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Peter W Gunning - One of the best experts on this subject based on the ideXlab platform.
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Actin Tropomyosin Assembly Intermediates
Biophysical Journal, 2015Co-Authors: Peyman Obeidy, Peter W Gunning, Thomas L. Sobey, Elvis Pandzic, Philip R. Nicovich, Adelle C. F. Coster, Till BöckingAbstract:Actin filaments play a critical role as part of the cytoskeleton, where they are involved in most cellular processes including determination of cell shape, cell migration, cell division, membrane function and intracellular transport. In muscle cells actin filaments are a key component of the contractile apparatus. This enormous functional specialisation is associated with differences in filament organisation, dynamics and interaction with actin-binding proteins. Recent evidence reveals that Tropomyosins are key players in this dynamic regulation of the functions of actin filaments. However, the molecular mechanisms underlying the assembly of tropomyosin strands on actin filaments, the competition between tropomyosin isoforms and the differences in stability are not known. The main roadblock to addressing these questions is a lack of techniques to follow the dynamics of the process at the molecular level.Here we have developed a single-molecule fluorescence imaging approach to visualise and quantify snapshots of the assembly process by reconstituting actin filaments in the presence of labelled tropomyosin isoforms. Cytoskeletal and skeletal tropomyosin isoforms were labelled using maleimide chemistry. Biochemical assays showed that labelled Tropomyosins bind cooperatively to actin filaments. We then competitively bound different tropomyosin isoforms to actin filaments in a microfluidic flow channel and visualised early assembly intermediates utilising TIRF microscopy. Our observations reveal nucleation of short stretches of tropomyosin polymers at multiple locations along the actin filaments. Our results have implications for the assembly pathways of specialised actin filaments in cells.
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Tropomyosins induce neuritogenesis and determine neurite branching patterns in b35 neuroblastoma cells
Molecular and Cellular Neuroscience, 2014Co-Authors: Nikki M Curthoys, Galina Schevzov, Hannah Freittag, Andrea Connor, Melissa Desouza, Merryn Brettle, Anne Poljak, Amelia Hall, Edna C Hardeman, Peter W GunningAbstract:Abstract Background The actin cytoskeleton is critically involved in the regulation of neurite outgrowth. Results The actin cytoskeleton-associated protein tropomyosin induces neurite outgrowth in B35 neuroblastoma cells and regulates neurite branching in an isoform-dependent manner. Conclusions Our data indicate that Tropomyosins are key regulators of the actin cytoskeleton during neurite outgrowth. Significance Revealing the molecular machinery that regulates the actin cytoskeleton during neurite outgrowth may provide new therapeutic strategies to promote neurite regeneration after nerve injury. Summary The formation of a branched network of neurites between communicating neurons is required for all higher functions in the nervous system. The dynamics of the actin cytoskeleton is fundamental to morphological changes in cell shape and the establishment of these branched networks. The actin-associated proteins Tropomyosins have previously been shown to impact on different aspects of neurite formation. Here we demonstrate that an increased expression of Tropomyosins is sufficient to induce the formation of neurites in B35 neuroblastoma cells. Furthermore, our data highlight the functional diversity of different tropomyosin isoforms during neuritogenesis. Tropomyosins differentially impact on the expression levels of the actin filament bundling protein fascin and increase the formation of filopodia along the length of neurites. Our data suggest that Tropomyosins are central regulators of actin filament populations which drive distinct aspects of neuronal morphogenesis.
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cytoskeletal Tropomyosins choreographers of actin filament functional diversity
Journal of Muscle Research and Cell Motility, 2013Co-Authors: Howard Vindin, Peter W GunningAbstract:The actin cytoskeleton plays a central role in many essential cellular processes. Its involvement requires actin filaments to form multiple populations with different structural and therefore functional properties in specific subcellular locations. This diversity is facilitated through the interaction between actin and a number of actin binding proteins. One family of proteins, the Tropomyosins, are absolutely essential in regulating actin’s ability to form such diverse structures. In this review we integrate studies from different organisms and cell types in an attempt to provide a unifying view of tropomyosin dependent regulation of the actin cytoskeleton.
