Structural determinants of cooperativity in acto-myosin interactions

Regulation of muscle contraction is a very cooperative process. The presence of tropomyosin on the thin filament is both necessary and sufficient for cooperativity to occur. Data recently obtained with various tropomyosin isoforms and mutants help us to understand better the structural requirements in the thin filament for cooperative protein interactions. Forming an end-to-end overlap between neighboring tropomyosin molecules is not necessary for the cooperativity of the thin filament activation. When direct contacts between tropomyosin molecules are disrupted, the conformational changes in the filament are most probably transmitted cooperatively through actin subunits, although the exact nature of these changes is not known. The function of tropomyosin ends, alternatively expressed in various isoforms, is to confer specific actin affinity. Tropomyosin's affinity or actin is directly related to the size of the apparent cooperative unit defined as the number of actin subunits turned into the active state by binding of one myosin head. Inner sequences of tropomyosin, particularly actin-binding periods 3 to 5, play crucial role in myosin-induced activation of the thin filament. A plausible mechanism of tropomyosin function in this process is that inner tropomyosin regions are either specifically recognized by myosin or they define the right actin conformation required for tropomyosin movement from its blocking position.     

Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments

Tropomyosin is present in virtually all eucaryotic cells, where it functions to modulate actin-myosin interaction and to stabilize actin filament structure. In striated muscle, tropomyosin regulates contractility by sterically blocking myosin-binding sites on actin in the relaxed state. On activation, tropomyosin moves away from these sites in two steps, one induced by Ca(2+) binding to troponin and a second by the binding of myosin to actin. In smooth muscle and non-muscle cells, where troponin is absent, the precise role and structural dynamics of tropomyosin on actin are poorly understood. Here, the location of tropomyosin on F-actin filaments free of troponin and other actin-binding proteins was determined to better understand the structural basis of its functioning in muscle and non-muscle cells. Using electron microscopy and three-dimensional image reconstruction, the association of a diverse set of wild-type and mutant actin and tropomyosin isoforms, from both muscle and non-muscle sources, was investigated. Tropomyosin position on actin appeared to be defined by two sets of binding interactions and tropomyosin localized on either the inner or the outer domain of actin, depending on the specific actin or tropomyosin isoform examined. Since these equilibrium positions depended on minor amino acid sequence differences among isoforms, we conclude that the energy barrier between thin filament states is small. Our results imply that, in striated muscles, troponin and myosin serve to stabilize tropomyosin in inhibitory and activating states, respectively. In addition, they are consistent with tropomyosin-dependent cooperative switching on and off of actomyosin-based motility. Finally, the locations of tropomyosin that we have determined suggest the possibility of significant competition between tropomyosin and other cellular actin-binding proteins. Based on these results, we present a general framework for tropomyosin modulation of motility and cytoskeletal modelling.     

Tropomyosin isoforms define distinct microfilament populations with different drug susceptibility

Two tropomyosin isoforms, human Tm5(NM1) and Tm3, were over-expressed in B35 rat neuro-epithelial cells to examine preferential associations between specific actin and tropomyosin isoforms and to determine the role tropomyosin isoforms play in regulating the drug susceptibility of actin filament populations. Immunofluorescence staining and Western blot analysis were used to study the organisation of specific filament populations and their response to treatment with two widely used actin-destabilising drugs, latrunculin A and cytochalasin D. In Tm5(NM1) cells, we observed large stress fibres which showed predominant co-localisation of beta-actin and low-molecular-weight gamma-tropomyosin isoforms. Tm3 cells had an abundance of cellular protrusions which contained both the beta- and gamma-actin isoforms, predominately populated by high-molecular-weight alpha- and beta-tropomyosin isoforms. The stress fibres observed in Tm5(NM1) cells were more resistant to both latrunculin A and cytochalasin D than filaments containing the high-molecular-weight tropomyosins observed in Tm3 cells. Knockdown of the over-expressed Tm5(NM1) isoform with a human-specific Tm5(NM1) siRNA reversed the phenotype and caused a reversal in the observed drug resistance. We conclude that there are preferential associations between specific actin and tropomyosin isoforms, which are cell type specific, but it is the tropomyosin composition of a filament population which determines the susceptibility to actin-targeting drugs.     

