Tropomodulin contains two actin filament pointed end-capping domains

Tropomodulin 1 (Tmod1) is a approximately 40-kDa tropomyosin binding and actin filament pointed end-capping protein that regulates pointed end dynamics and controls thin filament length in striated muscle. In vitro, the capping affinity of Tmod1 for tropomyosin-actin filaments (Kd approximately 50 pm) is several thousand-fold greater than for capping of pure actin filaments (Kd approximately 0.1 microM). The tropomyosin-binding region of Tmod1 has been localized to the amino-terminal portion between residues 1 and 130, but the location of the actin-capping domain is not known. We have now identified two distinct actin-capping regions on Tmod1 by testing a series of recombinant Tmod1 fragments for their ability to inhibit actin elongation from gelsolin-actin seeds using pyrene-actin polymerization assays. The carboxyl-terminal portion of Tmod1 (residues 160-359) contains the principal actin-capping activity (Kd approximately 0.4 microM), requiring residues between 323 and 359 for full activity, whereas the amino-terminal portion of Tmod1 (residues 1-130) contains a second, weaker actin-capping activity (Kd approximately 1.8 microM). Interestingly, 160-359 but not 1-130 enhances spontaneous actin nucleation, suggesting that the carboxyl-terminal domain may bind to two actin subunits across the actin helix at the pointed end, whereas the amino-terminal domain may bind to only one actin subunit. On the other hand, the actin-capping activity of the amino-terminal but not the carboxyl-terminal portion of Tmod1 is enhanced several thousand-fold in the presence of skeletal muscle tropomyosin. We conclude that the carboxyl-terminal capping domain of Tmod1 contains a TM-independent actin pointed end-capping activity, whereas the amino-terminal domain contains a TM-regulated pointed end actin-capping activity.     

Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, approximately 40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils.     

The N-terminal end of nebulin interacts with tropomodulin at the pointed ends of the thin filaments

Strict regulation of actin thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with actin filament capping proteins to regulate thin filament lengths. Nebulin is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. Tropomodulin (Tmod), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of nebulin colocalizes with Tmod at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of nebulin bind specifically to Tmod as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the nebulin molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two Tmod isoforms, erythrocyte Tmod (E-Tmod), expressed in embryonic and slow skeletal muscle, and skeletal Tmod (Sk-Tmod), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal nebulin fragments. Sk-Tmod binds nebulin with higher affinity than E-Tmod does, suggesting that the Tmod/nebulin interaction exhibits isoform specificity. These data provide evidence that Tmod and nebulin may work together as a linked mechanism to control thin filament lengths in skeletal muscle.     

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.     

Thin filaments elongate from their pointed ends during myofibril assembly in Drosophila indirect flight muscle.

Tropomodulin (Tmod) is an actin pointed-end capping protein that regulates actin dynamics at thin filament pointed ends in striated muscle. Although pointed-end capping by Tmod controls thin filament lengths in assembled myofibrils, its role in length specification during de novo myofibril assembly is not established. We used the Drosophila Tmod homologue, sanpodo (spdo), to investigate Tmod's function during muscle development in the indirect flight muscle. SPDO was associated with the pointed ends of elongating thin filaments throughout myofibril assembly. Transient overexpression of SPDO during myofibril assembly irreversibly arrested elongation of preexisting thin filaments. However, the lengths of thin filaments assembled after SPDO levels had declined were normal. Flies with a preponderance of abnormally short thin filaments were unable to fly. We conclude that: (a) thin filaments elongate from their pointed ends during myofibril assembly; (b) pointed ends are dynamically capped at endogenous levels of SPDO so as to allow elongation; (c) a transient increase in SPDO levels during myofibril assembly converts SPDO from a dynamic to a permanent cap; and (d) developmental regulation of pointed-end capping during myofibril assembly is crucial for specification of final thin filament lengths, myofibril structure, and muscle function.     