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high molecular weight Tropomyosins localize to the contractile rings of dividing cns cells but are absent from malignant pediatric and adult cns tumors
Glia, 2003Co-Authors: Julie A I Hughes, Claire Cookeyarborough, Nigel C Chadwick, Galina Schevzov, Susan Arbuckle, Peter W Gunning, Ron P WeinbergerAbstract:Tropomyosin has been implicated in the control of actin filament dynamics during cell migration, morphogenesis, and cytokinesis. In order to gain insight into the role of Tropomyosins in cell division, we examined their expression in developing and neoplastic brain tissue. We found that the high-molecular-weight Tropomyosins are downregulated at birth, which correlates with glial cell differentiation and withdrawal of most cells from the cell cycle. Expression of these isoforms was restricted to proliferative areas in the embryonic brain and was absent from the adult, where the majority of cells are quiescent. However, they were induced under conditions where glial cells became proliferative in response to injury. During cytokinesis, these tropomyosin isoforms were associated with the contractile ring. We also investigated tropomyosin expression in neoplastic CNS tissues. Low-grade astrocytic tumors expressed high-molecular-weight Tropomyosins, while highly malignant CNS tumors of diverse origin did not (P ≤ 0.001). Furthermore, high-molecular-weight Tropomyosins were absent from the contractile ring in highly malignant astrocytoma cells. Our findings suggest a role for high-molecular-weight Tropomyosins in astrocyte cytokinesis, although highly malignant CNS tumors are still able to undergo cell division in their absence. Additionally, the correlation between high-molecular-weight tropomyosin expression and tumor grade suggests that Tropomyosins are potentially useful as indicators of CNS tumor grade. GLIA 42:25–35, 2003. © 2003 Wiley-Liss, Inc.
David M. Helfman - One of the best experts on this subject based on the ideXlab platform.
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a critical role of Tropomyosins in tgf β regulation of the actin cytoskeleton and cell motility in epithelial cells
Molecular Biology of the Cell, 2004Co-Authors: Andrei V Bakin, Alfiya Safina, Cammie Rinehart, Cecilia M Daroqui, Huferesh Darbary, David M. HelfmanAbstract:We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight Tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, Tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of Tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of Tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of Tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of Tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of Tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of Tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.
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specificity of dimer formation in Tropomyosins influence of alternatively spliced exons on homodimer and heterodimer assembly
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Mario Gimona, Akiya Watakabe, David M. HelfmanAbstract:Tropomyosins consist of nearly 100% alpha-helix and assemble into parallel and in-register coiled-coil dimers. In vitro it has been established that nonmuscle as well as native muscle Tropomyosins can form homodimers. However, a mixture of muscle alpha and beta tropomyosin subunits results in the formation of the thermodynamically more stable alpha/beta heterodimer. Although the assembly preference of the muscle tropomyosin heterodimer can be understood thermodynamically, the presence of multiple tropomyosin isoforms expressed in nonmuscle cells points toward a more complex principle for determining dimer formation. We have investigated the dimerization of rat Tropomyosins in living cells by the use of epitope tagging with a 16-aa sequence of the influenza hemagglutinin. Employing transfection and immunoprecipitation techniques, we have analyzed the dimers formed by muscle and nonmuscle Tropomyosins in rat fibroblasts. We demonstrate that the information for homo- versus heterodimerization is contained within the tropomyosin molecule itself and that the information for the selectivity is conferred by the alternatively spliced exons. These results have important implications for models of the regulation of cytoskeletal dynamics.
