Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization.

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

Modulation of cardiac thin filament structure by phosphorylated troponin-I analyzed by protein-protein docking and molecular dynamics simulation.

Tropomyosin, controlled by troponin-linked Ca2+-binding, regulates muscle contraction by a macromolecular scale steric-mechanism that governs myosin-crossbridge-actin interactions. At low-Ca2+, C-terminal domains of troponin-I (TnI) trap tropomyosin in a position on thin filaments that interferes with myosin-binding, thus causing muscle relaxation. Steric inhibition is reversed at high-Ca2+ when TnI releases from F-actin-tropomyosin as Ca2+ and the TnI switch-peptide bind to the N-lobe of troponin-C (TnC). The opposite end of cardiac TnI contains a phosphorylation-sensitive ∼30 residue-long N-terminal peptide that is absent in skeletal muscle, and likely modifies these interactions in hearts. Here, PKA-dependent phosphorylation of serine 23 and 24 modulates Ca2+ and possibly switch-peptide binding to TnC, causing faster relaxation during the cardiac-cycle (lusitropy). The cardiac-specific N-terminal TnI domain is not captured in crystal structures of troponin or in cryo-EM reconstructions of thin filaments; thus, its global impact on thin filament structure and function is uncertain. Here, we used protein-protein docking and molecular dynamics simulation-based protocols to build a troponin model that was guided by and hence consistent with the recent seminal Yamada structure of Ca2+-activated thin filaments. We find that when present on thin filaments, phosphorylated Ser23/24 along with adjacent polar TnI residues interact closely with both tropomyosin and the N-lobe of TnC during our simulations. These interactions would likely bias tropomyosin to an off-state positioning on actin. In situ, such enhanced relaxation kinetics would promote cardiac lusitropy.

M8R tropomyosin mutation disrupts actin binding and filament regulation: The beginning affects the middle and end.

Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin-tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin's reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry, and function caused by the DCM-linked mutation, M8R, which is located at the overlap junction. Localized and nonlocalized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin filament regulatory function. Comparison of mutant and WT α-tropomyosin was carried out using in vitro motility assays, CD, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed that the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. The binding of troponin T's N terminus to the actin-mutant tropomyosin complex was also weakened. CD and molecular dynamics indicate that the M8R mutation disrupts the four-helix bundle at the head-to-tail junction, leading to weaker tropomyosin-tropomyosin binding and weaker tropomyosin-actin binding. Molecular dynamics revealed that altered end-to-end bond formation has effects extending toward the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin-thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM.

C-terminal troponin-I residues trap tropomyosin in the muscle thin filament blocked-state.

Tropomyosin and troponin regulate muscle contraction by participating in a macromolecular scale steric-mechanism to control myosin-crossbridge - actin interactions and consequently contraction. At low-Ca2+, the C-terminal 30% of troponin subunit-I (TnI) is proposed to trap tropomyosin in a position on thin filaments that sterically interferes with myosin-binding, thus causing muscle relaxation. In contrast, at high-Ca2+, inhibition is released after the C-terminal domains dissociate from F-actin-tropomyosin as its component switch-peptide domain binds to the N-lobe of troponin-C (TnC). Recent, paradigm-shifting, cryo-EM reconstructions by the Namba group have revealed density attributed to TnI along cardiac muscle thin filaments at both low- and high-Ca2+ concentration. Modeling the reconstructions showed expected high-Ca2+ hydrophobic interactions of the TnI switch-peptide and TnC. However, under low-Ca2+ conditions, sparse interactions of TnI and tropomyosin, and in particular juxtaposition of non-polar switch-peptide residues and charged tropomyosin amino acids in the published model seem difficult to reconcile with an expected steric-blocking conformation. This anomaly is likely due to inaccurate fitting of tropomyosin into the cryo-EM volume. In the current study, the low-Ca2+ cryo-EM volume was fitted with a more accurate tropomyosin model and representation of cardiac TnI. Our results show that at low-Ca2+ a cluster of hydrophobic residues at the TnI switch-peptide and adjacent H4 helix (Ala149, Ala151, Met 154, Leu159, Gly160, Ala161, Ala163, Leu167, Leu169, Ala171, Leu173) draw-in tropomyosin surface residues (Ile143, Ile146, Ala151, Ile154), presumably attracting the entire tropomyosin cable to its myosin-blocking position on actin. The modeling confirms that neighboring TnI "inhibitory domain" residues (Arg145, Arg148) bind to thin filaments at actin residue Asp25, as previously suggested. ClusPro docking of TnI residues 137-184 to actin-tropomyosin, including the TnI inhibitory-domain, switch-peptide and Helix H4, verified the modeled configuration. Our residue-to-residue contact-mapping of the TnI-tropomyosin association lends itself to experimental validation and functional localization of disease-bearing mutations.