Structural requirements of tropomodulin for tropomyosin binding and actin filament capping

Regulation of actin filament dynamics underlies many cellular functions. Tropomodulin together with tropomyosin can cap the pointed, slowly polymerizing, filament end, inhibiting addition or loss of actin monomers. Tropomodulin has an unstructured N-terminal region that binds tropomyosin and a folded C-terminal domain with six leucine-rich repeats. Of tropomodulin 1's 359 amino acids, an N-terminal fragment (Tmod1(1)(-)(92)) suffices for in vitro function, even though the C-terminal domain can weakly cap filaments independent of tropomyosin. Except for one short alpha-helix with coiled coil propensity (residues 24-35), the Tmod1(1)(-)(92) solution structure shows that the fragment is disordered and highly flexible. On the basis of the solution structure and predicted secondary structure, we have introduced a series of mutations to determine the structural requirements for tropomyosin binding (using native gels and CD) and filament capping (by measuring actin polymerization using pyrene fluorescence). Tmod1(1)(-)(92) fragments with mutations of an interface hydrophobic residue, L27G and L27E, designed to destroy the alpha-helix or coiled coil propensity, lost binding ability to tropomyosin but retained partial capping function in the presence of tropomyosin. Replacement of a flexible region with alpha-helical residues (residues 59-61 mutated to Ala) had no effect on tropomyosin binding but inhibited the capping function. A mutation in a region predicted to be an amphipathic helix (residues 65-75), L71D, destroyed the capping function. The results suggest that molecular flexibility and binding to actin via an amphipathic helix are both required for tropomyosin-dependent capping of the pointed end of the actin filament.     

Actin dynamics at pointed ends regulates thin filament length in striated muscle

Regulation of actin dynamics at filament ends determines the organization and turnover of actin cytoskeletal structures. In striated muscle, it is believed that tight capping of the fast-growing (barbed) ends by CapZ and of the slow-growing (pointed) ends by tropomodulin (Tmod) stabilizes the uniform lengths of actin (thin) filaments in myofibrils. Here we demonstrate for the first time that both CapZ and Tmod are dynamic on the basis of the rapid incorporation of microinjected rhodamine-labelled actin (rho-actin) at both barbed and pointed ends and from the photobleaching of green fluorescent protein (GFP)-labelled Tmod. Unexpectedly, the inhibition of actin dynamics at pointed ends by GFP-Tmod overexpression results in shorter thin filaments, whereas the inhibition of actin dynamics at barbed ends by cytochalasin D has no effect on length. These data demonstrate that the actin filaments in myofibrils are relatively dynamic despite the presence of capping proteins, and that regulated actin assembly at pointed ends determines the length of thin filaments.     

Tropomodulin 3 binds to actin monomers

Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.     

A Nebulin Ruler Does Not Dictate Thin Filament Lengths

To generate force, striated muscle requires overlap between uniform-length actin and myosin filaments. The hypothesis that a nebulin ruler mechanism specifies thin filament lengths by targeting where tropomodulin (Tmod) caps the slow-growing, pointed end has not been rigorously tested. Using fluorescent microscopy and quantitative image analysis, we found that nebulin extended 1.01-1.03 μm from the Z-line, but Tmod localized 1.13-1.31 μm from the Z-line, in seven different rabbit skeletal muscles. Because nebulin does not extend to the thin filament pointed ends, it can neither target Tmod capping nor specify thin filament lengths. We found instead a strong correspondence between thin filament lengths and titin isoform sizes for each muscle. Our results suggest the existence of a mechanism whereby nebulin specifies the minimum thin filament length and sarcomere length regulates and coordinates pointed-end dynamics to maintain the relative overlap of the thin and thick filaments during myofibril assembly.     

Pointed-end capping by tropomodulin3 negatively regulates endothelial cell motility

Actin filament pointed-end dynamics are thought to play a critical role in cell motility, yet regulation of this process remains poorly understood. We describe here a previously uncharacterized tropomodulin (Tmod) isoform, Tmod3, which is widely expressed in human tissues and is present in human microvascular endothelial cells (HMEC-1). Tmod3 is present in sufficient quantity to cap pointed ends of actin filaments, localizes to actin filament structures in HMEC-1 cells, and appears enriched in leading edge ruffles and lamellipodia. Transient overexpression of GFP-Tmod3 leads to a depolarized cell morphology and decreased cell motility. A fivefold increase in Tmod3 results in an equivalent decrease in free pointed ends in the cells. Unexpectedly, a decrease in the relative amounts of F-actin, free barbed ends, and actin-related protein 2/3 (Arp2/3) complex in lamellipodia are also observed. Conversely, decreased expression of Tmod3 by RNA interference leads to faster average cell migration, along with increases in free pointed and barbed ends in lamellipodial actin filaments. These data collectively demonstrate that capping of actin filament pointed ends by Tmod3 inhibits cell migration and reveal a novel control mechanism for regulation of actin filaments in lamellipodia.     

