Characterisation of the Rac/PAK pathway in Amoeba proteus.

Molecular mechanisms underlying the unique locomotion of the highly motile Amoeba proteus still remain poorly understood. Recently, we have shown that blocking the endogenous amoebal Rac-like protein(s) leads to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. To elucidate the mechanism of the Rac pathway in Amoeba proteus, we tested the effects of blocking the endogenous myosin I heavy chain kinase (MIHCK), one of the Rac effectors in Acanthamoeba castellanii and Dictyostelium discoideum, with anti-MIHCK antibodies in migrating amoebae, as well as the effect of inhibiting Rac and MIHCK on the actin polymerisation process. Antibodies against A. castellanii MIHCK detected an A. proteus protein with a molecular mass (ca. 95 kDa) similar to the A. castellanii kinase. The cellular distribution of MIHCK in A. proteus was very similar to those of Rac-like protein in amoebae and MIHCK in A. castellanii. Amoebae microinjected with anti-MIHCK antibodies moved slower and protruded fewer wide pseudopodia (5-6) than the control cells (9-10), resembling to some extent the phenotype of cells microinjected with anti-Rac antibodies. The in vitro studies indicate that the A. proteus Rac-like protein, but not the MIHCK isoform, is engaged in the regulation of the nucleation step of the actin polymerisation process. These observations suggest that MIHCK may be one of the effectors for Rac in these extremely large cells.

Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function.

Tropomyosin (Tm) binds along actin filaments, one molecule spanning four to seven actin monomers, depending on the isoform. Periodic repeats in the sequence have been proposed to correspond to actin binding sites. To learn the functional importance of length and the internal periods we made a series of progressively shorter Tms, deleting from two up to six of the internal periods from rat striated alpha-TM (dAc2--3, dAc2--4, dAc3--5, dAc2--5, dAc2--6, dAc1.5--6.5). Recombinant Tms (unacetylated) were expressed in Escherichia coli. Tropomyosins that are four or more periods long (dAc2--3, dAc2--4, and dAc3--5) bound well to F-actin with troponin (Tn). dAc2--5 bound weakly (with EGTA) and binding of shorter mutants was undetectable in any condition. Myosin S1-induced binding of Tm to actin in the tight Tm-binding "open" state did not correlate with actin binding. dAc3--5 and dAc2--5 did not bind to actin even when the filament was saturated with S1. In contrast, dAc2--3 and dAc2--4 did, like wild-type-Tm, requiring about 3 mol of S1/mol of Tm for half-maximal binding. The results show the critical importance of period 5 (residues 166--207) for myosin S1-induced binding. The Tms that bound to actin (dAc2--3, dAc2--4, and dAc3--5) all fully inhibited the actomyosin ATPase (+Tn) in EGTA. In the presence of Ca(2+), relief of inhibition by these Tms was incomplete. We conclude (1) four or more actin periods are required for Tm to bind to actin with reasonable affinity and (2) that the structural requirements of Tm for the transition of the regulated filament from the blocked-to-closed/open (relief of inhibition by Ca(2+)) and the closed-to-open states (strong Tm binding to actin-S1) are different.

Alteration of tropomyosin function and folding by a nemaline myopathy-causing mutation.

Mutations in the human TPM3 gene encoding gamma-tropomyosin (alpha-tropomyosin-slow) expressed in slow skeletal muscle fibers cause nemaline myopathy. Nemaline myopathy is a rare, clinically heterogeneous congenital skeletal muscle disease with associated muscle weakness, characterized by the presence of nemaline rods in muscle fibers. In one missense mutation the codon corresponding to Met-8, a highly conserved residue, is changed to Arg. Here, a rat fast alpha-tropomyosin cDNA with the Met8Arg mutation was expressed in Escherichia coli to investigate the effect of the mutation on in vitro function. The Met8Arg mutation reduces tropomyosin affinity for regulated actin 30- to 100-fold. Ca(2+)-sensitive regulatory function is retained, although activation of the actomyosin S1 ATPase in the presence of Ca(2+) is reduced. The poor activation may reflect weakened actin affinity or reduced effectiveness in switching the thin filament to the open, force-producing state. The presence of the Met8Arg mutation severely, but locally, destabilizes the tropomyosin coiled coil in a model peptide, and would be expected to impair end-to-end association between TMs on the thin filament. In muscle, the mutation may alter thin filament assembly consequent to lower actin affinity and altered binding of the N-terminus to tropomodulin at the pointed end of the filament. The mutation may also reduce force generation during activation.

Independent functions for the N- and C-termini in the overlap region of tropomyosin.

