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.     

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.     

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.     

Solubilization of native actin monomers from human erythrocyte membranes

Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I.     

Biochemical characteristics of functionally active actin-like protein from liver mitochondria

The actin-like protein with a molecular weight of 42 kDa was obtained from the preparation of freshly isolated mitochondria of the rat liver using the method of immobilized DNAse affinity chromatography. The inhibitory ability of the isolated protein with respect to pancreatic DNAse I was the same as that of muscular actin. The native structure of the mitochondria protein is confirmed by the data of spectral analysis and its ability to globular-fibrillar transformation with an increased ionic strength of the solution. The polymerization ability as well as a stimulating effect of the actin-like protein of mitochondria on the ATPase activity of myosin is much less pronounced as compared to actin of skeletal muscles.     

Actin in the ciliated protozoan Climacostomum virens: purification by DNAse I affinity chromatography, electrophoretic characterization, and immunological analysis.

The anti-actin monoclonal antibody (mab) JLA20 (Lin: Proc. Natl. Acad. Sci. U.S.A. 78:2335-2339, 1981) labels a 43 kD protein on Western blots of Climacostomum cell extracts; this protein does not react with an anti-alpha-smooth muscle actin mab (Skalli et al.: J. Cell Biol. 103:2787-2796, 1986) nor with an anti-alpha-sarcomeric actin mab (Skalli et al.: Am. J. Pathol. 130:515-531, 1988). This protein binds to DNAse I and can be purified by DNAse I affinity chromatography. The affinity-purified actin also reacts with mab JLA20. Two-dimensional gel analysis reveals that Climacostomum actin focuses as three spots which are more basic than the mammalian actin isoforms. After addition of KCl, the affinity-purified actin polymerizes into filaments as shown by electron microscopy after negative staining.     

Fluorescent staining of the actin cytoskeleton in human lymphocytes, monocytes and polymorphonuclear cells using a DNAse 1/anti-DNAse 1 immunoglobulin fluorescein conjugated system.

The actin associated with membrane-enriched extracts of leukocytes can be quantitated by DNAse 1 inhibition. Using this assay, we previously demonstrated that the actin level in monocytes was significantly higher than that in polymorphonuclear, T and B cells respectively. However, the extracellular location of the actin fraction detected by DNAse 1 inhibition (monomeric "G") remained unclear. This study using the DNAse 1/anti DNAse 1 immunoglobulin fluorescein conjugated system demonstrated that G-actin is present primarily in the cortical cell cytoplasm of leukocytes, in confirmation of our previous biochemical findings. Since the solubilized G-actin activities of membrane-rich lymphoid cell fractions, measured by DNAse 1 inhibition, are a reflection of the migratory potential, this immunofluorescent system may permit identification of the leukocytic cell subpopulations that have a potential for active circulation.     

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.     

Actin in mammalian lens.

In this paper evidence is provided that one of the protein components of the water-soluble fraction of the calf lens binds specifically to deoxyribonuclease I (DNAse I). On the basis of this property, the polypeptide could be purified by applying DNAse I affinity chromatography. Concomitantly a protein of Mr55000 and a rather large amount of alpha-crystallin copurify with this polypeptide, which has a molecular weight of 42000. Highly purified 42000-Mr protein was also obtained by extraction of the water-insoluble fraction of the calf lens with 2-([tris(hydroxymethyl)methyl]amino) ethanesulfonic acid followed by gel filtration. Amino acid analyses, peptide mapping and electron microscopy show that the protein obtained from both lens fractions is identical to non-muscle actin. Furthermore the amino acid composition of the 55000-Mr protein is identical to hog stomach skeletin and very similar to calf brain desmin.     

Fluorescent staining of the actin cytoskeleton in human lymphocytes,monocytes and polymorphonuclear cells using a DNAse 1/anti-DNAse 1 immunoglobulin fluorescein conjugated system

Summary The actin associated with membrane-enriched extracts of leukocytes can be quantitated by DNAse 1 inhibition. Using this assay, we previously demonstrated that the actin level in monocytes was significantly higher than that in polymorphonuclear, T and B cells respectively. However, the extracellular location of the actin fraction detected by DNAse 1 inhibition (monomeric G) remained unclear. This study using the DNAse 1/anti DNAse 1 immunoglobulin fluorescein conjugated system demonstrated that G-actin is present primarily in the cortical cell cytoplasm of leukocytes, in confirmation of our previous biochemical findings. Since the solubilized G-actin activities of membrane-rich lymphoid cell fractions, measured by DNAse 1 inhibition, are a reflection of the migratory potential, this immunofluorescent system may permit identification of the leukocytic cell subpopulations that have a potential for active circulation.     

