Capping complex formation at the slow-growing end of the actin filament

Actin filaments are polar; their barbed (fast-growing) and pointed (slow-growing) ends differ in structure and dynamic properties. The slow-growing end is regulated by tropomodulins, a family of capping proteins that require tropomyosins for optimal function. There are four tropomodulin isoforms; their distributions vary depending on tissue type and change during development. The C-terminal half of tropomodulin contains one compact domain represented by alternating α-helices and β-structures. The tropomyosin-independent actin-capping site is located at the C-terminus. The N-terminal half has no regular structure; however, it contains a tropomyosin-dependent actin-capping site and two tropomyosin-binding sites. One tropomodulin molecule can bind two tropomyosin molecules. Effectiveness of tropomodulin binding to tropomyosin depends on the tropomyosin isoform. Regulation of tropomodulin binding at the pointed end as well as capping effectiveness in the presence of specific tropomyosins may affect formation of local cytoskeleton and dynamics of actin filaments in cells.     

Tropomodulin: a cytoskeletal protein that binds to the end of erythrocyte tropomyosin and inhibits tropomyosin binding to actin

Human erythrocytes contain a Mr 43,000 tropomyosin-binding protein that is unrelated to actin and that has been proposed to play a role in modulating the association of tropomyosin with spectrin-actin complexes based on its stoichiometry in the membrane skeleton of one Mr 43,000 monomer per short actin filament (Fowler, V. M. 1987. J. Biol. Chem. 262:12792-12800). Here, we describe an improved procedure to purify milligram quantities to 98% homogeneity and we show that this protein inhibits tropomyosin binding to actin by a novel mechanism. We have named this protein tropomodulin. Unlike other proteins that inhibit tropomyosin-actin interactions, tropomodulin itself does not bind to F-actin. EM of rotary-shadowed tropomodulin-tropomyosin complexes reveal that tropomodulin (14.5 +/- 2.4 nm [SD] in diameter) binds to one of the ends of the rod-like tropomyosin molecules (33 nm long). In agreement with this observation, Dixon plots of inhibition curves demonstrate that tropomodulin is a non-competitive inhibitor of tropomyosin binding to F-actin (Ki = 0.7 microM). Hill plots of the binding of the tropomodulin-tropomyosin complex to actin indicate that binding does not exhibit any positive cooperativity (n = 0.9), in contrast to tropomyosin (n = 1.9), and that the apparent affinity of the complex for actin is reduced 20-fold with respect to that of tropomyosin. These results suggest that binding of tropomodulin to tropomyosin may block the ability of tropomyosin to self-associate in a head-to-tail fashion along the actin filament, thereby weakening its binding to actin. Antibodies to tropomodulin cross-react strongly with striated muscle troponin I (but not with troponin T) as well as with a nontroponin Mr 43,000 polypeptide in muscle and in other nonerythroid cells and tissues, including brain, lens, neutrophils, and endothelial cells. Thus, erythrocyte tropomodulin may be one member of a family of tropomyosin-binding proteins that function to regulate tropomyosin-actin interactions in non-muscle cells and tissues.     

Tropomodulin binds two tropomyosins: a novel model for actin filament capping

Tropomodulin, a tropomyosin-binding protein, caps the slow-growing (pointed) end of the actin filament regulating its dynamics. Tropomodulin, therefore, is important for determining cell morphology, cell movement, and muscle contraction. For the first time we show that one tropomodulin molecule simultaneously binds two tropomyosin molecules in a cooperative manner. On the basis of the tropomodulin solution structure and predicted secondary structure, we introduced a series of point mutations in regions important for tropomyosin binding and actin capping. Capping activity of these mutants was assayed by measuring actin polymerization using pyrene fluorescence. Using direct methods (circular dichroism and native gel electrophoresis) for detecting tropomodulin/tropomyosin binding, we localized the second tropomyosin-binding site to residues 109-144. Despite previous reports that the second binding site is for erythrocyte tropomyosin only, we found that both short nonmuscle and long muscle alpha-tropomyosins bind there as well, though with different affinities. We propose a model for actin capping where one tropomodulin molecule can bind to two tropomyosin molecules at the pointed end.     