Tropomyosin (TM) is a coiled-coil that binds head-to-tail along the helical actin filament. The ends of 284-residue tropomyosins are believed to overlap by about nine amino acids. The present study investigates the function of the N- and C-terminal overlap regions. Recombinant tropomyosins were produced in Escherichia coli in which nine amino acids were truncated from the N-terminal, C-terminal, or both ends of striated muscle alpha-tropomyosin (TM9a) and TM2 (TM9d), a nonmuscle alpha-tropomyosin expressed in many cells. The two isoforms are identical except for the C-terminal 27 amino acids encoded by exon 9a (striated) or exon 9d (TM2). Removal of either end greatly reduces the actin affinity of both tropomyosins in all conditions and the cooperativity with which myosin promotes tropomyosin binding to actin in the open state. N-Terminal truncations generally are more deleterious than C-terminal truncations. With TM9d, truncation of the N-terminus is as deleterious as both for myosin S1-induced binding. None of the TM9d variants binds well to actin with troponin (+/-Ca(2+)). TM9a with the truncated N-terminus binds more weakly to actin with troponin (-Ca(2+)) than when the C-terminus is removed but more strongly than when both ends are removed; the actin binding of all three forms is cooperative. The results show that the ends of TM9a, though important, are not required for cooperative function and suggest they have independent functions beyond formation of an overlap complex. The nonadditivity of the TM9d truncations suggests that the ends may primarily function as a complex in this isoform. A surprising result is that all variants bound with the same affinity, and noncooperatively, to actin saturated with myosin S1. Evidently, end-to-end interactions are not required for high-affinity binding to acto-myosin S1.

Proteolytic cleavage of actin within the DNase-I-binding loop changes the conformation of F-actin and its sensitivity to myosin binding.

Effects of subtilisin cleavage of actin between residues 47 and 48 on the conformation of F-actin and on its changes occurring upon binding of myosin subfragment-1 (S1) were investigated by measuring polarized fluorescence from rhodamine-phalloidin- or 1, 5-IAEDANS-labeled actin filaments reconstructed from intact or subtilisin-cleaved actin in myosin-free muscle fibers (ghost fibers). In separate experiments, polarized fluorescence from 1, 5-IAEDANS-labeled S1 bound to non-labeled actin filaments in ghost fibers was measured. The measurements revealed differences between the filaments of cleaved and intact actin in the orientation of rhodamine probe on the rhodamine-phalloidin-labeled filaments, orientation and mobility of the C-terminus of actin, filament flexibility, and orientation and mobility of the myosin heads bound to F-actin. The changes in the filament flexibility and orientation of the actin-bound fluorophores produced by S1 binding to actin in the absence of ATP were substantially diminished by subtilisin cleavage of actin. The results suggest that loop 38-52 plays an important role, not only in maintaining the F-actin structure, but also in the conformational transitions in actin accompanying the strong binding of the myosin heads that may be essential for the generation of force and movement during actin-myosin interaction.

Divalent cation-, nucleotide-, and polymerization-dependent changes in the conformation of subdomain 2 of actin.

Conformational changes in subdomain 2 of actin were investigated using fluorescence probes dansyl cadaverine (DC) or dansyl ethylenediamine (DED) covalently attached to Gln41. Examination of changes in the fluorescence emission spectra as a function of time during Ca2+/Mg2+ and ATP/ADP exchange at the high-affinity site for divalent cation-nucleotide complex in G-actin confirmed a profound influence of the type of nucleotide but failed to detect a significant cation-dependent difference in the environment of Gln41. No significant difference between Ca- and Mg-actin was also seen in the magnitude of the fluorescence changes resulting from the polymerization of these two actin forms. Evidence is presented that earlier reported cation-dependent differences in the conformation of the loop 38-52 may be related to time-dependent changes in the conformation of subdomain 2 in DED- or DC-labeled G-actin, accelerated by substitution of Mg2+ for Ca2+ in CaATP-G-actin and, in particular, by conversion of MgATP- into MgADP-G-actin. These spontaneous changes are associated with a denaturation-driven release of the bound nucleotide that is promoted by two effects of DED or DC labeling: lowered affinity of actin for nucleotide and acceleration of ATP hydrolysis on MgATP-G-actin that converts it into a less stable MgADP form. Evidence is presented that the changes in the environment of Gln41 accompanying actin polymerization result in part from the release of Pi after the hydrolysis of ATP on the polymer. A similarity of this change to that accompanying replacement of the bound ATP with ADP in G-actin is discussed.