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expression of smooth muscle and nonmuscle Tropomyosins in escherichia coli and characterization of bacterially produced Tropomyosins
Biochimica et Biophysica Acta, 1993Co-Authors: Robert E Novy, David M. HelfmanAbstract:The cDNA encoding the beta-tropomyosin isoform of chicken smooth muscle (CSMbeta) was constructed and expressed in Escherichia coli to produce recombinant, unacetylated beta-tropomyosin (rCSMbeta) and a mutant (rCSMbeta-7) with a 7-residue deletion at its amino-terminus. Furthermore, the cDNA coding for human fibroblast tropomyosin isoform 3 (hTM3) was also used to produce unacetylated hTM3 (called PEThTM3). All of bacterially-made Tropomyosins were high alpha-helical in structure as judged by CD analysis and resistant to heat denaturation. Both the rCSMbeta and PEThTM3 exhibited saturable binding to F-actin with apparent binding constants of 1.14 . 10(6) and 2.78 . 10(6) M-1, respectively. The bacterially made, unacetylated smooth muscle tropomyosin (rCSMbeta) appeared to have a comparable actin-binding affinity to that of gel-purified CSMbeta homodimer (1.25 . 10(6) M-1) but significantly lower than that for native gizzard tropomyosin (CSM-TM) heterodimer (1.28 . 10(7) M-1). The amino-terminal deletion mutant rCSMbeta-7 failed to bind to F-actin. Effects of gizzard caldesmon on the actin binding of these bacterially made Tropomyosins were also examined. Under the binding condition containing 0.5 mM MgCl2 and 30 mM KCl, caldesmon greatly enhanced the binding of rCSMbeta to F-actin. However, under the same condition, there was a slight enhancement in the actin-binding for gel-purified CSMbeta or PEThTM3 (1.2-1.6-fold stimulation) and no enhancement for native gizzard tropomyosin. Neither the presence of caldesmon nor native gizzard tropomyosin induced detectable binding of the amino-terminal deletion mutant rCSMbeta-7 to F-actin. These results clearly imply the importance of the amino-terminal 7 amino-acid residues of CSMbeta in the actin binding and the caldesmon enhancement.
Kazuo Shiomi - One of the best experts on this subject based on the ideXlab platform.
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Tropomyosins in gastropods and bivalves identification as major allergens and amino acid sequence features
Food Chemistry, 2009Co-Authors: Ai Emoto, Shoichiro Ishizaki, Kazuo ShiomiAbstract:Abstract Tropomyosin appears to be a major cross-reactive allergen of crustaceans and molluscs. In this study, four species of gastropods (disc abalone, turban shell, whelk and Middendorf’s buccinum) and seven species of bivalves (bloody cockle, Japanese oyster, Japanese cockle, surf clam, horse clam, razor clam and short-neck clam) were confirmed to be allergenic by ELISA and their major allergen identified as tropomyosin by immunoblotting. Inhibition immunoblotting data showed the cross-reactivity of gastropod and bivalve Tropomyosins with one another and also with cephalopod and crustacean Tropomyosins. Then, amino acid sequences of Tropomyosins from 10 species except for Middendorf’s buccinum were elucidated by cDNA cloning. The known amino acid sequence data including our results reveal that molluscan Tropomyosins share low sequence identities (about 60%) with crustacean Tropomyosins and that they are highly homologous with one another within the same group (same family or same order) but not among the groups.
