The interaction of bovine pancreatic deoxyribonuclease I and skeletal muscle actin

The rate of exchange of actin-bound nucleotide is decreased by a factor of about 20 when actin is complexed with DNAase I without affecting the binding constant of calcium for actin. Binding constants of DNAase I to monomeric and filamentous actin were determined to be 5 X 10(8) M-1 and 1.2 X 10(4) M-1 respectively. The depolymerisation of F-actin by DNAase I appears to be due to a shift in the G-F equilibrium of actin by DNAase I. Inhibition of the DNA-degrading activity of DNAase I by G-actin is of the partially competitive type.     

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.     

The inhibition of bovine and rat parotid deoxyribonuclease I by skeletal muscle actin. A biochemical and immunocytochemical study.

Rat and bovine parotid gland and pancreas contain deoxyribonuclease I (DNAase I) activities in different amounts. The DNAase I activity in tissue homogenates of bovine and rat parotid gland can be inhibited by addition of monomeric actin, as with the enzyme of bovine pancreas. The isolated DNAase I species from bovine and rat parotid gland differ in their molecular weights and also in their affinities for monomeric actin, being lowest for rat parotid DNAase I (5 X 10(6)M(-1). Antibodies raised against rat and bovine parotid and bovine pancreatic DNAase I can be used to study the subcellular localization of DNAase I in these tissues by indirect immunofluorescence. DNAase I was found to be confined solely to the secretory granules of the tissue from which it was isolated.     

Estimation of denaturation of actin in the actin-myosin complex treated with various conditions by DNAase I inhibition assay.

1. Experiments were conducted to evaluate whether DNAase I (EC 3.1.4.5) inhibition assay was a valuable tool to study the denaturation of actin in the actin-myosin complex treated with various conditions. 2. A sample containing F-actin or natural actomyosin(myosin B) was treated with KI-ATP solution to convert a form which inhibits DNAase I as effectively as G-actin, and the total amount of native actin was determined by DNAase I inhibition assay. 3. On the basis of the values for remaining native actin in the sample obtained by this assay, a percentage of denaturation of actin during treatment was calculated. 4. The present result demonstrated that DNAase I inhibition assay was easy to perform, very sensitive (0.5-2.0 microgram actin) and highly specific for estimating denaturation of actin in the actin-myosin complex treated with heat or high salt concentrations. 5. In addition, the use of DNAase I and standard G-actin preparations stored frozen at -80 degrees C for the assay was found to be possible within a fixed period of time (about 2 weeks), which was helpful in monitoring the denaturation process of actin treated under various conditions for a long period.     

The interaction of 5'-nucleotidase purified from chicken gizzard and actin, and the reversible loss of the inhibitory capacity of actin on deoxyribonuclease I

Evidence is presented for a direct interaction of the intrinsic membrane protein 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) purified from avian smooth muscle (chicken gizzard) and the cytoskeletal component actin. Two different modes of interaction can be discerned: firstly, an immediate inhibitory effect of preferentially filamentous actin (F-actin) on the enzymic (i.e., AMPase) activity of 5'-nucleotidase and a direct binding of this enzyme to immobilized F-actin. Since these effects are suppressed by the addition of myosin subfragment 1, binding of 5'-nucleotidase appears to occur along the F-actin filament axis. Secondly, a time- and 5'-nucleotidase concentration-dependent transformation of also preferentially F-actin into a form unable to inhibit the enzymic activity of deoxyribonuclease I (DNAase I). This desensitization of actin versus DNAase I is not due to a denaturation process and was found to be reversible after addition of ATP. Furthermore, it does not seem to effect the ability of actin to bind to DNAase I. The transformation is accompanied by the hydrolysis of actin-bound nucleotide into adenosine, which remains bound to actin. Therefore, the desensitization of actin versus DNAase I appears to be due to a nucleotide-dependent conformational change of actin. An unidentified contamination of the 5'-nucleotidase preparations to a varying degree with ADPase and ATPase activities appears to be responsible for the desensitization process, although a synergistic role of these activities and 5'-nucleotidase cannot be excluded.     