The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state.

Tropomyosin (TM) is thought to exist in equilibrium between two states on F-actin, closed and open [Geeves, M. A., and Lehrer, S. S. (1994) Biophys. J. 67, 273-282]. Myosin shifts the equilibrium to the open state in which myosin binds strongly and develops force. Tropomyosin isoforms, that primarily differ in their N- and C-terminal sequences, have different equilibria between the closed and open states. The aim of the research is to understand how the alternate ends of TM affect cooperative actin binding and the relationship between actin affinity and the cooperativity with which myosin S1 promotes binding of TM to actin in the open state. A series of rat alpha-tropomyosin variants was expressed in Escherichia coli that are identical except for the ends, which are encoded by exons 1a or 1b and exons 9a, 9c or 9d. Both the N- and C-terminal sequences, and the particular combination within a TM molecule, determine actin affinity. Compared to tropomyosins with an exon 1a-encoded N-terminus, found in long isoforms, the exon 1b-encoded sequence, expressed in 247-residue nonmuscle tropomyosins, increases actin affinity in tropomyosins expressing 9a or 9d but has little effect with 9c, a brain-specific exon. The relative actin affinities, in decreasing order, are 1b9d > 1b9a > acetylated 1a9a > 1a9d >> 1a9a > or = 1a9c congruent with 1b9c. Myosin S1 greatly increases the affinity of all tropomyosin variants for actin. In this, the actin affinity is the primary factor in the cooperativity with which myosin S1 induces TM binding to actin in the open state; generally, the higher the actin affinity, the lower the occupancy by myosin required to saturate the actin with tropomyosin: 1b9d >1a9d> 1b9a > or = acetylated 1a9a > 1a9a > 1a9c congruent with 1b9c.

Structural changes in subdomain 2 of G-actin observed by fluorescence spectroscopy.

The influence of DNase I binding to Ca-ATP-G-actin and of Ca2+/Mg2+ and ATP/ADP exchange on the conformation of G-actin were investigated by measuring the fluorescence of dansyl cadaverine (DC) conjugated to Gln41 in subdomain 2 of the protein. Fluorescence resonance energy transfer (FRET) between this probe and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide (DDPM) attached to Cys374 in subdomain 1 was also measured. Contrary to an earlier report [dos Remedios, Kiessling and Hambly (1994) in Synchrotron Radiation in the Biosciences (Chance, B., Deisenhofer, J., Ebashi, S., Goodhead, D. T., Helliwell, J. R., Huxley, H. E., Iizuka, T., Kirz, J., Mitsui, T., Rubenstein, E. et al., eds.), pp. 418-425, Oxford University Press, Oxford], the distance between these probes did not change significantly when DNase I was bound to actin. A small but reproducible increase in the quantum yield and a blue shift of the DC fluorescence maximum were observed when bound Ca2+ was replaced by Mg2+. A large increase (about 70%) in the quantum yield and an approx. 12 nm blue shift of the emission spectrum occurred when ATP in Mg-G-actin was replaced by ADP. These changes were not accompanied by any significant change in the FRET distance between the dansyl donor and DDPM acceptor probes. A substantial change in the fluorescence of DC-actin was observed after proteolytic removal of the last three residues of actin, in accordance with earlier evidence suggesting that there is a conformational coupling between subdomain 2 and the C-terminal segment in subdomain 1 of actin. The results are discussed in relation to recently published data obtained with another fluorescent probe and to earlier observations based on limited cleavage using proteolytic enzymes.

Long-range conformational effects of proteolytic removal of the last three residues of actin.

Truncated derivatives of actin devoid of either the last two (actin-2C) or three residues (actin-3C) were used to study the role of the C-terminal segment in the polymerization of actin. The monomer critical concentration and polymerization rate increased in the order: intact actin < actin-2C < actin-3C. Conversely, the rate of hydrolysis of actin-bound ATP during spontaneous polymerization of Mg-actin decreased in the same order, so that, for actin-3C, the ATP hydrolysis significantly lagged behind the polymer growth. Probing the conformation of the nucleotide site in the monomer form by measuring the rates of the bound nucleotide exchange revealed a similar change upon removal of either the two or three residues from the C-terminus. The C-terminal truncation also resulted in a slight decrease in the rate of subtilisin cleavage of monomeric actin within the DNAse-I binding loop, whereas in F-actin subunits the susceptibility of this and of another site within this loop, specifically cleaved by a proteinase from Escherichia coli A2 strain, gradually increased upon sequential removal of the two and of the third residue from the C-terminus. From these and other observations made in this work it has been concluded that perturbation of the C-terminal structure in monomeric actin is transmitted to the cleft, where nucleotide and bivalent cation are bound, and to the DNAse-I binding loop on the top of subdomain 2. Further changes at these sites, observed on the polymer level, seem to result from elimination of the intersubunit contact between the C-terminal residues and the DNAse-I binding loop. It is suggested that formation of this contact plays an essential role in regulating the hydrolysis of actin-bound ATP associated with the polymerization process.