Study of regulatory effect of tropomyosin on actin-myosin interaction in skeletal muscle by in vitro motility assay

The interaction between myosin and actin in striated muscle tissue is regulated by Ca²⁺ via thin filament regulatory proteins. Skeletal muscle possesses a whole pattern of myosin and tropomyosin isoforms. The regulatory effect of tropomyosin on actin-myosin interaction was investigated by measuring the sliding velocity of both actin and actin-tropomyosin filaments over fast and slow skeletal myosins using the in vitro motility assay. The actin-tropomyosin filaments were reconstructed with tropomyosin isoforms from striated muscle tissue. It was found that tropomyosins with different content of α-, β-, and γ-chains added to actin filaments affect the sliding velocity of filaments in different ways. On the other hand, the sliding velocity of filaments with the same content of α-, β-, and Γ-chains depends on myosin isoforms of striated muscle. The reciprocal effects of myosin and tropomyosin on actin-myosin interaction in striated muscle may play a significant role in maintenance of effective work of striated muscle both during ontogenesis and under pathological conditions.     

The Effect of Tropomyosin Mutations on Actin-Tropomyosin Binding: In Search of Lost Time

The initial binding of tropomyosin onto actin filaments and then its polymerization into continuous cables on the filament surface must be precisely tuned to overall thin-filament structure, function, and performance. Low-affinity interaction of tropomyosin with actin has to be sufficiently strong to localize the tropomyosin on actin, yet not so tight that regulatory movement on filaments is curtailed. Likewise, head-to-tail association of tropomyosin molecules must be favorable enough to promote tropomyosin cable formation but not so tenacious that polymerization precedes filament binding. Arguably, little molecular detail on early tropomyosin binding steps has been revealed since Wegner’s seminal studies on filament assembly almost 40 years ago. Thus, interpretation of mutation-based actin-tropomyosin binding anomalies leading to cardiomyopathies cannot be described fully. In vitro, tropomyosin binding is masked by explosive tropomyosin polymerization once cable formation is initiated on actin filaments. In contrast, in silico analysis, characterizing molecular dynamics simulations of single wild-type and mutant tropomyosin molecules on F-actin, is not complicated by tropomyosin polymerization at all. In fact, molecular dynamics performed here demonstrates that a midpiece tropomyosin domain is essential for normal actin-tropomyosin interaction and that this interaction is strictly conserved in a number of tropomyosin mutant species. Elsewhere along these mutant molecules, twisting and bending corrupts the tropomyosin superhelices as they “lose their grip” on F-actin. We propose that residual interactions displayed by these mutant tropomyosin structures with actin mimic ones that occur in early stages of thin-filament generation, as if the mutants are recapitulating the assembly process but in reverse. We conclude therefore that an initial binding step in tropomyosin assembly onto actin involves interaction of the essential centrally located domain.     

Tropomyosin assembly intermediates in the control of microfilament system turnover

Tropomyosin is a coiled-coil alpha-helical protein, which self-associates in a head-to-tail fashion along polymers of actin to produce thin filaments. Mammalian non-muscle cells express a large number of tropomyosin isoforms, which are differentially regulated during embryogenesis and associated with specialized actin microfilament ensembles in cells. The function of tropomyosin in specifying form and localization of these subcellular structures, and the precise mechanism(s) by which they carry out their functions, is unclear. This paper reports that, while the major fraction of non-muscle cell tropomyosin resides in actin thin filaments of the cytomatrix, the soluble part of the cytoplasm contains tropomyosins in the form of actin-free multimers, which are isoform specific and of high molecular weight (MW(app) 180,000-250,000). Stimulation of motile cells with growth factors induces a rapid, actin polymerization-dependent outgrowth of lamellipodia and filopodia. Concomitantly, the levels of tropomyosin isoform-specific multimers decrease, suggesting their involvement in actin thin filament formation. Malignant tumor cells have drastically altered levels and composition of tropomyosin isoform-specific multimers as well as tropomyosin in the cytomatrix.     

