed1980

Structure of crystalline actin sheets.

Although actin is one of the most abundant proteins found in nature, little detailed information about its molecular structure is available beyond the amino acid sequence. Electron microscopy of negatively stained filaments combined with three-dimensional image reconstruction techniques have revealed the overall size and shape of the actin monomer at 25 A resolution. Higher resolution structural data can be expected from electron microscopy of two-dimensional crystalline arrays and X-ray diffraction analysis of three-dimensional crystals, but only very preliminary results have been reported so far. The original finding by Dos Remedios and Dickens was that skeletal muscle actin forms microcrystals and tubes in the presence of the trivalent lanthanide gadolinium (Gd3+). We have modified and refined their conditions to obtain large crystalline sheets of Acanthamoeba actin and present here a model of the actin monomer in projection to 15 A resolution. We have found that, depending on the ionic strength used, these sheets occur in three different forms: 'cylinders', 'square type' sheets and 'rectangular type' sheets. These different polymorphic forms are built from the same fundamental two-dimensional crystalline actin lattice, which we call the 'basic sheet'. The present concerns the structural analysis of these basic sheets; the crystal polymorphism will be discussed in detail elsewhere (U.A. et al., in preparation). Furthermore, in addition to demonstrating that actin is an elongated globular molecule with a pronounced asymmetric shape in and perpendicular to the plane of the sheet, our results indicate that these crystalline actin sheets might be suitable for three-dimensional structure determination by low-dose electron microscopy of unstained specimens to at least 10 A resolution.

Actins↗