Structure of the F-actin-tropomyosin complex.

Filamentous actin (F-actin) is the major protein of muscle thin filaments, and actin microfilaments are the main component of the eukaryotic cytoskeleton. Mutations in different actin isoforms lead to early-onset autosomal dominant non-syndromic hearing loss, familial thoracic aortic aneurysms and dissections, and multiple variations of myopathies. In striated muscle fibres, the binding of myosin motors to actin filaments is mainly regulated by tropomyosin and troponin. Tropomyosin also binds to F-actin in smooth muscle and in non-muscle cells and stabilizes and regulates the filaments there in the absence of troponin. Although crystal structures for monomeric actin (G-actin) are available, a high-resolution structure of F-actin is still missing, hampering our understanding of how disease-causing mutations affect the function of thin muscle filaments and microfilaments. Here we report the three-dimensional structure of F-actin at a resolution of 3.7 Å in complex with tropomyosin at a resolution of 6.5 Å, determined by electron cryomicroscopy. The structure reveals that the D-loop is ordered and acts as a central region for hydrophobic and electrostatic interactions that stabilize the F-actin filament. We clearly identify map density corresponding to ADP and Mg(2+) and explain the possible effect of prominent disease-causing mutants. A comparison of F-actin with G-actin reveals the conformational changes during filament formation and identifies the D-loop as their key mediator. We also confirm that negatively charged tropomyosin interacts with a positively charged groove on F-actin. Comparison of the position of tropomyosin in F-actin-tropomyosin with its position in our previously determined F-actin-tropomyosin-myosin structure reveals a myosin-induced transition of tropomyosin. Our results allow us to understand the role of individual mutations in the genesis of actin- and tropomyosin-related diseases and will serve as a strong foundation for the targeted development of drugs.

Usability: Adoption, Measurement, Value.

OBJECTIVE: We searched for the application of usability in the literature with a focus on adoption, measurements employed, and demonstrated value. Five human factors domains served as a platform for our reflection, which included the last 20 years. BACKGROUND: As usability studies continue to accumulate, there has been only a little past reflection on usability and contributions across a variety of applications. Our research provides a background for general usability, and we target specific usability research subareas within transportation, aging populations, autistic populations, telehealth, and cybersecurity. METHOD: "Usability" research was explored across five different domains within human factors. The goal was not to perform an exhaustive review but, rather, sample usability practices within several specific subareas. We focused on answering three questions: How was usability adopted? How was it measured? How was it framed in terms of value? CONCLUSION: We found that usability is very domain specific. Usability benchmarking studies and empirical standards are rare. The value associated with improving usability ranged widely-from monetary benefits to saving lives. Thus, researchers are motivated to further improve usability practices. A number of data collection and interpretation challenges still call for solutions. APPLICATION: Findings offer insight into the development of usability, as applied across a variety of subdomains. Our reflection ought to inform future theory development efforts. We are concerned about the lack of established benchmarks, which can help ground data interpretation. Future research should address this gap in the literature. We note that our findings can be used to develop better training materials for future usability researchers.

Protein-Protein Docking Reveals Dynamic Interactions of Tropomyosin on Actin Filaments.

