Wild-type Actin Research Articles

A yeast based assay establishes the pathogenicity of novel missense ACTA2 variants associated with aortic aneurysms.

The ACTA2 gene codes for alpha-smooth muscle actin, a critical component of the contractile apparatus of the vascular smooth muscle cells. Autosomal dominant variants in the ACTA2 gene have been associated to familial non-syndromic thoracic aortic aneurysm/dissection (TAAD). They are thought to act through a dominant-negative mechanism. These variants display incomplete penetrance and variable expressivity, complicating the validation of ACTA2 variants pathogenicity by family segregation studies. In this study, we developed a yeast based assay to test putative TAAD-associated ACTA2 variants. We identified five new heterozygous ACTA2 missense variants in TAAD patients through next generation sequencing. We decided to test their pathogenicity in Saccharomyces cerevisiae, since yeast actin is very similar to human alpha-smooth muscle actin, and the residues at which the TAAD-associated variants occur in ACTA2 are well conserved. A wild type yeast strain was transformed with a vector expressing the different mutant alleles, to model the heterozygous condition of patients. Then, we evaluated yeast growth by spot test and cytoskeletal and mitochondrial morphology by fluorescence microscopy. We found that mutant yeast strains displayed only mild growth defects but a significant increase in the percentage of cells with abnormal mitochondrial distribution and abnormal organization of the actin cytoskeleton compared to controls. All variants appeared to interfere with the activity of wild type actin in yeast, suggesting a dominant-negative pathogenic mechanism. Our results demonstrate the utility of using the yeast actin model system to validate the pathogenicity of TAAD-associated ACTA2 variants.

Electrical impulse characterization along actin filaments in pathological conditions

We present an interactive Mathematica notebook that characterizes the electrical impulses along actin filaments in both muscle and non-muscle cells for a wide range of physiological and pathological conditions. The simplicity of the theoretical formulation, and high performance of the Mathematica software, enable the analysis of multiple conditions without computational restrictions. The program is based on a multi-scale (atomic → monomer → filament) approach capable of accounting for the atomistic details of a protein molecular structure, its biological environment, and their impact on the travel distance, velocity, and attenuation of monovalent ionic wave packets propagating along microfilaments. The interactive component allows investigators to choose the experimental conditions (intracellular Vs in vitro), nucleotide state (ATP Vs ADP), actin isoform (alpha, gamma, beta, and muscle or non-muscle cell), as well as a conformation model that covers a variety of mutants and wild-type (the control) actin filaments. We used the computational tool to analyze environmental changes such as temperature effects and pH changes of the surrounding solutions, as well as structural changes to an actin monomer due to radius changes. Additionally, we investigated for the first time the electrostatic consequences of actin mutations from different disease conditions. These studies may provide an unprecedented molecular understanding of why and how age, inheritance, and disease conditions induce dysfunctions in the biophysical mechanisms underlying the propagation of electrical signals along actin filaments. Program summaryProgram Title: EIAF (Electrical Impulse along Actin Filaments)CPC Library link to program files:https://doi.org/10.17632/k8vw4j6y75.1Developer's repository link:https://github.com/MarceloMarucho/SignalPropagationPathologicalConditionLicensing provisions: GPLv2Programming language: MathematicaNature of problem: Electrical properties of actin filaments in pathological conditions have yet to be studied, despite previous work showing influential consequences for physiological conditions on wild type actins.Solution method: The interactive Mathematica notebook is based on the Multi-scale Approach for electrical impulses along cytoskeletal filaments and uses non-linear considerations for the Boltzmann statistics of ionic distributions in the electrical double layer (EDL). Using the numerical methods incorporated in Mathematica’s default libraries, and an interactive GUI, the program allows users to conduct their own research by choosing the experimental conditions (intracellular Vs in vitro), nucleotide state (ATP Vs ADP), actin isoform (including muscle and non-muscles cells), as well as a conformation model that covers a variety of mutants and wild-type (the control) actin filament configurations.Additional comments including restrictions and unusual features: The program allows users to adjust pH values, input voltages, as well as study two default electrolyte conditions without rerunning the program. The program is fully editable, such that motivated users can change the default electrolytes to fit specific conditions in their research project in necessary.