The interaction of tropomodulin with tropomyosin stabilizes thin filaments in cardiac myocytes

Actin (thin) filament length regulation and stability are essential for striated muscle function. To determine the role of the actin filament pointed end capping protein, tropomodulin1 (Tmod1), with tropomyosin, we generated monoclonal antibodies (mAb17 and mAb8) against Tmod1 that specifically disrupted its interaction with tropomyosin in vitro. Microinjection of mAb17 or mAb8 into chick cardiac myocytes caused a dramatic loss of the thin filaments, as revealed by immunofluorescence deconvolution microscopy. Real-time imaging of live myocytes expressing green fluorescent protein-alpha-tropomyosin and microinjected with mAb17 revealed that the thin filaments depolymerized from their pointed ends. In a thin filament reconstitution assay, stabilization of the filaments before the addition of mAb17 prevented the loss of thin filaments. These studies indicate that the interaction of Tmod1 with tropomyosin is critical for thin filament stability. These data, together with previous studies, indicate that Tmod1 is a multifunctional protein: its actin filament capping activity prevents thin filament elongation, whereas its interaction with tropomyosin prevents thin filament depolymerization.     

Tissue-specific tropomyosin isoform composition.

Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells (Gunning et al. 1998b). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.     

Tropomyosin isoform 3 promotes the formation of filopodia by regulating the recruitment of actin-binding proteins to actin filaments

Tropomyosins are believed to function in part by stabilizing actin filaments. However, accumulating evidence suggests that fundamental differences in function exist between tropomyosin isoforms, which contributes to the formation of functionally distinct filament populations. We investigated the functions of the high-molecular-weight isoform Tm3 and examined the molecular properties of Tm3-containing actin filament populations. Overexpression of the Tm3 isoform specifically induced the formation of filopodia and changes in actin solubility. We observed alterations in actin-binding protein recruitment to filaments, co-incident with changes in expression levels, which can account for this functional outcome. Tm3-associated filaments recruit active actin depolymerizing factor and are bundled into filopodia by fascin, which is both up-regulated and preferentially associated with Tm3-containing filaments in the Tm3 overexpressing cells. This study provides further insight into the isoform-specific roles of different tropomyosin isoforms. We conclude that variation in the tropomyosin isoform composition of microfilaments provides a mechanism to generate functionally distinct filament populations.     

Study of reciprocal effects of cardiac myosin and tropomyosin isoforms on actin-myosin interaction with in vitro motility assay

Interaction of myosin with actin in striated muscle is controlled by Ca²⁺ via thin filament associated proteins: troponin and tropomyosin. In cardiac muscle there is a whole pattern of myosin and tropomyosin isoforms. The aim of the current work is to study regulatory effect of tropomyosin on sliding velocity of actin filaments in the in vitro motility assay over cardiac isomyosins. It was found that tropomyosins of different content of α- and β-chains being added to actin filament effects the sliding velocity of filaments in different ways. On the other hand the velocity of filaments with the same tropomyosins depends on both heavy and light chains isoforms of cardiac myosin.     

Creating intracellular structural domains: spatial segregation of actin and tropomyosin isoforms in neurons

Actin microfilaments play a direct role in a variety of cell processes. Distinct populations of microfilaments are associated with different cellular compartments, such as growth cones, filipodia, stress fibers, and lamellipodia. It is becoming clear that these different populations are often composed of different isoforms of the two core microfilament components, actin and tropomyosin. This is particularly true in neurons, where actin and tropomyosin isoforms are segregated into different intracellular compartments which correspond to functionally distinct regions of the neuron. Developmental regulation of this isoform sorting suggests a specific role for some isoforms in growth and for others in stabilization of neuronal structure. This provides a mechanism by which a neuron can create and independently regulate intracellular domains composed of microfilaments with different functional properties. BioEssays 20:892–900, 1998. © 1998 John Wiley & Sons, Inc.     

