Conformation changes of actin during formation of filaments and paracrystals and upon interaction with DNase I, cytochalasin B, and phalloidin

Spin labels attached to rabbit muscle actin became more immobilized upon conversion of actin from the G state to the F state with 50 mM KCl. Titration of G-actin with MgCl2 produced F-actin-like EPR spectra between 2 and 5 mM-actin filaments by electron microscopy. Higher concentrations of MgCl2 produced bundles of actin and eventually paracrystals, accompanied by further immobilization of spin labels. The effects of MgCl2 and KCl were competitive: addition of MgCl2 to 50 mM could convert F-actin (50 mM KCl) to paracrystalline (P) actin; the reverse titration (0 to 200 mM KCl in the presence of 20 mM MgCl2) was less complete. Addition of DNase I to G- or F-actin gave the expected amorphous electron micrographic pattern, and the actin was not sedimentable at (400,000 x g x h). EPR showed that the actin was in the G conformation. Addition of DNase I to paracrystalline actin gave the F conformation (EPR) but the actin was "G" by electron microscopy. Phalloidin converted G-actin to F-actin, had no effect on F-actin, and converted P-actin to the F state by electron microscopy but maintained the P conformation by EPR. Cytochalasin B produced no effects observable by EPR or centrifugation but "untwisted" paracrystals into nets. Since actin retained its P conformation by EPR in two states which were morphologically not P, we conclude that the P state is a distinct conformation of the actin molecule and that actin filaments aggregate to form bundles (and eventually paracrystals) when actin monomers are able to enter the P conformation.     

Association of deoxyribonuclease I with the pointed ends of actin filaments in human red blood cell membrane skeletons

We have characterized the interaction of bovine pancreatic deoxyribonuclease I (DNase I) with the filamentous (F-)actin of red cell membrane skeletons stabilized with phalloidin. The hydrolysis of [3H]DNA was used to assay DNase I. We found that DNase I bound to a homogenous class of approximately equal to 2.4 X 10(4) sites/skeleton with an association rate constant of approximately 1 X 10(6) M-1 S-1 and a KD of 1.9 X 10(-9) M at 20 degrees C. Phalloidin lowered the dissociation constant by approximately 1 order of magnitude. The DNase I which sedimented with the skeletons was catalytically inactive but could be reactivated by dissociation from the actin. Actin and DNA bound to DNase I in a mutually exclusive fashion without formation of a ternary complex. Phalloidin-treated red cell F-actin resembled rabbit muscle G-actin in all respects tested. Since the DNase I binding capacity of the skeletons corresponded to the number of actin protofilaments previously estimated by other methods, it seemed likely that the enzyme binding site was confined to one end of the filament. We confirmed this premise by showing that elongating the red cell filaments with rabbit muscle actin monomers did not appreciably add to their capacity to bind or inhibit DNase I. Saturation of skeletons with cytochalasin D or gelsolin, avid ligands for the barbed end of actin filaments, did not reduce their binding of DNase I. Furthermore, neither cytochalasin D nor DNase I alone blocked all of the sites for addition of monomeric pyrene-labeled rabbit muscle G-actin to phalloidin-treated skeletons; however, a combination of the two agents did so. In the presence of phalloidin, the polymerization of 300 nM pyrenyl actin on nuclei constructed from 5 nM gelsolin and 25 nM rabbit muscle G-actin was completely inhibited by 35 nM DNase I but not by 35 nM cytochalasin D. We conclude that DNase I associates uniquely with and caps the pointed (slow-growing or negative) end of F-actin. These results imply that the amino-terminal, DNase I-binding domain of the actin protomer is oriented toward the pointed end and is buried along the length of the actin filament.     