Tropomyosin ends determine the stability and functionality of overlap and troponin T complexes

Tropomyosin binds end to end along the actin filament. Tropomyosin ends, and the complex they form, are required for actin binding, cooperative regulation of actin filaments by myosin, and binding to the regulatory protein, troponin T. The aim of the work was to understand the isoform and structural specificity of the end-to-end association of tropomyosin. The ability of N-terminal and C-terminal model peptides with sequences of alternate alpha-tropomyosin isoforms, and a troponin T fragment that binds to the tropomyosin overlap, to form complexes was analyzed using circular dichroism spectroscopy. Analysis of N-terminal extensions (N-acetylation, Gly, AlaSer) showed that to form an overlap complex between the N-terminus and the C-terminus requires that the N-terminus be able to form a coiled coil. Formation of a ternary complex with the troponin T fragment, however, effectively takes place only when the overlap complex sequences are those found in striated muscle tropomyosins. Striated muscle tropomyosins with N-terminal modifications formed ternary complexes with troponin T that varied in affinity in the order: N-acetylated > Gly > AlaSer > unacetylated. The circular dichroism results were corroborated by native gel electrophoresis, and the ability of the troponin T fragment to promote binding of full-length tropomyosins to filamentous actin.     

Modulation of myosin function by isoform-specific properties of Saccharomyces cerevisiae and muscle tropomyosins.

Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments. The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2. Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding. These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin. Both of these effects are influenced by which tropomyosin isoform is present. Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function.     

Structural insights into the tropomodulin assembly at the pointed ends of actin filaments

Tropomodulins are a family of important regulators of actin dynamics at the pointed ends of actin filaments. Four isoforms of tropomodulin, Tmod1‐Tmod4, are expressed in vertebrates. Binding of tropomodulin to the pointed end is dependent on tropomyosin, an actin binding protein that itself is represented in mammals by up to 40 isoforms. The understanding of the regulatory role of the tropomodulin/tropomyosin molecular diversity has been limited due to the lack of a three‐dimensional structure of the tropomodulin/tropomyosin complex. In this study, we mapped tropomyosin residues interacting with two tropomyosin‐binding sites of tropomodulin and generated a three‐dimensional model of the tropomodulin/tropomyosin complex for each of these sites. The models were refined by molecular dynamics simulations and validated via building a self‐consistent three‐dimensional model of tropomodulin assembly at the pointed end. The model of the pointed‐end Tmod assembly offers new insights in how Tmod binding ensures tight control over the pointed end dynamics.     

Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin

Cultured rat cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J.J.-C. (1983) J. Biol. Chem. 258, 6636-6644). To explore the roles of the multiple tropomyosin isoforms in the microfilament organization of cultured cells, we have examined effects of tropomyosins on the bundling activity of the 55-kDa protein recently purified from HeLa cells (Yamashiro-Matsumura, S., and Matsumura, F. (1985) J. Biol. Chem. 260, 5087-5097). Maximum bundling of F-actin was observed at a molar ratio of 55-kDa protein to actin higher than 1:8. None of the isoforms of cultured rat cell tropomyosin significantly altered the F-actin-bundling activity of 55-kDa protein at this ratio, whereas skeletal muscle tropomyosin inhibited the bundling activity to about 50%. Also, cultured cell tropomyosins did not inhibit binding of 55-kDa protein to actin, whereas skeletal muscle tropomyosin inhibited it by 50%. The effect of 55-kDa protein on the binding of tropomyosin to actin varied with the isoform type of tropomyosin. Most (80%) of the tropomyosins with low Mr values (Mr 32,400 or 32,000) were caused to dissociate from actin by 55-kDa protein, but only 20% of tropomyosins with high Mr values (Mr 40,000 or 36,500) was dissociated from actin in these conditions. Immunofluorescence has shown that, while tropomyosin was localized in stress fibers, 55-kDa protein was found in microspikes as well as stress fibers, both of which are known to contain bundles of microfilaments. Therefore, we suggest that 55-kDa protein together with the multiple tropomyosin isoforms may regulate the formation of two types of actin-filament bundles, bundles containing tropomyosin and those without tropomyosin.     

Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83-kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.     

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.     

Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners

The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.     