A phage display-based method for determination of relative affinities of mutants. Application of the actin-binding motifs in thymosin beta 4 and the villin headpiece

We propose phage display combined with enzyme-linked immunosorbent assay as a tool for the systematic analysis of protein-protein interactions by investigating the binding behavior of variants to a partner protein. Via enzyme-linked immunosorbent assay we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate the relative affinities of a large number of mutants. As model systems, we investigated actin-binding motifs conserved in a number of proteins binding monomeric or filamentous actin. The hexapeptide motifs LKKTET, present in thymosin beta4, and LKKEKG, present in the villin headpiece, were mutated, and the variants were analyzed. Study of the positional tolerance allows postulating that the motifs, although similar in primary structures adopt different conformations when bound to actin. In addition, our data show that the second and the fourth amino acid of the thymosin beta4 motif and the first three residues of the villin headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.     

The 3' terminal sequence of chicken ovalbumin messenger RNA and its comparison with other messenger RNA molecules.

The subcellular distribution of actin in embryonic chick fibroblasts and brain was examined biochemically. Several gentle extraction procedures, which did not cause the breakdown of muscle filamentous actin, caused the release of large amounts of “cytoplasmic actin” in a monomeric form. This did not behave as a precursor or degradation product of filamentous actin in pulse label experiments and failed to form filaments under the same conditions as muscle actin. However, when it was purified and concentrated it was able to form aggregates which were very similar to paracrystals of muscle filamentous actin. These results suggest that cytoplasmic actin is at a higher concentration than muscle actin before it will polymerize, and that in the cell much of it is either monomeric or in a labile state.     

Filament assembly and regulation of the actin-activated ATPase activity of thymus myosin

The effects of light chain phosphorylation on the actin-activated ATPase activity and filament assembly of calf thymus cytoplasmic myosin were examined under a variety of conditions. When unphosphorylated and phosphorylated thymus myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, but when they were filamentous, their MgATPase activities were stimulated by actin. The phosphorylated myosin remained filamentous at lower Mg2+ concentrations and higher KC1 concentrations than did the unphosphorylated myosin, and the myosin concentration required for filament assembly was lower for phosphorylated myosin than for unphosphorylated myosin. By varying the myosin concentration, it was possible to have under the same assay conditions mostly monomeric myosin or mostly filamentous myosin; under these conditions, the actin-activated ATPase activities of the filamentous myosins were much greater than those of the monomeric myosins. The addition of phosphorylated myosin to unphosphorylated myosin promoted the assembly of unphosphorylated myosin into filaments. These results suggest that phosphorylation may regulate the actomyosin-based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly.     

Expression of a nonpolymerizable actin mutant in Sf9 cells

We have succeeded in expressing actin in the baculovirus/Sf9 cell system in high yield. The wild-type (WT) actin is functionally indistinguishable from tissue-purified actin in its ability to activate ATPase activity and to support movement in an in vitro motility assay. Having achieved this feat, we used a mutational strategy to express a monomeric actin that is incapable of polymerization. Native actin requires actin binding proteins or chemical modification to maintain it in a monomeric state. The mutant actin sediments in the analytical ultracentrifuge as a homogeneous monomeric species of 3.2 S in 100 mM KCl and 2 mM MgCl(2), conditions that cause WT actin to polymerize. The two point mutations that render actin nonpolymerizable are in subdomain 4 (A204E/P243K; "AP-actin"), distant from the myosin binding site. AP-actin binds to skeletal myosin subfragment 1 (S1) and forms a homogeneous complex as demonstrated by analytical ultracentrifugation. The ATPase activity of a cross-linked AP-actin.S1 complex is higher than that of S1 alone, although less than that supported by filamentous actin (F-actin). AP-Actin is an excellent candidate for structural studies of complexes of actin with motor proteins and other actin-binding proteins.     

Villin is a major protein of the microvillus cystoskeleton which binds both G and F actin in a calcium-dependent manner

The cores of the microvilli present on intestinal epithelial cells are currently the only microfilament arrangement which can be isolated ultrastructurally intact and in sufficient quantities for biochemical analysis. We have isolated and characterized villin, a major protein of the microvillus core. Using villin's ability to bind very tightly to immobilized monomeric actin in a calcium-dependent manner, we have developed a method for its rapid purification by affinity chromatography on G actin, which itself was bound to immobilized pancreatic deoxyribonuclease I (DNAase I). The villin-G actin complex on DNAase I is resistant to high ionic strength, and villin, but not actin, is released when the calcium concentration is less than 10⁶ M. Purified villin behaves as a globular monomeric protein of molecular weight 95,000, and is free of carbohydrate. Villin also interacts with F actin. In the absence of calcium, villin cross-links F actin having the properties of an F actin bundling or gelation factor. In the presence of calcium (>10^?7 M), villin apparently restricts the polymerization of actin to short filaments which cannot be readily sedimented. The properties of villin are not compatible with its previously suggested role as the cross-filament between the microvillus microfilament core and the plasma membrane, but rather indicate a function as a calcium-dependent F actin-bundling protein. The role of villin is discussed in terms of the other protein components of the microvillus core and in relation to recently described calcium-dependent gelation factors.     