Experimental approaches such as fiber diffraction and cryo-electron microscopy reconstruction have defined regulatory positions of tropomyosin on actin but have not, as yet, succeeded at determining key atomic-level contacts between these proteins or fully substantiated the dynamics of their interactions at a structural level. To overcome this deficiency, we have previously employed computational approaches to deduce global dynamics of thin filament components by energy landscape determination and molecular dynamics simulations. Still, these approaches remain computationally challenging for any complex and large macromolecular assembly like the thin filament. For example, tropomyosin cable wrapping around actin of thin filaments features both head-to-tail polymeric interactions and local twisting, both of which depart from strict superhelical symmetry. This produces a complex energy surface that is difficult to model and thus to evaluate globally. Therefore, at this stage of our understanding, assessing global molecular dynamics can prove to be inherently impractical. As an alternative, we adopted a "divide and conquer" protocol to investigate actin-tropomyosin interactions at an atomistic level. Here, we first employed unbiased protein-protein docking tools to identify binding specificity of individual tropomyosin pseudorepeat segments over the actin surface. Accordingly, tropomyosin "ligand" segments were rotated and translated over potential "target" binding sites on F-actin where the corresponding interaction energetics of billions of conformational poses were ranked by the programs PIPER and ClusPro. These data were used to assess favorable interactions and then to rebuild models of seamless and continuous tropomyosin cables over the F-actin substrate, which were optimized further by flexible fitting routines and molecular dynamics. The models generated azimuthally distinct regulatory positions for tropomyosin cables along thin filaments on actin dominated by stereo-specific head-to-tail overlap linkage. The outcomes are in good agreement with current cryo-electron microscopy topology and consistent with long-thought residue-to-residue interactions between actin and tropomyosin.

Cardiomyopathy Mutation Alters End-to-End Junction of Tropomyosin and Reduces Calcium Sensitivity.

Muscle contraction is governed by tropomyosin (Tpm) shifting azimuthally between three states on F-actin (B-, C-, and M-states) in response to calcium binding to troponin and actomyosin cross-bridge formation. The Tpm coiled coil polymerizes head to tail along the long-pitch helix of F-actin to form continuous superhelical cables that wrap around the actin filaments. The end-to-end bonds formed between the N- and C-terminus of adjacent Tpm molecules define Tpm continuity and play a critical role in the ability of Tpm to cooperatively bind to actin, thus facilitating Tpm conformational switching to cooperatively propagate along F-actin. We expect that a missense mutation in this critical overlap region associated with dilated cardiomyopathy, A277V, will alter Tpm binding and thin filament activation by altering the overlap structure. Here, we used cosedimentation assays and in vitro motility assays to determine how the mutation alters Tpm binding to actin and its ability to regulate actomyosin interactions. Analytical viscometry coupled with molecular dynamics simulations showed that the A277V mutation results in enhanced Tpm end-to-end bond strength and a reduced curvature of the Tpm overlap domain. The mutant Tpm exhibited enhanced actin-Tpm binding affinity, consistent with overlap stabilization. The observed A277V-induced decrease in cooperative activation observed with regulated thin filament motility indicates that increased overlap stabilization is not correlated with Tpm-Tpm overlap binding strength or mechanical rigidity as is often assumed. Instead, A277V-induced structural changes result in local and delocalized increases in Tpm flexibility and prominent coiled-coil twisting in pseudorepeat 4. An A277V-induced decrease in Ca2+ sensitivity, consistent with a mutation-induced bolstering of the B-state Tpm-actin electrostatic contacts and an increased Tpm troponin T1 binding affinity, was also observed.

Docking Troponin T onto the Tropomyosin Overlapping Domain of Thin Filaments.