Push and pull of tropomyosin's opposite effects on myosin attachment to actin. A chimeric tropomyosin host-guest study

Tropomyosin is a ubiquitous actin-binding protein with an extended coiled-coil structure. Tropomyosin-actin interactions are weak and loosely specific, but they potently influence myosin. One such influence is inhibitory and is due to tropomyosin's statistically preferred positions on actin that sterically interfere with actin's strong attachment site for myosin. Contrastingly, tropomyosin's other influence is activating. It increases myosin's overall actin affinity ～4-fold. Stoichiometric considerations cause this activating effect to equate to an ～4(7)-fold effect of myosin on the actin affinity of tropomyosin. These positive, mutual, myosin-tropomyosin effects are absent if Saccharomyces cerevisiae tropomyosin replaces mammalian tropomyosin. To investigate these phenomena, chimeric tropomyosins were generated in which 38-residue muscle tropomyosin segments replaced a natural duplication within S. cerevisiae tropomyosin TPM1. Two such chimeric tropomyosins were sufficiently folded coiled coils to allow functional study. The two chimeras differed from TPM1 but in opposite ways. Consistent with steric interference, myosin greatly decreased the actin affinity of chimera 7, which contained muscle tropomyosin residues 228-265. On the other hand, myosin S1 increased by an order of magnitude the actin affinity of chimera 3, which contained muscle tropomyosin residues 74-111. Similarly, myosin S1-ADP binding to actin was strengthened 2-fold by substitution of chimera 3 tropomyosin for wild-type TPM1. Thus, a yeast tropomyosin was induced to mimic the activating behavior of mammalian tropomyosin by inserting a mammalian tropomyosin sequence. The data were not consistent with direct tropomyosin-myosin binding. Rather, they suggest an allosteric mechanism, in which myosin and tropomyosin share an effect on the actin filament.     

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.     

Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes

A new Mr 43,000 tropomyosin-binding protein (TMBP) has been identified in erythrocyte membranes by binding of 125I-labeled Bolton-Hunter tropomyosin to nitrocellulose blots of membrane proteins separated by sodium dodecyl sulfate-gel electrophoresis. This protein is not actin, because 125I-tropomyosin does not bind to purified actin on blots. Binding of 125I-tropomyosin to this protein is specific because it is inhibited by excess unlabeled tropomyosin but not by F-actin or muscle troponins. This protein has been purified to 95% homogeneity from a 1 M Tris extract of tropomyosin-depleted erythrocyte membranes by DEAE-cellulose and hydroxylapatite chromatography, followed by gel filtration on Ultrogel AcA 44. The purified protein has a Stokes radius of 3.9 nm and a sedimentation coefficient of 2.8 S, corresponding to a native molecular weight of 43,000. Binding of 125I-tropomyosin to the purified TMBP saturates at one tropomyosin molecule (Mr 60,000) to two Mr 43,000 TMBPs, with an affinity of about 5 X 10(-7) M. The TMBP is associated with the membrane skeleton after extraction of membranes with the non-ionic detergent, Triton X-100, and is present with respect to tropomyosin at a ratio of about one for every two tropomyosin molecules. Because there is enough tropomyosin for two tropomyosin molecules to be associated with each of the short actin filaments in the membrane skeleton, the erythrocyte membrane TMBP, together with tropomyosin, could function to restrict the number of spectrin molecules attached to each of the short actin filaments and thus specify the hexagonal symmetry of the spectrin-actin lattice. Alternatively, this TMBP could be homologous to one of the muscle troponins and might function with tropomyosin to regulate erythrocyte actomyosin-ATPase activity and influence erythrocyte shape.     

Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate

Differential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five αTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform’s affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC–actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee–von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner.     

Low Mr tropomyosin isoforms from chicken brain and intestinal epithelium have distinct actin-binding properties

Tropomyosin isoforms of the low Mr class were isolated from chicken intestinal epithelium and brain, and their physical and functional properties were characterized. Tropomyosin from each tissue contains four distinct polypeptides, all of about 32,000 daltons. In two-dimensional gels, brain tropomyosin contains two major and two minor polypeptides; the major epithelium isoforms coelectrophorese with the two minor brain isoforms. Conversely, only small amounts of the major brain isoforms are detected in the epithelium. Actin-binding properties of brain tropomyosin isoforms are distinct from those of the intestinal epithelium. At 2.5 mM MgCl2 and physiological ionic strength, the intestinal epithelial tropomyosin binds to filamentous actin with an apparent Ka of 8 X 10(6) M-1 whereas brain tropomyosin has an apparent Ka of 8 X 10(5) M-1. Tropomyosin from either tissue binds actin cooperatively with a Hill coefficient of 2.3 for intestinal epithelial cell and 1.95 for brain tropomyosin. Isoforms from both tissues exhibit reduced head-to-tail polymerizability as compared to muscle tropomyosin. The actin-binding properties of intestinal epithelial cell tropomyosin are therefore similar to those of the muscle tropomyosins even though the isoforms have lower molecular weight, a paracrystal structure, and reduced head-to-tail polymerizability typical of the other nonmuscle tropomyosins. These results indicate that a heterogeneity of functional properties may be expressed among the low Mr tropomyosin isoforms.     