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identification of Tropomyosins as major allergens in antarctic krill and mantis shrimp and their amino acid sequence characteristics
Marine Biotechnology, 2008Co-Authors: Kanna Motoyama, Shoichiro Ishizaki, Yuji Nagashima, Yota Suma, Ying Lu, Hideki Ushio, Kazuo ShiomiAbstract:Tropomyosin represents a major allergen of decapod crustaceans such as shrimps and crabs, and its highly conserved amino acid sequence (>90% identity) is a molecular basis of the immunoglobulin E (IgE) cross-reactivity among decapods. At present, however, little information is available about allergens in edible crustaceans other than decapods. In this study, the major allergen in two species of edible crustaceans, Antarctic krill Euphausia superba and mantis shrimp Oratosquilla oratoria that are taxonomically distinct from decapods, was demonstrated to be tropomyosin by IgE-immunoblotting using patient sera. The cross-reactivity of the Tropomyosins from both species with decapod Tropomyosins was also confirmed by inhibition IgE immunoblotting. Sequences of the Tropomyosins from both species were determined by complementary deoxyribonucleic acid cloning. The mantis shrimp tropomyosin has high sequence identity (>90% identity) with decapod Tropomyosins, especially with fast-type Tropomyosins. On the other hand, the Antarctic krill tropomyosin is characterized by diverse alterations in region 13–42, the amino acid sequence of which is highly conserved for decapod Tropomyosins, and hence, it shares somewhat lower sequence identity (82.4–89.8% identity) with decapod Tropomyosins than the mantis shrimp tropomyosin. Quantification by enzyme-linked immunosorbent assay revealed that Antarctic krill contains tropomyosin at almost the same level as decapods, suggesting that its allergenicity is equivalent to decapods. However, mantis shrimp was assumed to be substantially not allergenic because of the extremely low content of tropomyosin.
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comparative analysis of barnacle tropomyosin divergence from decapod Tropomyosins and role as a potential allergen
Comparative Biochemistry and Physiology B, 2007Co-Authors: Yota Suma, Shoichiro Ishizaki, Yuji Nagashima, Ying Lu, Hideki Ushio, Kazuo ShiomiAbstract:Abstract Tropomyosin, a myofibrillar protein of 35–38 kDa, represents a major and cross-reactive allergen in decapod crustaceans. This study was initiated to clarify whether decapod-allergic patients also recognize Tropomyosins of barnacles, crustaceans phylogenetically remote from decapods, which are locally consumed as a delicacy. On SDS-PAGE, a 37 kDa protein was observed in all the heated extracts prepared from two species of decapods (American lobster Homarus americanus and black tiger prawn Penaeus monodon) and two species of barnacles (acorn barnacle Balanus rostratus and goose barnacle Capitulum mitella). In immunoblotting, the 37 kDa protein was found to react with monoclonal antibodies against American lobster tropomyosin and hence identified as tropomyosin. The patient sera reacted to Tropomyosins from both decapods and barnacles and the reactivity was abolished by preincubation with American lobster tropomyosin, demonstrating that barnacle Tropomyosins are allergens cross-reactive with decapod Tropomyosins. However, the amino acid sequence of acorn barnacle tropomyosin, deduced by cDNA cloning experiments, shares higher sequence identity with abalone Tropomyosins than with decapod Tropomyosins. In accordance with this, the phylogenetic tree made for Tropomyosins from various animals showed that the acorn barnacle tropomyosin is evolutionally classified not into the decapod tropomyosin family but into the molluscan tropomyosin family.
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molecular cloning of Tropomyosins identified as allergens in six species of crustaceans
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Kanna Motoyama, Shoichiro Ishizaki, Yuji Nagashima, Yota Suma, Kazuo ShiomiAbstract:Although tropomyosin is known to be a major allergen of crustaceans, its structural information is limited to only five species. In this study, tropomyosin was confirmed to be a major allergen in six species of crustaceans (black tiger prawn, kuruma prawn, pink shrimp, king crab, snow crab, and horsehair crab) by immunoblotting. Then, the amino acid sequences of Tropomyosins from these crustaceans were elucidated by a cDNA cloning technique. Sequence data for crustacean Tropomyosins including the obtained results reveal that fast Tropomyosins are contained in shrimps (or prawns) and lobsters, slow Tropomyosins in crabs, and both Tropomyosins in crayfishes and hermit crabs. Although fast and slow Tropomyosins share a high sequence identity (about 90%) with each other, significant differences are observed in specific regions between both Tropomyosins. Keywords: Allergen; cDNA cloning; crab; cross-reactivity; crustacean; prawn; shrimp; tropomyosin
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cephalopod Tropomyosins identification as major allergens and molecular cloning
Food and Chemical Toxicology, 2006Co-Authors: Kanna Motoyama, Shoichiro Ishizaki, Yuji Nagashima, Kazuo ShiomiAbstract:Heated extracts prepared from the mantle muscles (for decapods) or leg muscles (for octapods) of nine species of cephalopods were shown to be all reactive with serum IgE in crustacean-allergic patients. No marked difference in the reactivity with IgE was recognized among the cephalopods, suggesting that they are almost equally allergenic. Immunoblotting and inhibition immunoblotting data revealed that the major allergen is tropomyosin in common with the nine species of cephalopods and that the cephalopod Tropomyosins are cross-reactive with one another and also with crustacean Tropomyosins. Molecular cloning experiments first elucidated the primary structures of Tropomyosins from five species of cephalopods. The cephalopod Tropomyosins show high sequence identity (more than 92% identity) with one another, being the molecular basis for their cross-reactivity. Although the sequence identity between cephalopod and crustacean topomyosins is only about 63–64%, some of the IgE-binding epitopes proposed for brown shrimp Penaeus aztecus tropomyosin (Pen a 1) are well conserved in the cephalopod Tropomyosins, supporting the cross-reactivity between cephalopod and crustacean Tropomyosins.