Studies on cardiac myofibrillogenesis with antibodies to titin, actin, tropomyosin, and myosin

Cardiac myofibrillogenesis was examined in cultured chick cardiac cells by immunofluorescence using antibodies against titin, actin, tropomyosin, and myosin. Primitive cardiomyocytes initially contained stress fiber-like structures (SFLS) that stained positively for alpha actin and/or muscle tropomyosin. In some cases the staining for muscle tropomyosin and alpha actin was disproportionate; this suggests that the synthesis and/or assembly of these two isoforms into the SFLS may not be stoichiometric. The alpha actin containing SFLS in these myocytes could be classified as either central or peripheral; central SFLS showed developing sarcomeric titin while peripheral SFLS had weak titin fluorescence and a more uniform stain distribution. Sarcomeric patterns of titin and myosin were present at multiple sites on these structures. A pair of titin staining bands was clearly associated with each developing A band even at the two or three sarcomere stage, although occasional examples of a titin band being associated with a half sarcomere were noted. The appearance of sarcomeric titin patterns coincided or preceded sarcomere periodicity of either alpha actin or muscle tropomyosin. The early appearance of titin in myofibrillogenesis suggests it may have a role in filament alignment during sarcomere assembly.     

Evidence for a direct but sequential binding of titin to tropomyosin and actin filaments

Titin is a giant molecule that spans half a sarcomere, establishing several specific bindings with both structural and contractile myofibrillar elements. It has been demonstrated that this giant protein plays a major role in striated muscle cell passive tension and contractile filament alignment. The in vitro interaction of titin with a new partner (tropomyosin) reported here is reinforced by our recent in vitro motility study using reconstituted Ca-regulated thin filaments, myosin and a native 800-kDa titin fragment. In the presence of the tropomyosin-troponin complex, the actin filament movement onto coated S1 is improved by the titin fragment. Here, we found that two purified native titin fragments of 150 and 800 kDa, covering respectively the N1-line and the N2-line/PEVK region in the I-band and known to contain actin-binding sites, directly bind tropomyosin in the absence of actin. We have also shown that binding of the 800-kDa fragment with filamentous actin inhibited the subsequent interaction of tropomyosin with actin, as judged by cosedimentation. However, this was not the case if the complex of actin and tropomyosin was formed before the addition of the 800-kDa fragment. We thus conclude that a sequential arrangement of contacts exists between parts of the titin I-band region, tropomyosin and actin in the thin filament.     

Dual roles of tropomyosin as an F-actin stabilizer and a regulator of muscle contraction in Caenorhabditis elegans body wall muscle

Tropomyosin is a well-characterized regulator of muscle contraction. It also stabilizes actin filaments in a variety of muscle and non-muscle cells. Although these two functions of tropomyosin could have different impacts on actin cytoskeletal organization, their functional relationship has not been studied in the same experimental system. Here, we investigated how tropomyosin stabilizes actin filaments and how this function is influenced by muscle contraction in Caenorhabditis elegans body wall muscle. We confirmed the antagonistic role of tropomyosin against UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament organization using multiple UNC-60B mutant alleles. Tropomyosin was also antagonistic to UNC-78 (AIP1) in vivo and protected actin filaments from disassembly by UNC-60B and UNC-78 in vitro, suggesting that tropomyosin protects actin filaments from the ADF/cofilin-AIP1 actin disassembly system in muscle cells. A mutation in the myosin heavy chain caused greater reduction in contractility than tropomyosin depletion. However, the myosin mutation showed much weaker suppression of the phenotypes of ADF/cofilin or AIP1 mutants than tropomyosin depletion. These results suggest that muscle contraction has only minor influence on the tropomyosin's protective role against ADF/cofilin and AIP1, and that the two functions of tropomyosin in actin stability and muscle contraction are independent of each other.     

