Dissociation of the DNAse-I . actin complex by formamide

Rabbit skeletal muscle actin labeled with 125 iodine by an enzymic method is shown to be capable of polymerization and to bind to matrix-bound pancreatic DNAse I like unlabeled G-actin. It was used to demonstrate that actin can be released from DNAse-I-agarose by 35--40% formamide. Actin which was only shortly exposed to this solvent was able to bind again to DNAse I and to form filaments indicating that it has been recovered functionally intact from the affinity matrix.     

DNAse I—actin complex: An immunological study

DNAse I - actin complex formation is studied in the presence of different anti actin antibody populations. The binding of DNAse I to actin is shown to be affected by antibodies specific to a central region in actin sequence (168–226). The C- and N-extremities of actin are shown to be in spatial proximity at the surface of the actin monomer and far from the binding area of DNAse I.     

Association of rapidly-labelled RNAs with actin in nuclear matrix from mouse L5178Y cells

More than 90% of rapidly-labelled nuclear RNA was associated with a nuclear matrix prepared from mouse leukemia L5178Y cells. The binding was not affected with up to 4 M NaCl; however, these RNAs were released from the nuclear matrix by treatment with a low ionic strength buffer (5 mM Tris-HCl buffer, pH 7.5, containing 1 mM ATP, 1 mM dithiothreitol, 0.2 mM ethylenediaminetetraacetic acid (EDTA) and 0.4 mM calcium chloride), without destruction of the sphere of the nuclear matrix. Actin filaments in the nuclear matrix were depolymerized with this buffer accompanied with rapidly-labelled RNAs. When the depolymerization was inhibited by slight modifications of the low ionic strength buffer (replacement of ATP by the same concentration of GTP; replacement of calcium ion by the same concentration of magnesium ion; addition of 20 micrograms/ml of phalloidine, which is a specific inhibitor of actin depolymerization), the release of rapidly-labelled RNAs from the nuclear matrix was also inhibited. The complex containing rapidly-labelled RNAs and matrix proteins was solubilized by a sonication from the nuclear matrix, and subjected to cesium chloride equilibrium centrifugation. Rapidly-labelled RNAs were concentrated on the bottom of the gradient accompanied with a small number of proteins (68K, 60K, 43K and 40K). The 43K protein was identified as actin by immunoblotting. By RNase digestion before equilibrium centrifugation, actin in the bottom fractions disappeared. These results suggest that rapidly-labelled RNAs anchor on the actin filaments in the nuclear matrix.     

Depolymerization of F-actin by deoxyribonuclease I.

Deoxyribonuclease I causes depolymerization of filamentous muscle actin to form a stable complex of 1 mole DNAase I:1 mole actin. The regulatory proteins tropomyosin and troponin bind to filamentous actin and slow down but do not prevent the depolymerization. In the absense of ATP, heavy meromyosin binds tightly to actin filaments and blocks completely the DNAase I: actin filament interaction. Addition of ATP releases heavy meromyosin; DNAase I is then rapidly inhibited and the actin filaments are depolymerized.     

Partially polymerized erythrocyte actin obtained by affinity chromatography on DNAse sepharose. Comparison with rabbit skeletal muscle actin and role of calcium

A new procedure, consisting of affinity chromatography on DNAse sepharose, is worked out for the purification of human erythrocyte actin from an extract of acetone powder. Comparison of skeletal muscle and erythrocyte actin purified either by reversible polymerization or affinity chromatography on DNAse Sepharose led us to infer that the erythrocyte actin isolated by affinity chromatography was pure, devoid of spectrin, and was obtained in part under polymerized (di and tetrameric) forms. This partial polymerization is related to a loss of calcium bound to actin.     

Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.     

The contractile basis of ameboid movement. II. Structure and contractility of motile extracts and plasmalemma-ectoplasm ghosts

The role of calcium and magnesium-ATP on the structure and contractility in motile extracts of Amoeba proteus and plasmalemma-ectoplasm "ghosts" of Chaos carolinensis has been investigated by correlating light and electron microscope observations with turbidity and birefringence measurements. The extract is nonmotile and contains very few F-actin filaments and myosin aggregates when prepared in the presence of both low calcium ion and ATP concentrations at an ionic strength of I = 0.05, pH 6.8. The addition of 1.0 mM magnesium chloride, 1.0 mM ATP, in the presence of a low calcium ion concentration (relaxation solution) induced the formation of some fibrous bundles of actin without contracting, whereas the addition of a micromolar concentration of calcium in addition to 1.0 mM magnesium-ATP (contraction solution) (Taylor, D. L., J. S. Condeelis, P. L. Moore, and R. D. Allen. 1973. J. Cell Biol. 59:378-394) initiated the formation of large arrays of F-actin filaments followed by contractions. Furthermore, plasmalemma-ectoplasm ghosts prepared in the relaxation solution exhibited very few straight F-actin filaments and myosin aggregates. In contrast, plasmalemmaectoplasm ghosts treated with the contraction solution contained many straight F-actin filaments and myosin aggregates. The increase in the structure of ameba cytoplasm at the endoplasm-ectoplasm interface can be explained by a combination of the transformation of actin from a less filamentous to a more structured filamentous state possibly involving the cross-linking of actin to form fibrillar arrays (see above-mentioned reference) followed by contractions of the actin and myosin along an undetermined distance of the endoplasm and/or ectoplasm.     