Velia M. Fowler - One of the best experts on this subject based on the ideXlab platform.
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Tmod3 regulates polarized epithelial cell morphology.
Journal of cell science, 2007Co-Authors: Kari L Weber, Robert S Fischer, Velia M. FowlerAbstract:Although the role of the actin cytoskeleton in morphogenesis of polarized epithelial sheets is generally accepted as centrally important, the regulation of actin dynamics in this process remains unclear. Here, we show that the pointed-end capping protein Tmod3 contributes to epithelial cell shape within confluent monolayers of polarized epithelial cells. Tmod3 localizes to lateral cell membranes in polarized epithelia of several cell types. Reduction of Tmod3 levels by shRNA leads to a loss of F-actin and Tropomyosins from lateral cell membranes, and a decrease in epithelial cell height, without effects on localisation of tight junction or adherens junction proteins, or any apparent changes in cell-cell adhesion. Instead, distribution of alphaII-spectrin on lateral membranes is disrupted upon reduction of Tmod3 levels, suggesting that loss of Tmod3 function leads to destabilization and disassembly of tropomyosin-coated actin filaments followed by disorganization of the spectrin-based membrane skeleton on lateral membranes. These data demonstrate for the first time a role for pointed-end capping in morphology regulation of polarized epithelial cells through stabilization of F-actin on lateral membranes. We propose that Tmod3-capped tropomyosin-actin filaments provide crucial links in the spectrin membrane skeleton of polarized epithelial cells, enabling the membrane skeleton to maintain cell shape.
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isoform specific interaction of tropomodulin with skeletal muscle and erythrocyte Tropomyosins
Journal of Biological Chemistry, 1994Co-Authors: G G Babcock, Velia M. FowlerAbstract:Abstract Tropomodulin is a tropomyosin-binding protein that localizes to the pointed end of striated muscle thin filaments and caps the pointed end of tropomyosin-actin filaments in vitro. Results from previous studies have suggested that tropomyosin-tropomodulin interactions are isoform-specific. To investigate the molecular basis for the isoform specific interactions of tropomyosin and tropomodulin, we isolated a cDNA for chicken skeletal muscle tropomodulin. The derived amino acid sequence of muscle tropomodulin is 86% identical with that of human erythrocyte tropomodulin, indicating that tropomodulins are highly conserved proteins. Multiple mRNAs seen on Northern blots of chicken skeletal muscle mRNA can be accounted for by multiple polyadenylation signals in the 3'-untranslated region of the cDNA. 125I-Labeled skeletal muscle and erythrocyte Tropomyosins were assayed for binding bacterially expressed muscle tropomodulin using a solid phase binding assay. Unexpectedly, skeletal muscle and erythrocyte Tropomyosins bound with similar affinities to muscle tropomodulin (Kd values approximately 0.2 microM). However, cross-competition studies using erythrocyte and skeletal muscle Tropomyosins indicated that they bound different sites on tropomodulin. Competition studies using recombinant fragments of tropomodulin to map the binding domains for the two Tropomyosins demonstrated that residues 6-94 contained the skeletal muscle tropomyosin binding site and residues 90-184 contained the erythrocyte tropomyosin binding site. Binding of different regions of tropomodulin to muscle and non-muscle Tropomyosins may permit interaction of tropomodulin with tissue-specific components or may influence the pointed end capping activity of tropomodulin.