Accumulation of tropomyosin in developing chicken gizzard smooth muscle

Smooth muscle of chicken embryonic gizzards has been shown to contain 9 tropomyosin isoforms (E1, E2, E3, E4, E5, E6, E7, E8, and E9) in addition to alpha and beta isoforms (Hosoya et al. (1989) J. Biochem. 105, 712-717). At the early stages of development, the amount of these isoforms was larger than those of alpha and beta isoforms. However, they gradually decreased at later stages and finally disappeared completely after hatching. By using two-dimensional gel electrophoresis and an image analyzing system, we examined the process of tropomyosin accumulation in gizzard smooth muscle development. The accumulation patterns of tropomyosin isoforms and their relative molar ratios to actin in embryonic development were different from those in the stages after hatching. The relative molar ratio of tropomyosin to actin in the thin filament preparation of embryonic gizzards was lower than that of adult, and it gradually increased in the course of embryonic development.     

Assembly of different isoforms of actin and tropomyosin into the skeletal tropomyosin-enriched microfilaments during differentiation of muscle cells in vitro

We have used a monoclonal antibody (CL2) directed against striated muscle isoforms of tropomyosin to selectively isolate a class of microfilaments (skeletal tropomyosin-enriched microfilaments) from differentiating muscle cells. This class of microfilaments differed from the one (tropomyosin-enriched microfilaments) isolated from the same cells by a monoclonal antibody (LCK16) recognizing all isoforms of muscle and nonmuscle tropomyosin. In myoblasts, the skeletal tropomyosin-enriched microfilaments had a higher content of alpha-actin and phosphorylated isoforms of tropomyosin as compared with the tropomyosin-enriched microfilaments. Moreover, besides muscle isoforms of actin and tropomyosin, significant amounts of nonmuscle isoforms of actin and tropomyosin were found in the skeletal tropomyosin-enriched microfilaments of myoblasts and myotubes. These results suggest that different isoforms of actin and tropomyosin can assemble into the same set of microfilaments, presumably pre-existing microfilaments, to form the skeletal tropomyosin-enriched microfilaments, which will eventually become the thin filaments of myofibrils. Therefore, the skeletal tropomyosin-enriched microfilaments detected here may represent an intermediate class of microfilaments formed during thin filament maturation. Electron microscopic studies of the isolated microfilaments from myoblasts and myotubes showed periodic localization of tropomyosin molecules along the microfilaments. The tropomyosin periodicity in the microfilaments of myoblasts and myotubes was 35 and 37 nm, respectively, whereas the nonmuscle tropomyosin along chicken embryo fibroblast microfilaments had a 34-nm repeat.     

Tropomyosin isoform modulation of focal adhesion structure and cell migration

Orderly cell migration is essential for embryonic development, efficient wound healing and a functioning immune system and the dysregulation of this process leads to a number of pathologies. The speed and direction of cell migration is critically dependent on the structural organization of focal adhesions in the cell. While it is well established that contractile forces derived from the acto-myosin filaments control the structure and growth of focal adhesions, how this may be modulated to give different outcomes for speed and persistence is not well understood. The tropomyosin family of actin-associating proteins are emerging as important modulators of the contractile nature of associated actin filaments. The multiple non-muscle tropomyosin isoforms are differentially expressed between tissues and across development and are thought to be major regulators of actin filament functional specialization. In the present study we have investigated the effects of two splice variant isoforms from the same α-tropomyosin gene, TmBr1 and TmBr3, on focal adhesion structure and parameters of cell migration. These isoforms are normally switched on in neuronal cells during differentiation and we find that exogenous expression of the two isoforms in undifferentiated neuronal cells has discrete effects on cell migration parameters. While both isoforms cause reduced focal adhesion size and cell migration speed, they differentially effect actin filament phenotypes and migration persistence. Our data suggests that differential expression of tropomyosin isoforms may coordinate acto-myosin contractility and focal adhesion structure to modulate cell speed and persistence.Key words: focal adhesion, tropomyosin, actin, migration, persistence, speed, mesenchymal     

Movement of smooth muscle tropomyosin by myosin heads.