K336I mutant actin alters the structure of neighbouring protomers in filaments and reduces affinity for actin-binding proteins

Mutation of the Lys-336 residue of actin to Ile (K336I) or Asp (K336E) causes congenital myopathy. To understand the effect of this mutation on the function of actin filaments and gain insight into the mechanism of disease onset, we prepared and biochemically characterised K336I mutant actin from Dictyostelium discoideum. Subtilisin cleavage assays revealed that the structure of the DNase-I binding loop (D-loop) of monomeric K336I actin, which would face the adjacent actin-protomer in filaments, differed from that of wild type (WT) actin. Although K336I actin underwent normal salt-dependent reversible polymerisation and formed apparently normal filaments, interactions of K336I filaments with alpha-actinin, myosin II, and cofilin were disrupted. Furthermore, co-filaments of K336I and WT actins also exhibited abnormal interactions with cofilin, implying that K336I actin altered the structure of the neighbouring WT actin protomers such that interaction between cofilin and the WT actin protomers was prevented. We speculate that disruption of the interactions between co-filaments and actin-binding proteins is the primary reason why the K336I mutation induces muscle disease in a dominant fashion.

The Missed Role of Cytoskeletal Filaments in Information Processing

Recently, substantial experimental evidence on Cytoskeleton filaments have demonstrated that the concurrent propagation of both electrical signals along cytoskeleton filaments and electrochemical currents along the axonal membrane are highly possible. This discovery opened an unexplored frontier in understanding brain information processing, neural plasticity and changes in the cytoskeleton. However, the molecular mechanisms governing the bionanowire properties in cytoskeleton filaments under physiological and pathological conditions are still poorly understood. In this presentation, we introduce an innovative multi-scale approach able to account for the atomistic details of a protein molecular structure, its biological environment, and their impact on electrical impulses propagating along wild type Actin filaments. Our results for in-vitro and intracellular conditions show a significant influence of the voltage stimulus and biological environment on the electrical impulse shape, attenuation and propagation velocity. The filament is shown to sustain the ionic wave propagation at almost constant velocity for the in-vitro condition, whereas the intracellular condition displays a remarkable deceleration. As a unique feature, this multi-scale theory is able to account for molecular structure conformation (mutation) and biological environment changes (pH and ionic concentrations) often present in pathological conditions. Thus, it may be potentially useful in providing a molecular understanding for how and why age and inheritance conditions induce dysfunction and malformation in cytoskeleton filaments associated with a variety of neurological diseases. It is also applicable to other conducting biopolymers with relevance in biomedicine and biophysics and their potential nanotechnological applications.

Ate1-mediated posttranslational arginylation affects substrate adhesion and cell migration in Dictyostelium discoideum.

The highly conserved enzyme arginyl-tRNA-protein transferase (Ate1) mediates arginylation, a posttranslational modification that is only incompletely understood at its molecular level. To investigate whether arginylation affects actin-dependent processes in a simple model organism, Dictyostelium discoideum, we knocked out the gene encoding Ate1 and characterized the phenotype of ate1-null cells. Visualization of actin cytoskeleton dynamics by live-cell microscopy indicated significant changes in comparison to wild-type cells. Ate1-null cells were almost completely lacking focal actin adhesion sites at the substrate-attached surface and were only weakly adhesive. In two-dimensional chemotaxis assays toward folate or cAMP, the motility of ate1-null cells was increased. However, in three-dimensional chemotaxis involving more confined conditions, the motility of ate1-null cells was significantly reduced. Live-cell imaging showed that GFP-tagged Ate1 rapidly relocates to sites of newly formed actin-rich protrusions. By mass spectrometric analysis, we identified four arginylation sites in the most abundant actin isoform of Dictyostelium, in addition to arginylation sites in other actin isoforms and several actin-binding proteins. In vitro polymerization assays with actin purified from ate1-null cells revealed a diminished polymerization capacity in comparison to wild-type actin. Our data indicate that arginylation plays a crucial role in the regulation of cytoskeletal activities.