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tropomodulin binding to Tropomyosins
FEBS Journal, 1992Co-Authors: Mark A Sussman, Velia M. FowlerAbstract:Tropomodulin is a human erythrocyte membrane cytoskeletal protein that binds to one end of tropomyosin molecules and inhibits tropomyosin binding to actin filaments [Fowler, V. M. (1990) J. Cell Biol. 111, 471–482]. We have characterized the interaction of erythroid and non-erythroid Tropomyosins with tropomodulin by non-denaturing gel electrophoresis and by solid-phase binding assays using 125I-tropomyosin. Non-denaturing gel analysis demonstrates that all tropomodulin molecules are able to bind tropomyosin and that tropomodulin forms complexes with tropomyosin isoforms from erythrocyte, brain, platelet and skeletal muscle tissue. Scatchard analysis of binding data using tropomyosin isoforms from these tissues indicate that tropomodulin binds preferentially to erythrocyte tropomyosin. Specificity is manifested by decreases in the apparent affinity or the saturation binding capacity of tropomodulin for non-erythrocyte Tropomyosins. Erythrocyte tropomyosin saturates tropomodulin at approximate stoichiometric ratios of 1:2 and 1:4 tropomyosin/tropomodulin (apparent Kd= 14 nM−1 and 5 nM−1, respectively). Brain tropomyosin saturates tropomodulin at a 1:2 ratio of tropomyosin/tropomodulin, but with a threefold lower affinity than erythrocyte tropomyosin. Platelet tropomyosin saturates tropomodulin at a tropomyosin/tropomodulin ratio of 1:4, but with a sevenfold lower affinity than erythrocyte tropomyosin at the 1:4 ratio. These results correlate with oxidative cross-linking data which indicate that tropomodulin can self-associate to form dimers and tetramers in solution. Since tropomodulin interacts with one of the ends of tropomyosin, varying interactions of tropomyosin isoforms with tropomodulin probably reflect the heterogeneity in N-terminal or C-terminal sequences characteristic of the different tropomyosin isoforms. Isoform-specific interactions of tropomodulin with Tropomyosins may represent a novel mechanism for selective regulation of tropomyosin/actin interactions.
Larry S. Tobacman - One of the best experts on this subject based on the ideXlab platform.
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Atomic Model of F-Actin-Tropomyosin
Biophysical Journal, 2011Co-Authors: Xiaochuan Edward Li, Larry S. Tobacman, Roger Craig, Stefan Fischer, William LehmanAbstract:The presence of tropomyosin on the surface of actin filaments governs the access and hence the interactions and activity of numerous actin-binding proteins in muscle and non-muscle thin filaments. Electron microscopy and fiber diffraction studies of native and reconstituted F-actin-tropomyosin filaments reveal the azimuthal position of end-to-end linked tropomyosin molecules on the surface of actin. However, the longitudinal z-position and pseudo-rotation of tropomyosin along F-actin is still uncertain. Without this information, atomic models of F-actin-tropomyosin, unconstrained by troponin in striated muscle or other actin-binding proteins in smooth muscle or somatic cells, cannot be formulated, and thus the optimal interfacial contacts between actin and tropomyosin remain unknown. We have carried out a computational search assessing electrostatic interactions for multiple azimuthal locations, z-positions and pseudo-rotations of αα-tropomyosin on F-actin. The information gleaned was used to localize tropomyosin on F-actin, yielding an atomic model characterized by protein-protein contacts that primarily involve clusters of basic amino acids on actin subdomains 1 and 3 juxtaposed against acidic amino acids on successive quasi-repeating units of tropomyosin. A virtually identical model generated by docking F-actin and tropomyosin atomic structures into EM reconstructions of F-actin-tropomyosin validated the above solution. Here, the z-position of tropomyosin alongside F-actin was defined by matching the “broad” and “narrow” faces typifying tropomyosin's twisting superhelical coiled-coil to the twisting contours of the tropomyosin densities seen in F-actin-tropomyosin reconstructions. Our computational search indicates that, in the absence of troponin or other actin-binding proteins, tropomyosin occupies an optimal z-position and rotation in the surrounds of the closed C-state location on actin. However, no specific electrostatic minima were noted when tropomyosin was shifted computationally toward the open or blocked states on the troponin/myosin-free structure. The functional implications of the F-actin-tropomyosin model that has been determined will be discussed.