In vivo analysis of functional domains from villin and gelsolin

Transfected CV1 cells were used to compare the in vivo effects of various domains of villin and gelsolin. These two homologous actin modulating proteins both contain a duplicated severin-like sequence. Villin has in addition a carboxy-terminal domain, the headpiece, which accounts for its bundling activity. The effects of the villin-deleted mutants were compared with those of native villin. Our results show that essential domains of villin required to induce the growth of microvilli and F-actin redistribution are present in the first half of the core and in the headpiece. We also show that the second half of the villin core cannot be exchanged by its homolog in gelsolin. When expressed at high levels of CV1 cells, full length gelsolin completely disrupted stress fibers without change of the cell shape. Addition of the villin headpiece to gelsolin had no effect on the phenotype induced by gelsolin alone. Expression of the first half of gelsolin induced similar modifications as capping proteins and rapid cell mortality; this deleterious effect on the cell structure was also observed when the headpiece was linked to the first half of gelsolin. In cells expressing the second half of gelsolin, a dotted F-actin staining was often seen. Moreover elongated dorsal F-actin structures were observed when the headpiece was linked to the second gelsolin domain. These studies illustrate the patent in vivo severing activity of gelsolin as well as the distinct functional properties of villin core in contrast to gelsolin.     

Binding of pig plasma gelsolin to F-actin and partial fractionation into calcium-dependent and calcium-independent forms

The interaction of pig plasma gelsolin with F-actin has been studied by a sedimentation assay using 125I-gelsolin in a Beckman Airfuge. Over 90% of the gelsolin bound to F-actin in 0.1 mM CaCl2 in experiments using 24 microM actin and 2-10 nM 125I-gelsolin, but only 40-50% bound in 1 mM EGTA. Addition of more F-actin to the EGTA supernatant does not sediment this gelsolin. Demonstration of this partial calcium sensitivity depends critically on the use of F-actin that has been prepared in the absence of calcium ions. F-actin prepared from G-actin in calcium or pretreated with calcium, binds 125I-gelsolin more completely in EGTA. This suggests that gelsolin activity is influenced by transient exposure of actin to calcium. Further evidence for partial calcium sensitivity in the interactions between gelsolin and F-actin has been obtained by other methods, including viscometry and electron microscopy. The gelsolin present in the EGTA supernatant is complexed to G-actin, predominantly as binary complexes. Very low concentrations of these complexes reduce the viscosity of F-actin in calcium but not in EGTA. Whether this effect is due to severing activity, or capping with consequent depolymerization to establish the new critical concentration, is uncertain. The results suggest the presence of two types of gelsolin, one that requires micromolar concentrations of calcium for binding to F-actin and one that does not. Both bind to G-actin. Partial separation has been achieved using actin-Sepharose. Pig plasma gelsolin is heterogeneous on isoelectric focussing gels in urea, but the two types of gelsolin separated on actin-Sepharose do not correspond to specific isoelectric species.     

Unfractionated heparin reduces the elasticity of sputum from patients with cystic fibrosis

Mucus obstruction of the airway in patients with cystic fibrosis (CF) reduces lung function, invites infection, and limits delivery of inhaled drugs including gene therapy vectors to target cells. Not all patients respond to presently available mucolytics, and new approaches are needed. Our objectives were to investigate the in vitro effects of unfractionated heparin (UFH) on the morphology and rheology of sputum and the effect of UFH on diffusion of 200-nm nanospheres through sputum from adult CF patients. Confocal laser scanning microscopy was used to image fluorescently stained actin and DNA components of CF sputum, and atomic force microscopy was used to image isolated DNA networks. The viscoelasticity of CF sputum was measured using dynamic oscillatory rheometry. Nanosphere diffusion was measured through CF sputum using a Boyden chamber-based assay. Actin-DNA bundles in CF sputum were disaggregated by UFH at concentrations of 0.1-10 mg/ml, and UFH enhanced the endonuclease activity in sputum from patients on dornase alfa therapy. UFH significantly reduced the elasticity and yield stress, but not the viscosity, of CF sputum from patients not receiving dornase alfa therapy. Heparin dose-dependently significantly increased the diffusion of nanospheres through CF sputum from patients not on dornase alfa therapy from 10.5 +/- 2.5% at baseline to 36.9 +/- 4.4% at 10 mg/ml but was more potent, with maximal effect at 0.1 mg/ml, in patients who were on dornase alfa therapy. Thus the mucoactive properties of UFH indicate its potential as a new therapeutic approach in patients with cystic fibrosis.     