Different Localizations and Cellular Behaviors of Leiomodin and Tropomodulin in Mature Cardiomyocyte Sarcomeres

Leiomodin (Lmod) is a muscle-specific F-actin–nucleating protein that is related to the F-actin pointed-end–capping protein tropomodulin (Tmod). However, Lmod contains a unique ∼150-residue C-terminal extension that is required for its strong nucleating activity. Overexpression or depletion of Lmod compromises sarcomere organization, but the mechanism by which Lmod contributes to myofibril assembly is not well understood. We show that Tmod and Lmod localize through fundamentally different mechanisms to the pointed ends of two distinct subsets of actin filaments in myofibrils. Tmod localizes to two narrow bands immediately adjacent to M-lines, whereas Lmod displays dynamic localization to two broader bands, which are generally more separated from M-lines. Lmod''s localization and F-actin nucleation activity are enhanced by interaction with tropomyosin. Unlike Tmod, the myofibril localization of Lmod depends on sustained muscle contraction and actin polymerization. We further show that Lmod expression correlates with the maturation of myofibrils in cultured cardiomyocytes and that it associates with sarcomeres only in differentiated myofibrils. Collectively, the data suggest that Lmod contributes to the final organization and maintenance of sarcomere architecture by promoting tropomyosin-dependent actin filament nucleation.     

Alteration of Tropomyosin-binding Properties of Tropomodulin-1 Affects Its Capping Ability and Localization in Skeletal Myocytes

Tropomodulin (Tmod) is an actin-capping protein that binds to the two tropomyosins (TM) at the pointed end of the actin filament to prevent further actin polymerization and depolymerization. Therefore, understanding the role of Tmod is very important when studying actin filament dependent processes such as muscle contraction and intracellular transport. The capping ability of Tmod is highly influenced by TM and is 1000-fold greater in the presence of TM. There are four Tmod isoforms (Tmod1–4), three of which, Tmod1, Tmod3, and Tmod4, are expressed in skeletal muscles. The affinity of Tmod1 to skeletal striated TM (stTM) is higher than that of Tmod3 and Tmod4 to stTM. In this study, we tested mutations in the TM-binding sites of Tmod1, using circular dichroism (CD) and prediction analysis (PONDR). The mutations R11K, D12N, and Q144K were chosen because they decreased the affinity of Tmod1 to stTM, making it similar to that of affinity of Tmod3 and Tmod4 to stTM. Significant reduction of inhibition of actin pointed-end polymerization in the presence of stTM was shown for Tmod1 (R11K/D12N/Q144K) as compared with WT Tmod1. When GFP-Tmod1 and mutants were expressed in primary chicken skeletal myocytes, decreased assembly of Tmod1 mutants was revealed. This indicates a direct correlation between TM-binding and the actin-capping abilities of Tmod. Our data confirmed the hypothesis that assembly of Tmod at the pointed-end of the actin filament depends on its TM-binding affinity.     

Tropomodulin caps the pointed ends of actin filaments

Many proteins have been shown to cap the fast growing (barbed) ends of actin filaments, but none have been shown to block elongation and depolymerization at the slow growing (pointed) filament ends. Tropomodulin is a tropomyosin-binding protein originally isolated from red blood cells that has been localized by immunofluorescence staining to a site at or near the pointed ends of skeletal muscle thin filaments (Fowler, V. M., M. A., Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120: 411-420). Our experiments demonstrate that tropomodulin in conjunction with tropomyosin is a pointed end capping protein: it completely blocks both elongation and depolymerization at the pointed ends of tropomyosin-containing actin filaments in concentrations stoichiometric to the concentration of filament ends (Kd < or = 1 nM). In the absence of tropomyosin, tropomodulin acts as a "leaky" cap, partially inhibiting elongation and depolymerization at the pointed filament ends (Kd for inhibition of elongation = 0.1-0.4 microM). Thus, tropomodulin can bind directly to actin at the pointed filament end. Tropomodulin also doubles the critical concentration at the pointed ends of pure actin filaments without affecting either the rate of extent of polymerization at the barbed filament ends, indicating that tropomodulin does not sequester actin monomers. Our experiments provide direct biochemical evidence that tropomodulin binds to both the terminal tropomyosin and actin molecules at the pointed filament end, and is the long sought-after pointed end capping protein. We propose that tropomodulin plays a role in maintaining the narrow length distributions of the stable, tropomyosin-containing actin filaments in striated muscle and in red blood cells.     