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Push and pull of tropomyosin's opposite effects on myosin attachment to actin. A chimeric tropomyosin host-guest study.
Biochemistry, 2010Co-Authors: Joshua M. Cohen, Larry S. TobacmanAbstract:In the absence of Ca2+, muscle tropomyosin attaches to the outer domain of actin, where it blocks substantial portions of the actin site to which myosin otherwise binds strongly to actin (1–3). When Ca2+ binds to the thin filament regulatory protein troponin, tropomyosin shifts position on the filament surface to the outer edge of the inner domain of actin, i.e., to the edge of the myosin binding site. Tropomyosins in general are located in these positions on the actin filament, not limited to Tropomyosins that are regulated by troponin (4). Only when myosin S1 is attached to the actin filament does tropomyosin undergoes a further azimuthal shift across more of the actin inner domain. This additional shift fully exposes the myosin binding site on actin (3, 5). Despite this steric hindrance between the preferred binding sites for tropomyosin and myosin S1 when they attach to actin separately, there is a paradoxical, positive interaction between tropomyosin and myosin. Myosin S1 binds to actin-tropomyosin filaments, or to actin-troponin-tropomyosin filaments, with an affinity 3- to 7-fold higher than to bare actin (6–8). Results with tropomyosin alone are very similar to those with tropomyosin-troponin-Ca2+, indicating that this effect is primarily due to tropomyosin. This effect has a dramatic consequence that necessarily follows from equilibrium linkage principles. That is, because seven myosin heads can bind to actin for every tropomyosin, the implication is that myosin S1 must increase the actin-affinity of tropomyosin (or of troponin-tropomyosin) by ~ 37- to 77-fold. This very high affinity binding has firm experimental support (9–11). It is an activating effect that is directly opposite of what would be expected based upon steric hindrance. Its importance is supported by other evidence that troponin-tropomyosin enhance, albeit more modestly, force, sliding speed, the rate of ATP product release by myosin, and other aspects of actin-myosin interactions (12–18). The mechanism of these effects remains an open question. A fruitful, relatively recent approach to investigating tropomyosin’s actions is to investigate and exploit the divergent properties of yeast and mammalian Tropomyosins (11, 19–21). In the present work this is pursued for the first time in a ‘host-guest’ manner, so termed in analogy to helical propensity studies in which guest amino acids are inserted into a host helical segment (22). The predominant tropomyosin isoform of S. cerevisiae, TPM1, spans 5 actins and has two properties that lend it to an attempt at such an approach. First, it contains an internal duplication of 38 residues. This duplication is flanked by flexible sites in the heptad repeat of the coiled-coil. Thus, one might be able to replace this one actin segment (i.e., this evolutionary internal duplication) of yeast host tropomyosin with a sequence(s) from foreign guest tropomyosin. Second, yeast Tropomyosins have interesting functional differences compared to mammalian Tropomyosins, including different spectroscopic effects on actin (23). The most significant dissimilarity is that yeast Tropomyosins do not bind more strongly to actin-myosin than to actin (11, 19, 20), unlike all mammalian non-muscle and muscle Tropomyosins that have been tested (7–11, 24, 25). (Yeast myosin-yeast tropomyosin effects have not been tested yet in such work, however.) Therefore, in principle one might test whether an important aspect of mammalian tropomyosin function, i.e, the tendency of tropomyosin and myosin to promote each other’s attachment to actin, is produced when yeast tropomyosin hosts inserted guest sequences from mammalian tropomyosin. In practice, utility depends upon such Tropomyosins having 3-dimensional structures suitable for functional study, including proper folding and shape. Following this approach, a panel of several chimeric Tropomyosins were generated, by replacement of TPM1 residues 70–107 with various sequences from muscle tropomyosin. Actin binding function was detected for two chimeras with relatively high preservation of folding thermodynamics, but no actin binding was observed for four other chimeras that folded more poorly. As described below, the two functional chimeras had opposite properties. The chimera containing residues mostly from muscle tropomyosin’s 7th quasi-repeat could not bind to actin when myosin was present. In contrast, the chimera containing residues mostly from muscle tropomyosin’s 3rd repeat bound to actin much more strongly when myosin was present. The significance of these findings is discussed.