Cross-linked long-pitch actin dimer forms stoichiometric complexes with gelsolin segment 1 and/or deoxyribonuclease I that nonproductively interact with myosin subfragment 1

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.     

Enhanced viscoelasticity of human cystic fibrotic sputum correlates with increasing microheterogeneity in particle transport

Current biochemical characterizations of cystic fibrosis (CF) sputum do not address the high degree of microheterogeneity in the rheological properties of the mucosal matrix and only provide bulk-average particle diffusion coefficients. The viscoelasticity of CF sputum greatly reduces the diffusion rates of colloidal particles, limiting the effectiveness of gene delivery to underlying lung cells. We determine diffusion coefficients of hundreds of individual amine-modified and carboxylated polystyrene particles (diameter 100-500 nm) embedded in human CF sputum with 5 nm and 33 ms of spatiotemporal resolution. High resolution multiple particle tracking is used to calculate the effective viscoelastic properties of CF sputum at the micron scale, which we relate to its macroscopic viscoelasticity. CF sputum microviscosity, as probed by 100- and 200-nm particles, is an order of magnitude lower than its macroviscosity, suggesting that nanoparticles dispersed in CF sputum are transported primarily through lower viscosity pores within a highly elastic matrix. Multiple particle tracking provides a non-destructive, highly sensitive method to quantify the high heterogeneity of the mucus pore network. The mean diffusion coefficient becomes dominated by relatively few but fast-moving particles as particle size is reduced from 500 to 100 nm. Neutrally charged particles with a diameter <200 nm undergo more rapid transport in CF sputum than charged particles. Treatment with recombinant human DNase (Pulmozyme) reduces macroviscoelastic properties of CF sputum by up to 50% and dramatically narrows the distribution of individual particle diffusion rates but surprisingly does not significantly alter the ensemble-average particle diffusion rate.     

Counterion-induced actin ring formation

Actin filaments form rings and loops when > 20 mM divalent cations are added to very dilute solutions of phalloidin-stabilized filamentous actin (F-actin). Some rings consist of very long single actin filaments partially overlapping at their ends, and others are formed by small numbers of filaments associated laterally. In some cases, undulations of the rings are observed with amplitudes and dynamics similar to those of the thermal motions of single actin filaments. Lariat-shaped aggregates also co-exist with rings and rodlike bundles. These polyvalent cation-induced actin rings are analogous to the toroids of DNA formed by addition of polyvalent cations, but the much larger diameter of actin rings reflects the greater bending stiffness of F-actin. Actin rings can also be formed by addition of streptavidin to crosslink sparsely biotinylated F-actin at very low concentrations. The energy of bending in a ring, calculated from the persistence length of F-actin and the ring diameter, provides an estimate for the adhesion energy mediated by the multivalent counterions, or due to the streptavidin-biotin bonds, required to keep the ring closed.     