Mammalian Tropomodulins Nucleate Actin Polymerization via Their Actin Monomer Binding and Filament Pointed End-capping Activities

Many actin-binding proteins have been shown to possess multiple activities to regulate filament dynamics. Tropomodulins (Tmod1–4) are a conserved family of actin filament pointed end-capping proteins. Our previous work has demonstrated that Tmod3 binds to monomeric actin in addition to capping pointed ends. Here, we show a novel actin-nucleating activity in mammalian Tmods. Comparison of Tmod isoforms revealed that Tmod1–3 but not Tmod4 nucleate actin filament assembly. All Tmods bind to monomeric actin, and Tmod3 forms a 1:1 complex with actin. By truncation and mutagenesis studies, we demonstrated that the second α-helix in the N-terminal domain of Tmod3 is essential for actin monomer binding. Chemical cross-linking and LC-MS/MS further indicated that residues in this second α-helix interact with actin subdomain 2, whereas Tmod3 N-terminal domain peptides distal to this α-helix interact with actin subdomain 1. Mutagenesis of Leu-73 to Asp, which disrupts the second α-helix of Tmod3, decreases both its actin monomer-binding and -nucleating activities. On the other hand, point mutations of residues in the C-terminal leucine-rich repeat domain of Tmod3 (Lys-317 in the fifth leucine-rich repeat β-sheet and Lys-344 or Arg-345/Arg-346 in the C-terminal α6-helix) significantly reduced pointed end-capping and nucleation without altering actin monomer binding. Taken together, our data indicate that Tmod3 binds actin monomers over an extended interface and that nucleating activity depends on actin monomer binding and pointed end-capping activities, contributed by N- and C-terminal domains of Tmod3, respectively. Tmod3 nucleation of actin assembly may regulate the cytoskeleton in dynamic cellular contexts.     

Multifunctional roles of tropomodulin-3 in regulating actin dynamics

Tropomodulins (Tmods) are proteins that cap the slow-growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and is present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.     

Leiomodin and tropomodulin in smooth muscle

Evidence isaccumulating to suggest that actin filament remodeling is critical forsmooth muscle contraction, which implicates actin filament ends asimportant sites for regulation of contraction. Tropomodulin (Tmod) andsmooth muscle leiomodin (SM-Lmod) have been found in many tissuescontaining smooth muscle by protein immunoblot and immunofluorescencemicroscopy. Both proteins cofractionate with tropomyosin in theTriton-insoluble cytoskeleton of rabbit stomach smooth muscle and aresolubilized by high salt. SM-Lmod binds muscle tropomyosin, abiochemical activity characteristic of Tmod proteins. SM-Lmod stainingis present along the length of actin filaments in rat intestinal smoothmuscle, while Tmod stains in a punctate pattern distinct from that ofactin filaments or the dense body marker -actinin. After smoothmuscle is hypercontracted by treatment with 10 mM Ca²⁺,both SM-Lmod and Tmod are found near -actinin at the periphery ofactin-rich contraction bands. These data suggest that SM-Lmod is anovel component of the smooth muscle actin cytoskeleton and, furthermore, that the pointed ends of actin filaments in smooth musclemay be capped by Tmod in localized clusters.

     

Mechanisms of thin filament assembly in embryonic chick cardiac myocytes: tropomodulin requires tropomyosin for assembly