Complete description of thin filament conformational transitions accompanying muscle regulation requires ready access to atomic structures of actin-bound tropomyosin-troponin. To date, several molecular-docking protocols have been employed to identify troponin interactions on actin-tropomyosin because high-resolution experimentally determined structures of filament-associated troponin are not available. However, previously published all-atom models of the thin filament show chain separation and corruption of components during our molecular dynamics simulations of the models, implying artifactual subunit organization, possibly due to incorporation of unorthodox tropomyosin-TnT crystal structures and complex FRET measurements during model construction. For example, the recent Williams et al. (2016) atomistic model of the thin filament displays a paucity of salt bridges and hydrophobic complementarity between the TnT tail (TnT1) and tropomyosin, which is difficult to reconcile with the high, 20 nM Kd binding of TnT onto tropomyosin. Indeed, our molecular dynamics simulations show the TnT1 component in their model partially dissociates from tropomyosin in under 100 ns, whereas actin-tropomyosin and TnT1 models themselves remain intact. We therefore revisited computational work aiming to improve TnT1-thin filament models by employing unbiased docking methodologies, which test billions of trial rotations and translations of TnT1 over three-dimensional grids covering end-to-end bonded tropomyosin alone or tropomyosin on F-actin. We limited conformational searches to the association of well-characterized TnT1 helical domains and either isolated tropomyosin or actin-tropomyosin yet avoided docking TnT domains that lack known or predicted structure. The docking programs PIPER and ClusPro were used, followed by interaction energy optimization and extensive molecular dynamics. TnT1 docked to either side of isolated tropomyosin but uniquely onto one location of actin-bound tropomyosin. The antiparallel interaction with tropomyosin contained abundant salt bridges and intimately integrated hydrophobic networks joining TnT1 and the tropomyosin N-/C-terminal overlapping domain. The TnT1-tropomyosin linkage yields well-defined molecular crevices. Interaction energy measurements strongly favor this TnT1-tropomyosin design over previously proposed models.

Cryo-EM and Molecular Docking Shows Myosin Loop 4 Contacts Actin and Tropomyosin on Thin Filaments.

The motor protein myosin drives muscle and nonmuscle motility by binding to and moving along actin of thin filaments. Myosin binding to actin also modulates interactions of the regulatory protein, tropomyosin, on thin filaments, and conversely tropomyosin affects myosin binding to actin. Insight into this reciprocity will facilitate a molecular level elucidation of tropomyosin regulation of myosin interaction with actin in muscle contraction, and in turn, promote better understanding of nonmuscle cell motility. Indeed, experimental approaches such as fiber diffraction, cryoelectron microscopy, and three-dimensional reconstruction have long been used to define regulatory interaction of tropomyosin and myosin on actin at a structural level. However, their limited resolution has not proven sufficient to determine tropomyosin and myosin contacts at an atomic-level and thus to fully substantiate possible functional contributions. To overcome this deficiency, we have followed a hybrid approach by performing new cryogenic electron microscopy reconstruction of myosin-S1-decorated F-actin-tropomyosin together with atomic scale protein-protein docking of tropomyosin to the EM models. Here, cryo-EM data were derived from filaments reconstituted with α1-actin, cardiac αα-tropomyosin, and masseter muscle ß-myosin complexes; masseter myosin, which shares sequence identity with ß-cardiac myosin-heavy chain, was used because of its stability in vitro. The data were used to build an atomic model of the tropomyosin cable that fits onto the actin filament between the tip of the myosin head and a cleft on the innermost edge of actin subunits. The docking and atomic scale fitting showed multiple discrete interactions of myosin loop 4 and acidic residues on successive 39-42 residue-long tropomyosin pseudorepeats. The contacts between S1 and tropomyosin on actin appear to compete with and displace ones normally found between actin and tropomyosin on myosin-free thin filaments in relaxed muscle, thus restructuring the filament during myosin-induced activation.

A new twist on tropomyosin binding to actin filaments: perspectives on thin filament function, assembly and biomechanics.