Viscoelasticity of F-actin and F-actin/gelsolin complexes

Actin is the major protein of eukaryote peripheral cytoplasm where its mechanical effects could determine cell shape and motility. The mechanical properties of purified F-actin, whether it is a viscoelastic fluid or an elastic solid, have been a subject of controversy. Mainstream polymer theory predicts that filaments as long as those found in purified F-actin are so interpenetrated as to appear immobile in measurements over a reasonable time with available instrumentation and that the fluidity of F-actin could only be manifest if the filaments were shortened. We show that the static and dynamic elastic moduli below a critical degree of shear strain are much higher than previously reported, consistent with extreme interpenetration, but that higher strain or treatment with very low concentrations of the F-actin severing protein gelsolin greatly diminish the moduli and cause F-actin to exhibit rheologic behavior expected for independent semidilute rods, and defined by the dimensions of the filaments, including shear rate independent viscosity below a critical shear rate. The findings show that shortening of actin filaments sufficiently to permit reasonable measurements brings out their viscoelastic fluid properties. Since gelsolin shortens F-actin, it is likely that the effect of high strain is also to fragment a population of long actin filaments. We confirmed recent findings that the viscosity of F-actin is inversely proportional to the shear rate, consistent with an indeterminate fluid, but found that gelsolin abolishes this unusual shear rate dependence, indicating that it results from filament disruption during the viscosity measurements.(ABSTRACT TRUNCATED AT 250 WORDS)     

Controlled self-assembly of actin filaments for dynamic biodevices

We investigated the immobilization of actin filaments and its self-assembly in vitro in a continuous-flow system on poly(styrene-maleic acid) (PSMA), poly(methyl methacrylate) (PMMA), poly(t-butyl methacrylate) [P(tBuMA)] polymeric surfaces and along the linear channels. Among these polymeric surfaces, PSMA appeared to be more suitable for supramolecular manipulations as it lacked inherent fluorescence, had good biocompatibility with actin-myosin, and provided sufficient amounts of binding sites for the covalent immobilization of actin. Covalent attachment of G-actin on PSMA polymeric surfaces resulted in stable polymerization followed by alignment of filaments over 1.5 h, along with a greater surface density of the proteins. It is shown that electrostatic condensation of intact F-actin filaments and F-actin/gelsolin filaments with Ba²⁺ can be successfully used for progressive bundle formation and alignment in the constant flow. Actin bundles retained their ability to support HMM-anti-HMM bead translocation. Long-range cooperative transitions in actin induced by gelsolin represent a structural perturbation of the barbed end and presumably result in regularly organized bundles that secure directional movement. This simple technique for fabrication of self-assembled and aligned F-actin/gelsolin bundles provides a convenient experimental system for nanotechnological applications.     

Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

Background

To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA).

Methodology

Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated.

Results

The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations - DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold.

Conclusions

Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections.     

Thioredoxin and dihydrolipoic acid inhibit elastase activity in cystic fibrosis sputum

Excessive neutrophil elastase activity within airways of cystic fibrosis (CF) patients results in progressive lung damage. Disruption of disulfide bonds on elastase by reducing agents may modify its enzymatic activity. Three naturally occurring dithiol reducing systems were examined for their effects on elastase activity: 1) Escherichia coli thioredoxin (Trx) system, 2) recombinant human thioredoxin (rhTrx) system, and 3) dihydrolipoic acid (DHLA). The Trx systems consisted of Trx, Trx reductase, and NADPH. As shown by spectrophotometric assay of elastase activity, the two Trx systems and DHLA inhibited purified human neutrophil elastase as well as the elastolytic activity present in the soluble phase (sol) of CF sputum. Removal of any of the three Trx system constituents prevented inhibition. Compared with the monothiols N-acetylcysteine and reduced glutathione, the dithiols displayed greater elastase inhibition. To streamline Trx as an investigational tool, a stable reduced form of rhTrx was synthesized and used as a single component. Reduced rhTrx inhibited purified elastase and CF sputum sol elastase without NADPH or Trx reductase. Because Trx and DHLA have mucolytic effects, we investigated changes in elastase activity after mucolytic treatment. Unprocessed CF sputum was directly treated with reduced rhTrx, the Trx system, DHLA, or DNase. The Trx system and DHLA did not increase elastase activity, whereas reduced rhTrx treatment increased sol elastase activity by 60%. By contrast, the elastase activity after DNase treatment increased by 190%. The ability of Trx and DHLA to limit elastase activity combined with their mucolytic effects makes these compounds potential therapies for CF.     