Calmodulin dissociation regulates brush border myosin I (110-kD- calmodulin) mechanochemical activity in vitro

110-kD-calmodulin, when immobilized on nitrocellulose-coated coverslips, translocates actin filaments at a maximal rate of 0.07-0.1 micron/s at 37 degrees C. Actin activates MgATPase activity greater than 40-fold, with a Km of 40 microM and Vmax of 0.86 s-1 (323 nmol/min/mg). The rate of motility mediated by 110-kD-calmodulin is dependent on temperature and concentration of ATP, but independent of time, actin filament length, amount of enzyme, or ionic strength. Tropomyosin inhibits actin binding by 110-kD-calmodulin in MgATP and inhibits motility. Micromolar calcium slightly increases the rate of motility and increases the actin-activated MgATP hydrolysis of the intact complex. In 0.1 mM or higher calcium, motility ceases and actin-dependent MgATPase activity remains at a low rate not activated by increasing actin concentration. Correlated with these inhibitions of activity, a subset of calmodulin is dissociated from the complex. To determine if calmodulin loss is the cause of calcium inhibition, we assayed the ability of calmodulin to rescue the calcium-inactivated enzyme. Readdition of calmodulin to the nitrocellulose-bound, calcium-inactivated enzyme completely restores motility. Addition of calmodulin also restores actin activation to MgATPase activity in high calcium, but does not affect the activity of the enzyme in EGTA. These results demonstrate that in vitro 110-kD-calmodulin functions as a calcium-sensitive mechanoenzyme, a vertebrate myosin I. The properties of this enzyme suggest that despite unique structure and regulation, myosins I and II share a molecular mechanism of motility.     

Modulation of cross-bridge affinity for MgGTP by Ca2+ in skinned fibers of rabbit psoas muscle.

Previously we reported that saturation of cross-bridges with MgATP gamma S in skinned muscle fibers was calcium sensitive. In the present study we investigate whether this observation can be generalized to other nucleotides by studying saturation of cross-bridges with MgGTP. In solution, myosin-subfragment 1 (S1) in the presence of 10 mM MgGTP was found to bind to actin with low affinity, similar to that in the presence of MgATP and MgATP gamma S. In EGTA buffer, the equatorial x-ray diffraction intensity ratio I11/I10 recorded in single skinned fibers decreased upon increasing MgGTP concentration from 0 to 10 mM (1 degree C and 170 mM ionic strength). The I11/I10 ratio leveled off at 10 mM MgGTP, indicating full saturation of cross-bridges with the nucleotide. Under these conditions, the value of I11/I10 is indistinguishable from that obtained in the presence of saturating [MgATP]. In CaEGTA buffer, however, the decrease in I11/I10 occurs over a wider range of concentrations, and there is no indication of I11/I10 leveling off at 10 mM MgGTP, suggesting that full saturation is not reached. The Ca2+ dependence of GTP binding appears to be a direct consequence of the differences in the affinities of the strongly bound cross-bridges to actin versus weakly bound cross-bridges to actin. A biochemical scheme that could qualitatively explain the titration behavior of ATP gamma S and GTP is presented.     

Polymerization of ADP-actin

Using hexokinase, glucose, and ATP to vary reversibly the concentrations of ADP and ATP in solution and bound to Acanthamoeba actin, I measured the relative critical concentrations and elongation rate constants for ATP-actin and ADP-actin in 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.1 mM nucleotide, 0.1 mM CaCl2, 10 mM imidazole, pH 7. By both steady-state and elongation rate methods, the critical concentrations are 0.1 microM for ATP-actin and 5 microM for ADP-actin. Consequently, a 5 microM solution of actin can be polymerized, depolymerized, and repolymerized by simply cycling from ATP to ADP and back to ATP. The critical concentrations differ, because the association rate constant is 10 times higher and the dissociation rate constant is five times lower for ATP-actin than ADP-actin. These results show that ATP-actin occupies both ends of actin filaments growing in ATP. The bound ATP must be split on internal subunits and the number of terminal subunits with bound ATP probably depends on the rate of growth.     