The ACTC M305L Hypertrophic Cardiomyopathy Mutation Results in Hypercontractility and Impaired Relaxation of Drosophila Muscles

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle characterized by ventricular wall thickening, hyperdynamic contractile properties, and impaired relaxation. It is commonly caused by mutations in sarcomeric proteins. The alpha-cardiac actin ACTC M305L HCM mutation is located near the nucleotide-binding site and residues predicted to help confine tropomyosin to an inhibitory position along thin filaments. We generated several Drosophila models of the disorder to evaluate the effects of the lesion from the organ through molecular level. In all muscles tested, phenotypic disturbances correlated with mutant protein load. For example, relative to wildtype actin, when highly overexpressed in the fly heart, the variant significantly reduced cardiac output, prolonged systole, and restricted diastolic volumes. Preliminary force measurements from jump muscle fibers corroborated a dose-dependent, M305L actin-induced pathological effect. Moreover, we observed greater incremental decreases in flight ability with increasing amounts of mutant versus wildtype actin expression in indirect flight muscles (IFM). Flight was abolished when the mutant was highly overexpressed and destructive myosin-induced hypercontraction was evident, suggesting disrupted contractile regulation and poorly inhibited actomyosin associations. However, regulated in vitro motility parameters, including Vmax, nH, and pCa50, of thin filaments reconstituted from M305L or wildtype IFM actin and bovine cardiac troponin-tropomyosin, were indistinguishable. Likewise, no overt differences in computationally-derived electrostatic interaction energy landscapes between M305L and wildtype actin-tropomyosin filaments were observed, suggesting unperturbed tropomyosin-positioning in the absence of troponin. Nonetheless, over sparsely populated beds of myosin, a significantly higher percentage of troponin-free M305L versus wildtype actin-tropomyosin filaments were motile, implying tropomyosin is less effective at blocking actomyosin associations on mutant filaments. Mispositioning of tropomyosin may contribute to the supranormal contraction and compromised relaxation observed in our in vivo models and, potentially, in human patients.

A multi-scale approach to describe electrical impulses propagating along actin filaments in both intracellular and in vitro conditions.

An accurate and efficient characterization of the polyelectrolyte properties for cytoskeleton filaments are key to the molecular understanding of electrical signal propagation, bundle and network formation, as well as their potential nanotechnological applications. In this article, we introduce an innovative multi-scale approach able to account for the atomistic details of a protein molecular structure, its biological environment, and their impact on electrical impulses propagating along wild type actin filaments. The formulation includes non-trivial contributions to the ionic electrical conductivity and capacitance coming from the diffuse part of the electrical double layer of G-actins. We utilize this monomer characterization in a non-linear inhomogeneous transmission line prototype model to account for the monomer–monomer interactions, dissipation and damping perturbations along the filament length. A novel, simple, accurate, approximate analytic expression has been obtained for the transmission line model. Our results reveal the propagation of electrical signal impulses in the form of solitons for the range of voltage stimulus and electrolyte solutions typically present for intracellular and in vitro conditions. The approach predicts a lower electrical conductivity with higher linear capacitance and non-linear accumulation of charge for intracellular conditions. Our results show a significant influence of the voltage input on the electrical impulse shape, attenuation and kern propagation velocity. The filament is able to sustain the soliton propagation at almost constant kern velocity for the in vitro condition, whereas the intracellular condition displays a remarkable deceleration. Additionally, the solitons are narrower and travel faster at higher voltage input. As a unique feature, this multi-scale theory is able to account for molecular structure conformation (mutation) and biological environment (protonations/deprotonations) changes often present in pathological conditions. It is also applicable to other highly charged rod-like polyelectrolytes with relevance in biomedicine and biophysics.