Tropomodulin is a pointed end capping protein for tropomyosin-coated actin filaments that is hypothesized to play a role in regulating the precise lengths of striated muscle thin filaments (Fowler, V. M., M. A. Sussman, P. G. Miller, B. E. Flucher, and M. P. Daniels. 1993. J. Cell Biol. 120:411-420; Weber, A., C. C. Pennise, G. G. Babcock, and V. M. Fowler. 1994, J. Cell Biol. 127:1627-1635). To gain insight into the mechanisms of thin filament assembly and the role of tropomodulin therein, we have characterized the temporal appearance, biosynthesis and mechanisms of assembly of tropomodulin onto the pointed ends of thin filaments during the formation of striated myofibrils in primary embryonic chick cardiomyocyte cultures. Our results demonstrate that tropomodulin is not assembled coordinately with other thin filament proteins. Double immunofluorescence staining and ultrastructural immunolocalization demonstrate that tropomodulin is incorporated in its characteristic sarcomeric location at the pointed ends of the thin filaments after the thin filaments have become organized into periodic I bands. In fact, tropomodulin assembles later than all other well characterized myofibrillar proteins studied including: actin, tropomyosin, alpha-actinin, titin, myosin and C-protein. Nevertheless, at steady state, a significant proportion (approximately 39%) of tropomodulin is present in a soluble pool throughout myofibril assembly. Thus, the absence of tropomodulin in some striated myofibrils is not due to limiting quantities of the protein. In addition, kinetic data obtained from [35S]methionine pulse-chase experiments indicate that tropomodulin assembles more slowly into myofibrils than does tropomyosin. This observation, together with results obtained using a novel permeabilized cell model for thin filament assembly, indicate that tropomodulin assembly is dependent on the prior association of tropomyosin with actin filaments. We conclude that tropomodulin is a late marker for the assembly of striated myofibrils in cardiomyocytes; its assembly appears to be linked to their maturity. We propose that tropomodulin is involved in maintaining and stabilizing the final lengths of thin filaments after they are assembled.     

Tropomyosin prevents depolymerization of actin filaments from the pointed end

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.     

Tropomyosin regulates elongation by formin at the fast-growing end of the actin filament

The balance between dynamic and stable actin filaments is essential for the regulation of cellular functions including the determination of cell shape and polarity, cell migration, and cytokinesis. Proteins that regulate polymerization at the filament ends and filament stability confer specificity to actin filament structure and cellular function. The dynamics of the barbed, fast-growing end of the filament are controlled in space and time by both positive and negative regulators of actin polymerization. Capping proteins inhibit the addition and loss of subunits, whereas other proteins, including formins, bind at the barbed end and allow filament growth. In this work, we show that tropomyosin regulates dynamics at the barbed end. Tropomyosin binds to constructs of FRL1 and mDia2 that contain the FH2 domain and modulates formin-dependent capping of the barbed end by relieving inhibition of elongation by FRL1-FH1FH2, mDia1-FH2, and mDia2-FH2 in an isoform-dependent fashion. In this role, tropomyosin functions as an activator of formin. Tropomyosin also inhibits the binding of FRL1-FH1FH2 to the sides of actin filaments independent of the isoform. In contrast, tropomyosin does not affect the ability of capping protein to block the barbed end. We suggest that tropomyosin and formin act together to ensure the formation of unbranched actin filaments, protected from severing, that could be capped in stable cellular structures. This role, in addition to its cooperative control of myosin function, establishes tropomyosin as a universal regulator of the multifaceted actin cytoskeleton.     

Tropomyosin requires an intact N-terminal coiled coil to interact with tropomodulin

Tropomodulins (Tmods) are tropomyosin (TM) binding proteins that bind to the pointed end of actin filaments and modulate thin filament dynamics. They bind to the N termini of both "long" TMs (with the N terminus encoded by exon 1a of the alpha-TM gene) and "short" nonmuscle TMs (with the N terminus encoded by exon 1b). In this present study, circular dichroism was used to study the interaction of two designed chimeric proteins, AcTM1aZip and AcTM1bZip, containing the N terminus of a long or a short TM, respectively, with protein fragments containing residues 1 to 130 of erythrocyte or skeletal muscle Tmod. The binding of either TMZip causes similar conformational changes in both Tmod fragments promoting increases in both alpha-helix and beta-structure, although they differ in binding affinity. The circular dichroism changes in the Tmod upon binding and modeling of the Tmod sequences suggest that the interface between TM and Tmod includes a three- or four-stranded coiled coil. An intact coiled coil at the N terminus of the TMs is essential for Tmod binding, as modifications that disrupt the N-terminal helix, such as removal of the N-terminal acetyl group from AcTM1aZip or striated muscle alpha-TM, or introduction of a mutation that causes nemaline myopathy, Met-8-Arg, into AcTM1aZip destroyed Tmod binding.