The complex of actin and deoxyribonuclease I as a model system to study the interactions of nucleotides, cations and cytochalasin D with monomeric actin

The stoichiometric actin--DNase-I complex was used to study the actin--nucleotide and actin--divalent-cation interactions and its ATPase activity in the presence of MgCl2 and cytochalasin D. Treatment of actin--DNase-I complex with 1 mM EDTA results in almost complete restoration of its otherwise inhibited DNase I activity, although the complex does not dissociate, as verified by size-exclusion chromatography. This effect is due to a loss of actin-bound nucleotide but is prevented by the presence of 0.1-0.5 mM ATP, ADP and certain ATP analogues. In this case no increase in DNase I activity occurs, even in the presence of EDTA. At high salt concentrations and in the presence of Mg2+ ('physiological conditions') the association rate constants for ATP, ADP and epsilon ATP (1,N6-ethenoadenosine 5'-triphosphate) and the dissociation rate constant for epsilon ATP were determined. Both the on and off rates were found to be reduced by a factor of about 10 when compared to uncomplexed actin. Thus the binding constant of epsilon ATP to actin is almost unaltered after complexing to DNase I (2.16 x 10(8) M-1). Titrating the increase in DNase I activity of the actin--DNase I complex against nucleotide concentration in the presence of EDTA, the association constant of ATP to the cation-free form of actin--DNase I complex was found to be 5 x 10(3) M-1, which is many orders of magnitude lower than in the presence of divalent metal ions. The binding constant of Ca2+ to the high-affinity metal-binding site of actin was found not to be altered when complexed to DNase I, although the rate of Ca2+ release decreases by a factor of 8 after actin binding to DNase I. The rate of denaturation of nucleotide-free and metal-ion-free actin--DNase I complex was found to be reduced by a factor of about 15. The ATPase activity of the complex is stimulated by addition of Mg2+ and even more effectively by cytochalasin D, proving that this drug is able to interact with monomeric actin.     

The effects of platelet tropomyosin on the ATPase activities of muscle actomyosin subfragment 1 in the absence and presence of troponin, its components, and calmodulin

The effect of platelet tropomyosin on the ATPase activity of a muscle actin-myosin subfragment 1 system has been examined in 30 mM KCl, 5 mM MgCl2, 2 mM ATP, 0.1 mM EGTA, 2 mM Tris, pH 7.8. Whereas muscle tropomyosin inhibits the activity by 60%, the platelet protein had no effect. Addition of muscle troponin in the absence of Ca2+ to the system inhibited the activity by up to 80% irrespective of whether muscle or platelet tropomyosin was used. The release of this inhibition by the addition of Ca2+ was much less in the case of platelet tropomyosin. This may result from the fact that platelet tropomyosin aggregates poorly in a head-to-tail manner and interacts only weakly with muscle troponin-T. In the presence of troponin-I and platelet tropomyosin, inhibition of the ATPase activity was 80%. This inhibition was largely released by the addition of troponin-C irrespective of the presence of Ca2+. The addition of brain calmodulin, however, released the inhibition in the presence of calcium but not in its absence. These effects can be correlated with the binding or lack of binding of the platelet tropomyosin to the actin filament.     

Actin content and synthesis in differentiating Drosophila cells in culture

In response to the addition of 20-hydroxyecdysone, Drosophila line Kc cells extend filopodia, become motile and aggregate. An investigation was carried out to determine whether the appearance of motility was correlated with an increase in intracellular actin content or actin synthesis, or a decrease in actin degradation. With the exception of actin content, measured by DNAse I inactivation, treated and untreated cells were indistinguishable for all parameters. DNAse I inactivation studies indicated a three- to four-fold increase in actin content during the two days following hormone exposure. These data are interpreted by a model in which an inactive pool of actin becomes available for microfilament assembly.     

Regulation of actin polymerization by membrane fraction of platelets

We studied the interaction between the purified membrane fraction of human platelets and the polymerization of skeletal actin. The viscosity of actin was measured by the falling ball method. The fraction suppressed the polymerization of actin in the presence of 20 mM KCl and 0.4 mM EGTA. The addition of calcium ion or thrombin to the fraction did not cause suppression. A DNase I affinity column bound the membrane fraction in the presence of calcium ion. The frozen membrane fraction and the vesicles reconstituted with lipids from the platelet membrane enhanced the polymerization of actin. Trypsinized membrane fraction and the membrane fraction treated with phospolipase A2 enhanced the polymerization of actin, but membrane fraction treated with phospholipase C had no effect. The reconstituted membrane vesicles mentioned above lowered the critical concentration for actin polymerization. These findings suggested that the polymerization of intracellular actin is enhanced not only by the mobilization of calcium ion, but also by biochemical changes in the membrane lipids.     