Actin polymerization induced by pulsed electric stimulation of bone cells in vitro

Electric field pulses, capacitively applied to tissue cultures of embryonic bone cells, were shown to induce changes in the state of cellular actin. Three actin states could be defined by DNAase I inhibition. A rapidly (20-30 s) inhibiting fraction, attributed to monomeric G-actin, amounts to 55% of total actin in nonstimulated cells. An additional fraction of 8% required approx. 20 min to reach full inhibition and was tentatively defined as polymeric 'F'-actin. The remaining 37% could be detected only after treatment of the cells with 0.75 M guanidine hydrochloride, which dissociates actin from all its protein interactions. This fraction, N-actin (network actin) is believed to represent F-actin integrated into some supramolecular structure, where it is not accessible to DNAase I. Upon short electric stimulation the distribution changed to 40% G-actin, 12% F-actin and 48% N-actin. 3-Isobutyl-1-methylxanthine (IBMX; an inhibitor of cAMP phosphodiesterase), depletion of extracellular calcium, and calmodulin inhibitors abolished this field effect.     

Aging and lymphocyte cytoskeleton: age-related decline in the state of actin polymerization in T lymphocytes from Fischer F344 rats

T cell functions are known to decline with age, but the underlying cause of the decline is unclear. Because of the importance of cytoskeletal elements in cellular functions, we examined the content and the state of polymerization of actin in lymphocytes from Fischer F344 rats of four different ages (6, 14, 23, and 31 mo). The cellular actin content was determined by a DNAase I inhibition assay. Our results indicate that the total actin content of spleen lymphocytes did not change significantly with age; however, polymeric actin content, particularly in T cells, decreased with age, which might be a result of the shift from the polymeric actin pool to the monomeric pool. Similar changes also occurred in B cells but to a lesser extent. We conclude that the state of polymerization of lymphocytes changed drastically with age, and that this might be an important factor in the age-related decline in the cellular functions of lymphocytes.     

Effects of the actin-binding protein DNAase I on cytoplasmic streaming and ultrastructure of Amoeba proteus

Microinjection of DNAase I, which is known to form a specific complex with G-actin, induces characteristic changes in cytoplasmic streaming, locomotion and morphology of the contractile apparatus of A. proteus. Light microscopical studies show pronounced streaming originating from the uroid and/or the retracting pseudopods, which ceases 10--15 min after injection of DNAase I, at a time when ultrasctructural studies show that the actin filament system is very much reduced. These results suggest that a controlled reversible equilibrium between soluble and polymerized forms of actin is a necessary requirement for amoeboid movement. The topographic distribution of contractile filaments beneath the plasma membrane visualized by correlated light- and electron microscopy of DNAase I-injected cells establishes the importance of the membrane-bound filamentous layer for three major aspects of streaming: (1) Streaming originates by local contractions of a cell membrane-associated filament layer at the uroid and/or retracting pseudopods, creating a pressure flow. (2) This flow continues beneath the membrane, which is stabilized by filaments in the lateral regions between the posterior end, with a high hydrostatic pressure, and the anterior end, with a low hydrostatic pressure. (3) Pseudopods or extending areas are created by a local destabilization of the cell periphery caused by the separation of the filamentous layer from the plasma membrane.     

Actin in B16 melanoma cells of differing metastatic potential : Effects of trypsin and serum

Actin is present in cells in monomeric and polymeric (filamentous) forms. Filamentous actin is distributed in Triton-soluble (cytosolic) and Triton-insoluble (cytoskeletal core) fractions. We have used the DNase 1 inhibition assay and immunofluorescence to investigate the distribution of actin in monomeric and polymeric forms in cloned B16 murine melanoma cell lines of low and high metastatic capacity. The protease trypsin caused rounding up and detachment of both cell lines within 5 min. This was associated with almost complete depolymerization of cytosolic actin filaments but the Triton-insoluble cytoskeleton was not quantitatively affected by trypsin treatment. There were quantitative differences between the clones in their response to incubation in the presence or absence of 10% serum. The highly metastatic cell line contained 35% more actin when incubated in the presence of 10% serum, almost completely distributed to the Triton-insoluble cytoskeleton, an effect not seen in the low metastatic cells.     