Tropomyosin, best known for its role in the steric regulation of muscle contraction, polymerizes head-to-tail to form cables localized along the length of both muscle and non-muscle actin-based thin filaments. In skeletal and cardiac muscles, tropomyosin, under the control of troponin and myosin, moves in a cooperative manner between blocked, closed and open positions on filaments, thereby masking and exposing actin-binding sites necessary for myosin crossbridge head interactions. While the coiled-coil signature of tropomyosin appears to be simple, closer inspection reveals surprising structural complexity required to perform its role in steric regulation. For example, component α-helices of coiled coils are typically zippered together along a continuous core hydrophobic stripe. Tropomyosin, however, contains a number of anomalous, functionally controversial, core amino acid residues. We argue that the atypical residues at this interface, including clusters of alanines and a charged aspartate, are required for preshaping tropomyosin to readily fit to the surface of the actin filament, but do so without compromising tropomyosin rigidity once the filament is assembled. Indeed, persistence length measurements of tropomyosin are characteristic of a semi-rigid cable, in this case conducive to cooperative movement on thin filaments. In addition, we also maintain that tropomyosin displays largely unrecognized and residue-specific torsional variance, which is involved in optimizing contacts between actin and tropomyosin on the assembled thin filament. Corresponding twist-induced stiffness may also enhance cooperative translocation of tropomyosin across actin filaments. We conclude that anomalous core residues of tropomyosin facilitate thin filament regulatory behavior in a multifaceted way.

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.

Precise Binding of Tropomyosin on Actin Involves Sequence-Dependent Variance in Coiled-Coil Twisting.

Often considered an archetypal dimeric coiled coil, tropomyosin nonetheless exhibits distinctive "noncanonical" core residues located at the hydrophobic interface between its component α-helices. Notably, a charged aspartate, D137, takes the place of nonpolar residues otherwise present. Much speculation has been offered to rationalize potential local coiled-coil instability stemming from D137 and its effect on regulatory transitions of tropomyosin over actin filaments. Although experimental approaches such as electron cryomicroscopy reconstruction are optimal for defining average tropomyosin positions on actin filaments, to date, these methods have not captured the dynamics of tropomyosin residues clustered around position 137 or elsewhere. In contrast, computational biochemistry, involving molecular dynamics simulation, is a compelling choice to extend the understanding of local and global tropomyosin behavior on actin filaments at high resolution. Here, we report on molecular dynamics simulation of actin-free and actin-associated tropomyosin, showing noncanonical residue D137 as a locus for tropomyosin twist variation, with marked effects on actin-tropomyosin interactions. We conclude that D137-sponsored coiled-coil twisting is likely to optimize electrostatic side-chain contacts between tropomyosin and actin on the assembled thin filament, while offsetting disparities between tropomyosin pseudorepeat and actin subunit periodicities. We find that D137 has only minor local effects on tropomyosin coiled-coil flexibility, (i.e., on its flexural mobility). Indeed, D137-associated overtwisting may actually augment tropomyosin stiffness on actin filaments. Accordingly, such twisting-induced stiffness of tropomyosin is expected to enhance cooperative regulatory translocation of the tropomyosin cable over actin.

HCM and DCM cardiomyopathy-linked α-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments.

Calcium regulation of cardiac muscle contraction is controlled by the thin-filament proteins troponin and tropomyosin bound to actin. In the absence of calcium, troponin-tropomyosin inhibits myosin-interactions on actin and induces muscle relaxation, whereas the addition of calcium relieves the inhibitory constraint to initiate contraction. Many mutations in thin filament proteins linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we tested perturbations caused by mutant tropomyosins (E40K, DCM; and E62Q, HCM) on intra-filament interactions affecting acto-myosin interactions including those induced further by myosin association. Comparison of wild-type and mutant human α-tropomyosin (Tpm1.1) behavior was carried out using in vitro motility assays and molecular dynamics simulations. Our results show that E62Q tropomyosin destabilizes thin filament off-state function by increasing calcium-sensitivity, but without apparent affect on global tropomyosin structure by modifying coiled-coil rigidity. In contrast, the E40K mutant tropomyosin appears to stabilize the off-state, demonstrates increased tropomyosin flexibility, while also decreasing calcium-sensitivity. In addition, the E40K mutation reduces thin filament velocity at low myosin concentration while the E62Q mutant tropomyosin increases velocity. Corresponding molecular dynamics simulations indicate specific residue interactions that are likely to redefine underlying molecular regulatory mechanisms, which we propose explain the altered contractility evoked by the disease-causing mutations.

Switching Muscles On and Off in Steps: The McKillop-Geeves Three-State Model of Muscle Regulation.

BJ Classic highlighting the article "Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament."

