Sodium fluoride as a nucleating factor for Mg-actin polymerization.

Dynamic instability of actin filaments can be inhibited by Pi analogs beryllium fluoride and aluminium fluoride that mimic the intermediate ADP-Pi state and stabilize actin filaments. On the other hand, the phosphoryl transfer enzymes can be activated in the absence of aluminium by magnesium fluoride if magnesium ions and sodium fluoride (NaF) were present in the solution. Whether magnesium fluoride promotes functional activities of actin is not known. Here we show, for the first time, that sodium fluoride strongly accelerates polymerization of highly dynamic Mg-F-actin assembled from the monomers proteolytically cleaved between Gly42 and Val43 within the D-loop with actin-specific protease protealysin (Pln-actin), apparently due to stabilization of nuclei formed at the initial step of actin polymerization. Thereby, NaF did not inhibit the ATPase activity (subunit exchange) on Pln-F-actin, did not increase the amount of Pln-F-actin sedimented by ultracentrifugation, and did not stabilize the inter-strand contacts of Pln-F-actin. On the other hand, NaF diminished accessibility of the nucleotide binding cleft of Mg-G-actin to trypsin, pointing to an additional cleft closure, and additionally protected the D-loop from the protealysin cleavage in Mg-F-actin, thus indicating that the longitudinal contacts are stabilized. We also demonstrate that in cultured cells NaF can directly promote assembly of F-actin structures under conditions when the corresponding activity of the RhoA pathway is inhibited. These data suggest that the NaF-induced assembly of actin filaments is promoted by magnesium fluoride that can be formed by the NaF-originating fluoride and the actin tightly bound magnesium.

Tropomyosin as a Regulator of Actin Dynamics.

Tropomyosin is a major regulatory protein of contractile systems and cytoskeleton, an actin-binding protein that positions laterally along actin filaments and modulates actin-myosin interaction. About 40 tropomyosin isoforms have been found in a variety of cytoskeleton systems, not necessarily connected with actin-myosin interaction and contraction. Involvement of specific tropomyosin isoforms in the regulation of key cell processes was shown, and specific features of tropomyosin genes and protein structure have been investigated with molecular biology and genetics approaches. However, the mechanisms underlying the effects of tropomyosin on cytoskeleton dynamics are still unclear. As tropomyosin is primarily an F-actin-binding protein, it is important to understand how it interacts both with actin and actin-binding proteins functioning in muscles and cytoskeleton to regulate actin dynamics. This review focuses on biochemical data on the effects of tropomyosin on actin assembly and dynamics, as well as on the modulation of these effects by actin-binding proteins. The data indicate that tropomyosin can efficiently regulate actin dynamics via allosteric conformational changes within actin filaments.

Draft Genome Sequence of Serratia grimesii Strain A2.

We report the first draft genome assembly of Serratia grimesii strain A2, previously identified as Escherichia coli strain A2, which produces protease ECP32 with a high specificity toward actin. S. grimesii strain A2 has multidrug resistance associated with a number of efflux pump genes.

Bacterial invasion of eukaryotic cells can be mediated by actin-hydrolysing metalloproteases grimelysin and protealysin.

Earlier, we have shown that spontaneously isolated non-pathogenic bacteria Serratia grimesii and Serratia proteamaculans invade eukaryotic cells, provided that they synthesize thermolysin-like metalloproteases ECP32/grimelysin or protealysin characterized by high specificity towards actin. To address the question of whether the proteases are active players in entry of these bacteria into host cells, in this work, human larynx carcinoma Hep-2 cells were infected with recombinant Escherichia coli expressing grimelysin or protealysin. Using confocal and electron microscopy, we have found that the recombinant bacteria, whose extracts limitedly cleaved actin, were internalized within the eukaryotic cells residing both in vacuoles and free in cytoplasm. The E. coli-carrying plasmids without inserts of grimelysin or protealysin gene did not enter Hep-2 cells. Moreover, internalization of non-invasive E. coli was not observed in the presence of protealysin introduced into the culture medium. These results are consistent with the direct participation of ECP32/grimelysin and protealysin in entry of bacteria into the host cells. We assume that ECP32/grimelysin and protealysin mediate invasion being injected into the eukaryotic cell and that the high specificity of the enzyme towards actin may be a factor contributed to the bacteria internalization.

Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actin.

Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNaseI-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases, subtilisin or ECP32/grimelysin (ECP), respectively. The results obtained show that both cleavages strongly decreased the thermal stability of monomeric actin with either ATP or ADP as a bound nucleotide. An even more pronounced difference in the thermal stability between the cleaved and intact actin was observed when both actins were polymerized into filaments. Similar to intact F-actin, both cleaved F-actins were significantly stabilized by phalloidin and aluminum fluoride; however, in all cases, the thermal stability of the cleaved F-actins was much lower than that of intact F-actin, and the stability of ECP-cleaved F-actin was lower than that of subtilisin-cleaved F-actin. These results confirm that the DNaseI-binding loop is involved in the stabilization of the actin structure, both in monomers and in the filament subunits, and suggest that the thermal stability of actin depends, at least partially, on the conformation of the nucleotide-binding cleft. Moreover, an additional destabilization of the unstable cleaved actin upon ATP/ADP replacement provides experimental evidence for the highly dynamic actin structure that cannot be simply open or closed, but rather should be considered as being able to adopt multiple conformations.