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Cooperative Binding of Tropomyosin to Actin
Advances in Experimental Medicine and Biology, 2008Co-Authors: Larry S. TobacmanAbstract:Tropomyosin molecules attach to the thin filament conjointly rather than separately, in a pattern indicating very high cooperativity. The equilibrium process drawing Tropomyosins together on the actin filament can be measured by application of a linear lattice model to binding isotherm data and hypotheses on the mechanism of cooperativity can be tested. Each end of tropomyosin overlaps and attaches to the end of a neighboring tropomyosin, facilitating the formation of continuous tropomyosin strands, without gaps between neighboring molecules along the thin filament. Interestingly, the overlap complexes vary greatly in size and composition among tropomyosin isoforms, despite consistently cooperative binding to actin. Also, the tendency of tropomyosin to bind to actin cooperatively rather than randomly does not correlate with the strength of end-to-end binding. By implication, tropomyosin’s actin-binding cooperativity likely involves effects on the actin filament, as well as direct interactions between adjacent Tropomyosins.
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modulation of myosin function by isoform specific properties of saccharomyces cerevisiae and muscle Tropomyosins
Journal of Biological Chemistry, 2001Co-Authors: James Strand, Mahta Nili, Earl Homsher, Larry S. TobacmanAbstract:Abstract 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.
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Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules.
Journal of Biological Chemistry, 1992Co-Authors: Laura E. Hill, John P. Mehegan, Carol A. Butters, Larry S. TobacmanAbstract:Abstract The binding of tropomyosin to actin and troponin-tropomyosin to actin was analyzed according to a linear lattice model which quantifies two parameters: Ko, the affinity of the ligand for an isolated site on the actin filament, and gamma, the fold increase in affinity when binding is contiguous to an occupied site (cooperativity). Tropomyosin-actin binding is very cooperative (gamma = 90-137). Troponin strengthens tropomyosin-actin binding greatly but, surprisingly, does so solely by an 80-130-fold increase in Ko, while cooperativity actually decreases. Additionally, troponin complexes containing TnT subunits with deletions of either amino acids 1-69 (troponin70-259) or 1-158 (troponin159-259) were examined. Deletion of amino acids 1-69 had only small effects on Ko and y, despite this peptide's location spanning the joint between adjacent Tropomyosins. Ca2+ reduced Ko by half for both troponin and troponin70-159 and had no detectable effect on cooperativity. Troponin159-259 had much weaker effects on tropomyosin-actin binding than did troponin70-259 and had no effect at all in the presence of Ca2+. This suggests the importance of Ca(2+)-insensitive interactions between tropomyosin and troponin T residues 70-159. Cooperativity was slightly lower for troponin159-259 than tropomyosin alone, suggesting that the globular head region of troponin affects tropomyosin-tropomyosin interactions along the thin filament.