Tropomyosin Must Interact Weakly with Actin to Effectively Regulate Thin Filament Function.

Elongated tropomyosin, associated with actin-subunits along the surface of thin filaments, makes electrostatic interactions with clusters of conserved residues, K326, K328, and R147, on actin. The association is weak, permitting low-energy cost regulatory movement of tropomyosin across the filament during muscle activation. Interestingly, acidic D292 on actin, also evolutionarily conserved, lies adjacent to the three-residue cluster of basic amino acids and thus may moderate the combined local positive charge, diminishing tropomyosin-actin interaction and facilitating regulatory-switching. Indeed, charge neutralization of D292 is connected to muscle hypotonia in individuals with D292V actin mutations and linked to congenital fiber-type disproportion. Here, the D292V mutation may predispose tropomyosin-actin positioning to a myosin-blocking state, aberrantly favoring muscle relaxation, thus mimicking the low-Ca2+ effect of troponin even in activated muscles. To test this hypothesis, interaction energetics and in vitro function of wild-type and D292V filaments were measured. Energy landscapes based on F-actin-tropomyosin models show the mutation localizes tropomyosin in a blocked-state position on actin defined by a deeper energy minimum, consistent with augmented steric-interference of actin-myosin binding. In addition, whereas myosin-dependent motility of troponin/tropomyosin-free D292V F-actin is normal, motility is dramatically inhibited after addition of tropomyosin to the mutant actin. Thus, D292V-induced blocked-state stabilization appears to disrupt the delicately poised energy balance governing thin filament regulation. Our results validate the premise that stereospecific but necessarily weak binding of tropomyosin to F-actin is required for effective thin filament function.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation.

Regulation of dynamin oligomerization in cells: the role of dynamin-actin interactions and its GTPase activity.

Dynamin is a 96-kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain-containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin's GTPase activity in the regulation of dynamin oligomerization in cells.

Structural determinants of muscle thin filament cooperativity.

End-to-end connections between adjacent tropomyosin molecules along the muscle thin filament allow long-range conformational rearrangement of the multicomponent filament structure. This process is influenced by Ca(2+) and the troponin regulatory complexes, as well as by myosin crossbridge heads that bind to and activate the filament. Access of myosin crossbridges onto actin is gated by tropomyosin, and in the case of striated muscle filaments, troponin acts as a gatekeeper. The resulting tropomyosin-troponin-myosin on-off switching mechanism that controls muscle contractility is a complex cooperative and dynamic system with highly nonlinear behavior. Here, we review key information that leads us to view tropomyosin as central to the communication pathway that coordinates the multifaceted effectors that modulate and tune striated muscle contraction. We posit that an understanding of this communication pathway provides a framework for more in-depth mechanistic characterization of myopathy-associated mutational perturbations currently under investigation by many research groups.

The propensity for tropomyosin twisting in the presence and absence of F-actin.

A canonical model of muscle α-tropomyosin (Tpm1.1), based on molecular-mechanics and electron microscopy of different contractile states, shows that the two-stranded coiled-coiled is pre-bent to present a specific molecular-face to the F-actin filament. This conformation is thought to facilitate both filament assembly and tropomyosin sliding across actin to modulate myosin-binding. However, to bind effectively to actin filaments, the 42 nm-long tropomyosin coiled-coil is not strictly canonical. Here, the mid-region of tropomyosin twists an additional ∼20° in order to better match the F-actin helix. In addition, the N- and C-terminal regions of tropomyosin polymerize head-to-tail to form continuous super-helical cables. In this case, 9 to 10 residue-long overlapping domains between adjacent molecules untwist relative to each other to accommodate orthogonal interactions between chains in the junctional four-helix nexus. Extensive molecular dynamics simulations show that the twisting and untwisting motions of tropomyosin vary appreciably along tropomyosin length, and in particular that substantial terminal domain winding and unwinding occurs whether tropomyosin is bound to F-actin or not. The local and regional twisting and untwisting do not appear to proceed in a concerted fashion, resembling more of a "wringing-type" behavior rather than a rotation.