It has been proposed that during the activation of muscle contraction the initial binding of myosin heads to the actin thin filament contributes to switching on the thin filament and that this might involve the movement of actin-bound tropomyosin. The movement of smooth muscle tropomyosin on actin was investigated in this work by measuring the change in distance between specific residues on tropomyosin and actin by fluorescence resonance energy transfer (FRET) as a function of myosin head binding to actin. An energy transfer acceptor was attached to Cys374 of actin and a donor to the tropomyosin heterodimer at either Cys36 of the beta-chain or Cys190 of the alpha-chain. FRET changed for the donor at both positions of tropomyosin upon addition of skeletal or smooth muscle myosin heads, indicating a movement of the whole tropomyosin molecule. The changes in FRET were hyperbolic and saturated at about one head per seven actin subunits, indicating that each head cooperatively affects several tropomyosin molecules, presumably via tropomyosin's end-to-end interaction. ATP, which dissociates myosin from actin, completely reversed the changes in FRET induced by heads, whereas in the presence of ADP the effect of heads was the same as in its absence. The results indicate that myosin with and without ADP, intermediates in the myosin ATPase hydrolytic pathway, are effective regulators of tropomyosin position, which might play a role in the regulation of smooth muscle contraction.     

Effect of the structure of the N terminus of tropomyosin on tropomodulin function

Tropomodulins (Tmod) bind to the N terminus of tropomyosin and cap the pointed end of actin filaments. Tropomyosin alone also inhibits the rate of actin depolymerization at the pointed end of filaments. Here we have defined 1) the structural requirements of the N terminus of tropomyosin important for regulating the pointed end alone and with erythrocyte Tmod (Tmod1), and 2) the Tmod1 subdomains required for binding to tropomyosin and for regulating the pointed end. Changes in pyrene-actin fluorescence during polymerization and depolymerization were measured with actin filaments blocked at the barbed end with gelsolin. Three tropomyosin isoforms differently influence pointed end dynamics. Recombinant TM5a, a short non-muscle alpha-tropomyosin, inhibited depolymerization. Recombinant (unacetylated) TM2 and N-acetylated striated muscle TM (stTM), long alpha-tropomyosin isoforms with the same N-terminal sequence, different from TM5a, also inhibited depolymerization but were less effective than TM5a. All blocked the pointed end with Tmod1 in the order of effectiveness TM5a >stTM >TM2, showing the importance of the N-terminal sequence and modification. Tmod1-(1-344), lacking the C-terminal 15 residues, did not nucleate polymerization but blocked the pointed end with all three tropomyosin isoforms as does a shorter fragment, Tmod1-(1-92), lacking the C-terminal "capping" domain though higher concentrations were required. An even shorter fragment, Tmod1-(1-48), bound tropomyosin but did not influence actin filament elongation. Tropomyosin-Tmod may function to locally regulate cytoskeletal dynamics in cells by stabilizing actin filaments.     

Troponin I: Inhibitor or facilitator

TN-I occurs as a homologous group of proteins which form part of the regulatory system of vertebrate and invertebrate striated muscle. These proteins are present in vertebrate muscle as isoforms, Mr 21000-24000, that are specific for the muscle type and under individual genetic control. TN-I occupies a central position in the chain of events starting with the binding of calcium to troponin C and ending with activation of the Ca2+ stimulated MgATPase of the actomyosin filament in muscle. The ability of TN-I to inhibit the MgATPase of actomyosin in a manner that is accentuated by tropomyosin is fundamental to its role but the molecular mechanism involved is not yet completely understood. For the actomyosin ATPase to be regulated the interaction of TN-I with actin, TN-C and TN-T must undergo changes as the calcium concentration in the muscle cell rises, which result in the loss of its inhibitory activity. A variety of techniques have enabled the sites of interaction to be defined in terms of regions of the polypeptide chain that must be intact to preserve the biological properties of TN-I. There is also evidence for conformational changes that occur when the complex with TN-C binds calcium. Nevertheless a detailed high resolution structure of the troponin complex and its relation to actin/tropomyosin is not yet available. TN-I induces changes in those proteins with which it interacts, that are essential for their function. In the special case of cardiac TN-I its effect on the calcium binding properties of TN-C is modulated by phosphorylation. It has yet to be determined whether TN-I acts directly as an inhibitor or indirectly by interacting with associated proteins to facilitate their role in the regulatory system.