Effects of semi-dilute actin solutions on the mobility of fibrin protofibrils during clot formation

Low concentrations of actin filaments (F-actin) inhibit the rate and extent of turbidity developed during polymerization of purified fibrinogen by thrombin. Actin incorporates into the fibrin clot in a concentration-dependent manner that does not reach saturation, indicating nonspecific trapping of actin filaments in the fibrin network. Actin does not retard activation of fibrinogen by thrombin, but rather the alignment of fibrin protofibrils into bundles which constitute the coarse clot. In contrast, equivalent F-actin concentrations have little or no effect on the turbidity of plasma clots. The difference is attributed to the presence of a plasma protein, gelsolin, that severs actin filaments. Purified gelsolin greatly reduces the effect of F-actin on the turbidity of a pure fibrin clot and decreases the fraction of actin incorporated by the clot. A calculation of the extent to which the gelsolin concentrations used in these experiments reduce the fraction of actin filaments which are long enough to impede each other's rotational diffusion indicates that it is the overlapping actin filaments which retard the association of fibrin protofibrils. The findings suggest that one role for the F-actin depolymerizing and particularly actin severing activities in blood is to prevent actin filaments released by tissue injury from interfering with the formation of coarse fibrin clots.     

Synthetic chloride channel restores glutathione secretion in cystic fibrosis airway epithelia

Cystic fibrosis (CF), an inherited disease characterized by defective epithelial Cl- transport, damages lungs via chronic inflammation and oxidative stress. Glutathione, a major antioxidant in the epithelial lung lining fluid, is decreased in the apical fluid of CF airway epithelia due to reduced glutathione efflux (Gao L, Kim KJ, Yankaskas JR, and Forman HJ. Am J Physiol Lung Cell Mol Physiol 277: L113-L118, 1999). The present study examined the question of whether restoration of chloride transport would also restore glutathione secretion. We found that a Cl- channel-forming peptide (N-K4-M2GlyR) and a K+ channel activator (chlorzoxazone) increased Cl- secretion, measured as bumetanide-sensitive short-circuit current, and glutathione efflux, measured by high-performance liquid chromatography, in a human CF airway epithelial cell line (CFT1). Addition of the peptide alone increased glutathione secretion (181 +/- 8% of the control value), whereas chlorzoxazone alone did not significantly affect glutathione efflux; however, chlorzoxazone potentiated the effect of the peptide on glutathione release (359 +/- 16% of the control value). These studies demonstrate that glutathione efflux is associated with apical chloride secretion, not with the CF transmembrane conductance regulator per se, and the defect of glutathione efflux in CF can be overcome pharmacologically.     

The N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin complex

Apoptosis is essential in embryonic development, clonal selection of cells of the immune system and in the prevention of cancer. Apoptotic cells display characteristic changes in morphology that precede the eventual fragmentation of nuclear DNA resulting in cell death. Current evidence implicates DNase I as responsible for hydrolysis of DNA during apoptosis. In vivo, it is likely that cytoplasmic actin binds and inhibits the enzymatic activity and nuclear translocation of DNase I and that disruption of the actin-DNase I complex results in activation of DNase I. In this report we demonstrate that the N-terminal fragment of gelsolin (N-gelsolin) disrupts the actin-DNase I interaction. This provides a molecular mechanism for the role of the N-gelsolin in regulating DNase I activity. We also show that cofilin stabilises the actin-DNase I complex by forming a ternary complex that prevents N-gelsolin from releasing DNase I from actin. We suggest that both cofilin and gelsolin are essential in modulating the release of DNase I from actin.     