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

Pseudo-acetylation of K326 and K328 of actin disrupts Drosophila melanogaster indirect flight muscle structure and performance.

In striated muscle tropomyosin (Tm) extends along the length of F-actin-containing thin filaments. Its location governs access of myosin binding sites on actin and, hence, force production. Intermolecular electrostatic associations are believed to mediate critical interactions between the proteins. For example, actin residues K326, K328, and R147 were predicted to establish contacts with E181 of Tm. Moreover, K328 also potentially forms direct interactions with E286 of myosin when the motor is strongly bound. Recently, LC-MS/MS analysis of the cardiac acetyl-lysine proteome revealed K326 and K328 of actin were acetylated, a post-translational modification (PTM) that masks the residues' inherent positive charges. Here, we tested the hypothesis that by removing the vital actin charges at residues 326 and 328, the PTM would perturb Tm positioning and/or strong myosin binding as manifested by altered skeletal muscle function and structure in the Drosophila melanogaster model system. Transgenic flies were created that permit tissue-specific expression of K326Q, K328Q, or K326Q/K328Q acetyl-mimetic actin and of wild-type actin via the UAS-GAL4 bipartite expression system. Compared to wild-type actin, muscle-restricted expression of mutant actin had a dose-dependent effect on flight ability. Moreover, excessive K328Q and K326Q/K328Q actin overexpression induced indirect flight muscle degeneration, a phenotype consistent with hypercontraction observed in other Drosophila myofibrillar mutants. Based on F-actin-Tm and F-actin-Tm-myosin models and on our physiological data, we conclude that acetylating K326 and K328 of actin alters electrostatic associations with Tm and/or myosin and thereby augments contractile properties. Our findings highlight the utility of Drosophila as a model that permits efficient targeted design and assessment of molecular and tissue-specific responses to muscle protein modifications, in vivo.

G.O.3: Severe congenital actin related myofibrillar myopathy

Mutations in ACTA1 have been associated with nemaline myopathy, intranuclear rod myopathy, actin myopathy, cap myopathy, and congenital fiber type disproportion. Myofibrillar myopathies (MFM) are morphologically distinct but genetically heterogeneous muscular dystrophies arising from mutations in Z-disk related proteins. A 26-month-old boy was hypotonic and weak at birth, had significantly delayed motor development, and was mechanically ventilated and tube-fed since birth. At age 26 months he had cup-shaped low-set ears, a high-arched palate, contractures of the metacarpophalangeal and proximal interphalangeal joints, and facial diplegia with diffuse muscle weakness and reduced tendon reflexes, but had good head control and could sit with support. The EMG showed prominent fibrillation potentials and myopathic motor unit potentials. The muscle biopsy revealed abnormal variation in fiber size, fiber splitting, increased internal nuclei, vacuolar change, myofibrillar disorganization, hyaline structures, focal decreases of oxidative enzyme activity as well as scattered necrotic and regenerating fibers with marked increase of endomysial and perimysial connective tissue. Many structurally abnormal fibers displayed ectopic or abnormal expression of desmin, alphaB-crystallin, myotilin, dystrophin, and NCAM. Whole exome sequencing demonstrated a previously published in-frame insertion of two amino acids in ACTA1 . The mutant actin was expressed at ∼11% of wild-type in COS7 and C2 cells. C2 cells expressing mutant actin displayed cytoplasmic actin aggregates of different sizes whereas cells expressing wild-type actin displayed mostly filamentous actin. We conclude that (1) mutations in ACTA1 can cause MFM pathology; (2) MFM can be present at birth; (3) mutations in ACTA1 should be considered in patients with severe congenital hypotonia associated with muscle weakness and MFM pathology.