Role of actin DNase-I-binding loop in myosin subfragment 1-induced polymerization of G-actin: implications for the mechanism of polymerization.

Proteolytic cleavage of actin between Gly(42) and Val(43) within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess. The most significant effect of the cleavage, revealed by measuring the fluorescence of pyrene-actin and light-scattering intensities as a function of actin concentration at saturating concentrations of S1, is strong inhibition of association of G-actin-S1 complexes into oligomers. Measurements of the fluorescence of dansyl cadaverine attached to Gln(41) indicate substantial inhibition of the initial association of G-actin-S1 into longitudinal dimers. The data provide experimental evidence for the critical role of D-loop conformation in both longitudinal and lateral, cross-strand actin-actin contact formation in the nucleation reaction. Electron microscopic analysis of the changes in filament-length distribution during polymerization of actin by S1(A1) and S1(A2) suggests that the mechanism of S1-induced polymerization is not substantially different from the nucleation-elongation scheme of spontaneous actin polymerization.

The DNase-I binding loop of actin may play a role in the regulation of actin-myosin interaction by tropomyosin/troponin.

Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation. Possible participation of loop 38-52 in this communication has recently been questioned based on unimpaired Ca(2+) regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met(47) and Gly(48). We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly(42) and Val(43), on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin. Both individual modifications reduced activation of S1 ATPase by actin to a similar extent. The effect of ECP cleavage, but not of subtilisin cleavage, was partially reversed by stabilization of interprotomer contacts with phalloidin, indicating different pathways of signal transmission from the N- and C-terminal parts of loop 38-52 to myosin binding sites. ECP cleavage diminished the affinity to tropomyosin and reduced its inhibition of acto-S1 ATPase at low S1 concentrations, but increased the tropomyosin-mediated cooperative enhancement of the ATPase by S1 binding to actin. These effects were reversed by phalloidin. Subtilisin-cleaved actin more closely resembled unmodified actin than the ECP-modified actin. Limited proteolysis of the modified and unmodified F-actins revealed an allosteric effect of ECP cleavage on the conformation of the actin subdomain 4 region that is presumably involved in tropomyosin binding. Our results point to a possible role of the N-terminal part of loop 38-52 of actin in communication between tropomyosin and myosin through changes in actin structure.

Non-hydrolyzable analog of GTP induces activity of Na+ channels via disassembly of cortical actin cytoskeleton.

The role of G proteins in regulation of non-voltage-gated Na+ channels in human myeloid leukemia K562 cells was studied by inside-out patch-clamp method. Na+ channels were activated by non-hydrolyzable analog of guanosine triphosphate (GTP), GTPgammaS, known to activate both heterotrimeric and small G proteins. Channel activity was not affected by aluminum fluoride that indiscriminately activates heterotrimeric G proteins. The effect of GTPgammaS was prevented by phalloidin and by G-actin, both interfering with actin disassembly, which indicates that GTPgammaS-induced channel activation was likely due to microfilament disruption. GTPgammaS-activated channels were inactivated by polymerizing actin. These data show, for the first time, that small G proteins can regulate Na+ channels, and an intracellular mechanism mediating their effect involves actin cytoskeleton rearrangements.

Regulation of sodium channel activity by capping of actin filaments.

Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells.

Role of the DNase-I-binding loop in dynamic properties of actin filament.

Effects of proteolytic modifications of the DNase-I-binding loop (residues 39-51) in subdomain 2 of actin on F-actin dynamics were investigated by measuring the rates of the polymer subunit exchange with the monomer pool at steady state and of ATP hydrolysis associated with it, and by determination of relative rate constants for monomer addition to and dissociation from the polymer ends. Cleavage of actin between Gly-42 and Val-43 by protease ECP32 resulted in enhancement of the turnover rate of polymer subunits by an order of magnitude or more, in contrast to less than a threefold increase produced by subtilisin cleavage between Met-47 and Gly-48. Probing the structure of the modified actins by limited digestion with trypsin revealed a correlation between the increased F-actin dynamics and a change in the conformation of subdomain 2, indicating a more open state of the filament subunits relative to intact F-actin. The cleavage with trypsin and steady-state ATPase were cooperatively inhibited by phalloidin, with half-maximal effects at phalloidin to actin molar ratio of 1:8 and full inhibition at a 1:1 ratio. The results support F-actin models in which only the N-terminal segment of loop 39-51 is involved in monomer-monomer contacts, and suggest a possibility of regulation of actin dynamics in the cell through allosteric effects on this segment of the actin polypeptide chain.