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.     

Overexpression of cofilin stimulates bundling of actin filaments, membrane ruffling, and cell movement in Dictyostelium

Cofilin is a low molecular weight actin-modulating protein whose structure and function are conserved among eucaryotes. Cofilin exhibits in vitro both a monomeric actin-sequestering activity and a filamentous actin-severing activity. To investigate in vivo functions of cofilin, cofilin was overexpressed in Dictyostelium discoideum cells. An increase in the content of D. discoideum cofilin (d-cofilin) by sevenfold induced a co-overproduction of actin by threefold. In cells over-expressing d-cofilin, the amount of filamentous actin but not that of monomeric actin was increased. Overexpressed d-cofilin co-sedimented with actin filaments, suggesting that the sequestering activity of d- cofilin is weak in vivo. The overexpression of d-cofilin increased actin bundles just beneath ruffling membranes where d-cofilin was co- localized. The overexpression of d-cofilin also stimulated cell movement as well as membrane ruffling. We have demonstrated in vitro that d-cofilin transformed latticework of actin filaments cross-linked by alpha-actinin into bundles probably by severing the filaments. D. discoideum cofilin may sever actin filaments in vivo and induce bundling of the filaments in the presence of cross-linking proteins so as to generate contractile systems involved in membrane ruffling and cell movement.     

Rabbit skeletal muscle F-actin can be stable at low ionic strength, provided trace amounts of Ca2+ are absent.

Addition of low concentrations (0.2--2.0 mM) of EGTA to rabbit skeletal muscle G-actin in the presence of ATP caused increase in viscosity. The effect is probably due to chelation of Ca2+. EGTA-polymerized actin was sedimented in the ultracentrifuge as a pellet which could be depolymerized in the presence of Ca2+ and then repolymerized. Electron microscopy indicated that formation of filamentous actin which appears to be somewhat more flexible than F-actin obtained by polymerization with KCl. The EGTA-polymerized actin was dissociated by DNAase I faster than KCl-polymerized actin. F-Actin can thus be stable also in very low ionic strength media if Ca2+ is removed whereas for G-actin to be the only form of the protein in such media, micromolar concentrations of Ca2+ must be present.     

Crystallization of cytoplasmic actin in complex with deoxyribonuclease I.

Crystals of cytoplasmic (porcine liver) actin in complex with deoxyribonuclease I (DNAase I) were prepared for structural determination by X-ray-diffraction analysis. The crystallization of porcine liver actin-DNAase I complex is preceded by a brief treatment with immobilized trypsin, whereby a C-terminal tri- or di-peptide including cysteine-374 is removed from the actin without any noticeable degradation of both proteins as judged by sodium dodecyl-sulphate/polyacrylamide-gel electrophoresis. Analysis of the crystals obtained does not reveal any differences in the three-dimensional structure of porcine liver actin from its skeletal compartment at up to 0.6 nm resolution. However, in contrast with crystalline skeletal-muscle actin-DNAase I complex, heavy-atom substitution of crystals of porcine liver actin-DNAase I complex could not be achieved with methyl mercuriacetate. Evidence is presented that, in porcine liver actin, the N-terminal cysteine residue is not located at position no. 10, as in skeletal- and smooth-muscle actin, but most probably at position no. 17. Thus, because this site is covered by DNAase I, the cysteine becomes inaccessible to titration with 5,5'-dithiobis-(2-nitrobenzoic acid) after complex-formation with DNAase I.     

Cycling of actin assembly in synaptosomes and neurotransmitter release

We have investigated the regulation of actin assembly in whole mouse brain synaptosomes and how that regulation modulates neurotransmitter release. During a 30 s depolarization with high K+, filamentous actin (F-actin) levels, monitored by staining with rhodamine phalloidin, increase dramatically (up to 300% in 3 s), decrease, and increase once again. This F-actin cycling is regulated by pathways both dependent and independent of Ca2+ influx and is markedly affected by exposing synaptosomes to Li+, tetrodotoxin, and diacylglycerol. Measurement of [3H]norepinephrine release from synaptosomes containing entrapped agents that modulate actin assembly (DNAase I or phalloidin) indicates that actin depolymerization is necessary for normal release and that repolymerization limits release.     

Regulation of the actin-activated ATPase and in vitro motility activities of monomeric and filamentous Acanthamoeba myosin II.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.