Tropomodulin increases the critical concentration of barbed end-capped actin filaments by converting ADP.P(i)-actin to ADP-actin at all pointed filament ends

The pointed end capping protein, tropomodulin, increases the critical concentration of barbed end capped actin, i.e. it lowers the apparent affinity of pointed ends for actin monomers. We show here that this is due to the conversion of pointed end ADP. P(i)-actin (low critical concentration) to ADP-actin (high critical concentration) when 70-98% of the ends are capped by tropomodulin. We propose that this is due to the low affinity of tropomodulin for pointed ends (K(d) approximately 0.3 microM), which allows tropomodulin to rapidly exchange binding sites and transiently block access of actin monomers to all pointed ends. This leaves time for ATP hydrolysis and phosphate release to go to completion between successive monomer additions to the pointed end. When the affinity of tropomodulin for pointed ends was increased about 1000-fold by the presence of tropomyosin (K(d) < 0.05 nM), capping of 95% of the ends by tropomodulin did not alter the critical concentration. However, the critical concentration did increase when the tropomodulin concentration was raised to the high values effective in the absence of tropomyosin. This may reflect transient tropomodulin binding to tropomyosin-free actin molecules at the pointed ends of the tropomyosin-actin filaments without a high affinity tropomodulin cap, i.e. the ends that determine the value of the actin critical concentration.     

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.     

Tropomyosin and gelsolin cooperate in controlling the microfilament system

Tropomyosin has been shown to cause annealing of gelsolin-capped actin filaments. Here we show that tropomyosin is highly efficient in transforming even the smallest gelsolin-actin complexes into long actin filaments. At low concentrations of tropomyosin, the effect of tropomyosin depends on the length of the actin oligomer, and the cooperative nature of the process is a direct indication that tropomyosin induces a conformational change in the gelsolin-actin complexes, altering the structure at the actin (+) end such that capping by gelsolin is abolished. At increased concentrations of tropomyosin, heterodimers, trimers, and tetramers are converted to actin filaments. In addition, evidence is presented demonstrating that gelsolin, once removed from the (+) end of the actin, can reassociate with the newly formed tropomyosin-decorated actin filaments. Interestingly, the binding of gelsolin to the tropomyosin-actin filament complexes saturates at 2 gelsolin molecules per 14 actin and 2 tropomyosins, i.e. two gelsolins per tropomyosin-regulatory unit along the filament. These observations support the view that both tropomyosin and gelsolin are likely to have important functions in addition to those proposed earlier.     

Modulation of the actin-activated adenosinetriphosphatase activity of myosin by tropomyosin from vascular and gizzard smooth muscles

Tropomyosins from bovine aorta and pulmonary artery exhibit identical electrophoretic patterns in sodium dodecyl sulfate but differ from tropomyosins of either chicken gizzard or rabbit skeletal muscle. Each of the four tropomyosins binds readily to skeletal muscle F-actin as indicated by their sedimentation with actin and by their ability to maximally stimulate or inhibit actin-activated ATPase activity at a molar ratio of one tropomyosin per seven actin monomers. Smooth and skeletal muscle tropomyosins differ in their effects on activity of skeletal myosin or heavy meromyosin (HMM); the former can enhance activity under conditions in which the latter inhibits. Gizzard and arterial tropomyosins are usually equally effective in stimulating ATPase activity of skeletal acto-HMM, but at high concentrations of Mg2+ gizzard tropomyosin is more effective, a result that cannot be attributed to differences in the binding of the two tropomyosins to F-actin. The effects of tropomyosin also depend on the type of myosin; tropomyosin enhances activity of gizzard myosin under conditions in which it inhibits that of skeletal myosin. Increasing the pH or the Mg2+ concentration can reverse the effect of tropomyosin on actin-stimulated ATPase activity of skeletal HMM from activation to inhibition, but this reversal is not found with gizzard myosin. Activity in the absence of tropomyosin is independent of pH, and the loss of activation with increasing pH is not accompanied by loss of binding of tropomyosin to actin.