Nuclear actin and transport of RNA

The role of nuclear actin filaments in the RNA transport was investigated. Mouse lymphoma cells, L5178Y, were labeled for 20 min with 3H-uridine, and the isolated nuclei were incubated in a medium consisting of 0.25 M sucrose, 10 mM Tris-HCl (pH 7.5), 2 mM CaCl2, 1 mM ATP and 1mM PMSF. Release of the rapidly labeled RNA from the nuclei was temperature-dependent and was stimulated by ATP. Phalloidin, an inhibitor of actin filament depolymerization, had no effect on the system at 10 or 100 micrograms/ml. Therefore, actin filament depolymerization may not be involved in the transport of RNA.     

Crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution

A high-affinity complex formed between G-actin and plasma vitamin D-binding protein (DBP) is believed to form part of a scavenging system in the plasma for removing actin released from damaged cells. In the study presented here, we describe the crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution. The complex contains one molecule of each protein bound together by extensive ionic, polar, and hydrophobic interactions. It includes an ATP and a calcium ion bound to actin, but no evidence of vitamin D metabolites bound to the DBP. Both actin and DBP are multidomain molecules, two major domains in actin and three in DBP. All of these domains contribute to the interaction between the molecules. DBP enfolds the end of the actin molecule, principally in actin subdomain 3 but with additional interactions in actin subdomain 1. This orientation is similar to the binding of profilin to actin, as predicted from previous studies. The more extensive interactions of DBP give an affinity for actin some 3 orders of magnitude higher than that for profilin. The larger "footprint" of DBP on actin also leads to an overlap with the actin-binding site of gelsolin domain I.     

Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network

Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. Kitanishi-Yumura. 1990. Cell Struct. Funct. 15:355-364). Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCl2, which stabilized myosin II filaments, myosin II remained at the foci. Ultrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin II filaments remained in the foci in the presence of 10 mM MgCl2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form. Using [gamma 32P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin II ATPase) and/or ATP gamma S (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the foci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm.     

Preparation and purification of polymerized actin from sea urchin egg extracts

Isotonic extracts of the soluble cytoplasmic proteins of sea urchin eggs, containing sufficient EGTA to reduce the calcium concentration to low levels, form a dense gel on warming to 35-40 degrees C. Although this procedure is similar to that used to polymerize tubulin from mammalian brain, sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows this gel to have actin as a major component and to contain no tubulin. If such extracts are dialyzed against dilute salt solution, they no longer respond to warming, but gelation will occur if they are supplemented with 1 mM ATP and 0.020 M KCl before heating. Gelation is not temperature reversible, but the gelled material can be dissolved in 0.6-1 M KCl and these solutions contain F- actin filaments. These filaments slowly aggregate to microscopic, birefringent fibrils when 1 mM ATP is added to the solution, and this procedure provides a simple method for preparing purified actin. the supernate remaining after actin removal contains the other two components of the gel, proteins of approximately 58,000 and 220,000 mol wt. These two proteins plus actin recombine to form the original gel material when the ionic strength is reduced. This reaction is reversible at 0 degrees C, and no heating is required.     

The Isolation of Actin from Pea Roots by DNase I Affinity Chromatography

Native actin can be isolated from pea (Pisum sativum L.) roots by DNase I affinity chromatography, but the resulting yields and quality of actin are variable. By use of two assays for actin, a DNase I inhibition assay and a gel scanning assay, we identified several factors that increased actin yield. ATP is required for the actin in crude pea root extracts to bind to immobilized DNase I. Low amounts of ATP are hydrolyzed rapidly by an endogenous ATPase in the extract, and the actin then irreversibly loses the ability to bind to DNase I. High ATP concentrations (5-10 mm) or inhibition of the ATPase (with 10 mm pyrophosphate) are required for pea actin to retain DNase I binding ability. When adequate amounts of ATP are present, actin binding from the extract is further enhanced by basic pH, formamide, and soluble polyvinyl-pyrrolidone. Once actin is bound to the DNase I-agarose and washed free of extract, high ATP concentrations are not required to keep actin bound. Actin eluted from the DNase I-agarose with formamide retained its ability to polymerize into filaments with the addition of KCl and Mg²⁺. The advantages and disadvantages of this procedure and its application to other plant materials are discussed.     

Is the cytoskeleton-plasma membrane complex involved in lens protein biosynthesis?

Calf lens fiber cells contain a population of polyribosomes that direct, at leastin vitro, the synthesis of a specific plasma membrane protein MP26. This protein may serve as a marker in terminal differentiation, since it is absent in the lens epithelium but appears in lens fiber plasma membranes. The MP26 manufacturing polyribosomes are found to be associated with a structural complex in which also the cytoskeleton and plasma membranes participate. They can be released from the complex by treatment with DNAse I. This result presumably reflects the involvement of actin in the linkage of the MP26 synthesizing polyribosomes to the cytoskeleton-membrane complex.