Actin polymerization is essential for pollen tube growth

Actin microfilaments, which are prominent in pollen tubes, have been implicated in the growth process; however, their mechanism of action is not well understood. In the present work we have used profilin and DNAse I injections, as well as latrunculin B and cytochalasin D treatments, under quantitatively controlled conditions, to perturb actin microfilament structure and assembly in an attempt to answer this question. We found that a approximately 50% increase in the total profilin pool was necessary to half-maximally inhibit pollen tube growth, whereas a approximately 100% increase was necessary for half-maximal inhibition of cytoplasmic streaming. DNAse I showed a similar inhibitory activity but with a threefold more pronounced effect on growth than streaming. Latrunculin B, at only 1--4 nM in the growth medium, has a similar proportion of inhibition of growth over streaming to that of profilin. The fact that tip growth is more sensitive than streaming to the inhibitory substances and that there is no correlation between streaming and growth rates suggests that tip growth requires actin assembly in a process independent of cytoplasmic streaming.     

Inhibition of DNAse I activity in vivo]

Immune gamma-globulins containing antibodies to bovine DNAse I inhibit activities of bovine and mouse DNAse I both in vitro and in vivo. Bovine DNAse I was used as exogenous DNAse I in the in vivo studies and was injected to mice intraperitoneally in combination with gamma-globulins. The serous fluid of mice was used as a source of endogenous DNAse I. Both in in vitro and in vivo studies immune gamma-globulins caused a practically complete inhibition of bovine DNAse I activity, whereas the activity of mouse DNAse I (endogenous) was inhibited by 60-80%. Nonimmune gamma-globulins had no inhibitory effects whatsoever.     

Depolymerization of actin microfilaments in the I--Z--I brushes of transverse striated muscles by using DNAse I

To release Z-discs of skeletal muscles myofibrils from actin microfilaments, I--Z--I-brushes (complexes of Z-discs and thin filaments) were treated with DNAse I-both in suspension and on electron microscopical grids. It was shown that such a treatment resulted in depolymerization of actin filaments. The preparations obtained were heterogeneous and contained I--Z--I-brushes with shorter actin filaments and single Z-discs. The structure of Z-discs released from actin filament remained intact. Therefore these preparations may be used in studies on regulation of actin microfilaments assembly.     

Characterization of a DNA binding activity in DNAse I hypersensitive site 4 of the human globin locus control region.

A portion of the beta-globin Locus Control Region (LCR), which included DNAse I hypersensitive site 4 (HS4), was analyzed for its interactions with nuclear extracts and its contribution to LCR activity in a functional assay. In gel retardation assays, a short fragment from HS4 formed complexes with nuclear extracts from both erythroid and nonerythroid cells, and a core protected sequence 5'GACTGGC3' was revealed by DNAse I protection and methylation interference studies. This sequence resembles the binding sites of CCAAT-family members. Purified CP-2 but not CP-1 was shown to bind this HS4 sequence in a gel shift reaction, suggesting that the HS4 binding activity shares some sequence specificity with the CCAAT-factor family. Utilizing a transient expression assay in murine erythroleukemia cells, steady-state RNA levels were measured from pairs of LCR constructs linked to distinguishable beta-globin reporter genes. A short DNA fragment from HS4 which included the binding site for this novel binding activity accounted for most of the contribution to high level expression made by the entire HS4 region.     

In situ nick translation at the electron microscopic level: a tool for studying the location of DNAse I-sensitive regions within the cell

The in situ nick translation method was adapted to the ultrastructural level, to study the location of DNAse I-sensitive sequences within the cell. Ultra-thin sections of Lowicryl-embedded cells were incubated in a medium containing DNAse I, DNA polymerase I, and all four deoxyribonucleotides, some being biotinylated. The nick-translated sites were then visualized by an indirect immunogold labeling technique. The resulting labeling pattern is closely dependent on the DNAse I concentration in the nick-translation medium. The method reveals with great precision the specific DNAse I-sensitive regions within the nucleus. This technique can be used to discriminate between active and inactive regions of interphase chromatin.     

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.     

Supercontraction in crayfish muscle: correlation with a peculiar actin localization.

Crayfish muscle, like muscles from some other invertebrates, can supercontract. This muscle shortening is characterized by an overlap of thin filaments with crossing of thick filaments through the Z discs. In intact muscle cells, supercontraction does not seem to induce irreversible structural modifications in the tissue. Isolated crayfish myofibrils in the relaxed state cannot be distinguished from vertebrate myofibrils under light microscope, either by phase contrast or by immunofluorescence, with antiactin antibodies, actin being localized in the I bands. However, when isolated crayfish myofibrils are supercontracted, irreversible dammage occurs, most thin filaments being lost. Actin becomes then hardly detectable, being visible, by immunofluorescence, either in the Z discs or evenly distributed in the whole myofibril. During myofibril supercontraction, high amounts of denatured actin, become soluble as shown by SDS-PAGE, by double immunodiffusion, and by DNAse inhibition.     

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.