Common gene therapy viral vectors do not efficiently penetrate sputum from cystic fibrosis patients

Norwalk virus and human papilloma virus, two viruses that infect humans at mucosal surfaces, have been found capable of rapidly penetrating human mucus secretions. Viral vectors for gene therapy of Cystic Fibrosis (CF) must similarly penetrate purulent lung airway mucus (sputum) to deliver DNA to airway epithelial cells. However, surprisingly little is known about the rates at which gene delivery vehicles penetrate sputum, including viral vectors used in clinical trials for CF gene therapy. We find that sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus (d～80 nm) and adeno-associated virus (AAV serotype 5; d～20 nm), leading to average effective diffusivities that are ～3,000-fold and 12,000-fold slower than their theoretical speeds in water, respectively. Both viral vectors are slowed by adhesion, as engineered muco-inert nanoparticles with diameters as large as 200 nm penetrate the same sputum samples at rates only ～40-fold reduced compared to in pure water. A limited fraction of AAV exhibit sufficiently fast mobility to penetrate physiologically thick sputum layers, likely because of the lower viscous drag and smaller surface area for adhesion to sputum constituents. Nevertheless, poor penetration of CF sputum is likely a major contributor to the ineffectiveness of viral vector based gene therapy in the lungs of CF patients observed to date.     

Plasma gelsolin caps and severs actin filaments

Plasma gelsolin caps actin filaments at their 'barbed' ends and severs them along their length. Capping has been demonstrated both by direct visualization using gold-labeled gelsolin and by inhibition of actin polymerization onto the barbed ends of fragments of the acrosomal process of Limulus sperm. Severing activity is demonstrated by the fact that actin filaments nucleated off acrosomal fragments are shortened or removed within a few seconds by added plasma gelsolin without any obvious disruption of the actin bundles in the acrosomal processes themselves.     

Activation of cytosolic Slingshot-1 phosphatase by gelsolin-generated soluble actin filaments

Slingshot-1 (SSH1) is a protein phosphatase that dephosphorylates and activates cofilin, an actin-severing and -disassembling protein. SSH1 is bound to and activated by F-actin, but not G-actin. SSH1 is accumulated in the F-actin-rich lamellipodium but is also diffusely distributed in the cytoplasm. It remains unknown whether SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm. In this study, we show that SSH1 binds to gelsolin via actin filaments in the cytosolic fraction. Gelsolin promoted solubilization of actin filaments and SSH1 in cell-free assays and in cultured cells. SSH1 was activated by gelsolin-generated soluble actin filaments. Furthermore, gelsolin enhanced cofilin dephosphorylation in neuregulin-stimulated cells. Our results suggest that cytosolic SSH1 forms a complex with gelsolin via soluble actin filaments and is activated by gelsolin-generated soluble actin filaments and that gelsolin promotes stimulus-induced cofilin dephosphorylation through increasing soluble actin filaments, which support SSH1 activation in the cytoplasm.     

Actin deoxyroboncuclease I interaction. Depolymerization and nucleotide exchange

Deoxyribonuclease I (DNase I) forms a 1:1 complex with globular actin (G-actin) and also will depolymerize filamentous actin (F-actin) to form a 1:1 complex. The effect of DNase I on the exchange of the actin nucleotide has been investigated. When DNase I is added to G-actin, the rate of nucleotide exchange is decreased from 1.16 +/- 0.25 X 10(-4) s-1 to 0.28 +/- 0.09 X 10(-4) s-1 (0 degrees C). The presence of ATP or ADP in the actin has little effect on the rate of exchange of the nucleotide for ATP. This suggests that the weaker affinity of ADP than ATP for actin is due to a slower association rate of ADP. The rate of the nucleotide exchange in the actinDNase I complex is increased by the addition of NaCl or MgCl2. When DNase I is added to F-actin, the rate of nucleotide exchange (6.2 +/- 1.6 X 10(-4) x-1, 0 degrees C) is similar to the rate of depolymerization as measured by loss of viscosity. The actinDNase I complex formed by depolymerization of F-actin exchanges nucleotide at a 4-fold faster rate than the G-actinDNase I complex in the same ionic conditions. This and other experiments suggest that DNase I binds first to F-actin before dissociating the monomer from the filament. These results are discussed in terms of possible mechanisms of action depolymerization.     

Motion and enzymatic degradation of DNA in the atomic force microscope.

The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length.