The actin/actin interactions involving the N-terminus of the DNase-I-binding loop are crucial for stabilization of the actin filament.

Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, I.M., Pershina, V.P., Synakevich, I.G., Turoverov, K.K. & Usmanova, A.M. (1991) FEBS Lett. 279, 49-51]. The resulting C-terminal and N-terminal fragments remained associated to one another in the presence of either Ca2+ or Mg2+. The protease-treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Ca2+ was replaced with Mg2+. Substitution of Mg2+ for the bound Ca2+ was also necessary to partially restore the ability of the protease-treated actin to inhibit the DNase I activity. The critical concentration for KCl-induced polymerization of ECP-treated ATP-Mg-G-actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisin, and 36-fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphologically, the filaments of ECP-treated actin were indistinguishable from those of intact actin. Comparison of the fluorescence spectra of pyrenyl-labelled actins and chemical cross-linking with N,N'-1,2-phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP-treated actin and those of intact as well as subtilisin-treated actin. Moreover, the filaments of ECP-treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin. The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F-actin [Holmes, K.C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44-49] and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament. The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.

Proteolytic removal of three C-terminal residues of actin alters the monomer-monomer interactions.

Homogeneous preparations of actin devoid of the three C-terminal residues were obtained by digestion of G-actin with trypsin after blocking proteolysis at other sites by substitution of Mg2+ for the tightly bound Ca2+. Removal of the C-terminal residues resulted in the following: an enhancement of the Mg(2+)-induced hydrolysis of ATP in low-ionic-strength solutions of actin; an increase in the critical concentration for polymerization; a decrease in the initial rate of polymerization; and an enhancement of the steady-state exchange of subunits in the polymer. Electron microscopy indicated an increased fragility of the filaments assembled from truncated actin. The results suggest that removal of the C-terminal residues increases the rate constants for monomer dissociation from the polymer ends and from the oligomeric species.

Localization of the tightly bound divalent-cation-dependent and nucleotide-dependent conformation changes in G-actin using limited proteolytic digestion.

Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca2+ by Mg2+ in ATP-actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C-terminal segment. Conversion of ATP-actin to ADP-actin (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39-51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39-51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.

Biochemical and theoretical approach to localization of metal-ion-binding sites in the actin primary structure.

The number of Ca2+ ions bound at sites other than the single high-affinity site in CaCl2-induced polymers of rabbit skeletal muscle, chicken gizzard, and bovine aorta actin was determined. The polymer of skeletal muscle and aorta actin contained 4 mol Ca2+/mol, whereas gizzard actin only 3 mol weakly bound Ca2+/mol monomer. This difference correlates with the deletion in smooth muscle gamma-actin of one out of four NH2-terminal acidic residues typical of skeletal and smooth muscle alpha-actin isoforms, suggesting that this additional acidic residue in alpha-actins is involved in the weak binding of cations which is essential for polymerization. This experimental result, as well as a theoretical analysis of the actin primary structure, argue against the implication of the NH2-terminal acidic residues in the high-affinity site for divalent cation. The analysis of the actin primary structure aimed at identification of sequences resembling the known Ca2+-binding patterns has revealed the absence of an EF-hand Ca2+-binding site. The best match was obtained between the sequence of the 292-301 segment and that of Ca2+ site in lectins. However, in the light of experimental data discussed, it is more plausible that the actual high-affinity Ca2+ site in actin involves sequentially distant residues from the NH2- and COOH-terminal portions of the polypeptide chain.

Distribution of postsynthetic methylation sites in Physarum histone H1.

Using limited chymotrypsin and trypsin digestion of isolated Physarum histone H1 labeled in vivo in postsynthetically added N epsilon-methyl groups of lysine we show that: --there is no postsynthetic methylation in the central globular domain of H1, --a moderate number of methylated sites occurs in the N-terminal fragment and the part of the C-terminal fragment directly adjacent to the globular domain (the main site of interphase phosphorylation), --the most intensively methylated region occurs within the sequence located in an extended part of the C-terminal fragment, distant to the globular domain and the main site of interphase phosphorylation.