The ACTC A295S Mutation Increases Heart and Skeletal Muscle Contractile Activity in Drosophila Models of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease characterized by thickened heart walls, cardiac arrhythmias, myocyte disarray and interstitial fibrosis. Over 10 mutations in alpha-cardiac actin (ACTC) are associated with HCM. The ACTC Ala295Ser substitution causes a highly penetrant disease with diverse phenotypes. To investigate the in vivo consequences of the ACTC mutation we generated Drosophila lines with muscle-specific expression of A295S actin. Our unique models limit genetic diversity and pathological complexity to help resolve molecular mechanisms of diseases. For expression in the fly heart we used UAS-Act57BWT and UAS-Act57BA295S actin transgenes in conjunction with the HandGal4 cardiac-specific driver. Confocal microscopy confirmed heart-restricted expression of UAS-Act57BWTGFP and UAS-Act57BA295SGFP and co-polymerization of transgenic and endogenous actin. High-speed video microscopy and motion analysis revealed A295S actin expression significantly reduced diastolic volumes and increased systolic intervals consistent with elevated contractile properties at rest and during contraction. For indirect flight muscle (IFM)-specific actin expression, we used the IFM targeted Act88FWTand Act88FA295S transgenes, in an IFM actin null background. Muscle mechanics on heterozygous IFM fibers revealed that the A295S actin mutation increased power, the frequency of maximum power generation (fmax), and stiffness relative to control fibers. The higher fmax and stiffness are likely causes of an observed increase in wing beat frequency (WBF) of Act88FA295Sheterozygous flies. The WBF increase had a negative impact on flight ability which decreased further as mutant: wildtype actin ratios were increased. Overall, enhanced contractile activity of actin HCM cardiac myocytes and IFM fibers is consistent with the hypercontractile properties frequently associated with HCM mutations.

Bulkiness or aromatic nature of tyrosine-143 of actin is important for the weak binding between F-actin and myosin-ADP-phosphate

Actin filaments (F-actin) interact with myosin and activate its ATPase to support force generation. By comparing crystal structures of G-actin and the quasi-atomic model of F-actin based on high-resolution cryo-electron microscopy, the tyrosine-143 was found to be exposed more than 60Å2 to the solvent in F-actin. Because tyrosine-143 flanks the hydrophobic cleft near the hydrophobic helix that binds to myosin, the mutant actins, of which the tyrosine-143 was replaced with tryptophan, phenylalanine, or isoleucine, were generated using the Dictyostelium expression system. It polymerized significantly poorly when induced by NaCl, but almost normally by KCl. In the presence of phalloidin and KCl, the extents of the polymerization of all the mutant actins were comparable to that of the wild-type actin so that the actin-activated myosin ATPase activity could be reliably compared. The affinity of skeletal heavy meromyosin to F-actin and the maximum ATPase activity (Vmax) were estimated by a double reciprocal plot. The Tyr143Trp-actin showed the higher affinity (smaller Kapp) than that of the wild-type actin, with the Vmax being almost unchanged. The Kapp and Vmax of the Tyr143Phe-actin were similar to those of the wild-type actin. However, the activation by Tyr143Ile-actin was much smaller than the wild-type actin and the accurate determination of Kapp was difficult. Comparison of the myosin ATPase activated by the various mutant actins at the same concentration of F-actin showed that the extent of activation correlates well with the solvent-accessible surface areas (ASA) of the replaced amino acid molecule. Because 1/Kapp reflects the affinity of F-actin for the myosin–ADP-phosphate intermediate (M.ADP.Pi) through the weak binding, these data suggest that the bulkiness or the aromatic nature of the tyrosin-143 is important for the initial binding of the M.ADP.Pi intermediate with F-actin but not for later processes such as the phosphate release.

Analysis of nuclear actin by overexpression of wild-type and actin mutant proteins

Compared to the cytoplasmic F-actin abundance in cells, nuclear F-actin levels are generally quite low. However, nuclear actin is present in certain cell types including oocytes and under certain cellular conditions including stress or serum stimulation. Currently, the architecture and polymerization status of nuclear actin networks has not been analyzed in great detail. In this study, we investigated the architecture and functions of such nuclear actin networks. We generated nuclear actin polymers by overexpression of actin proteins fused to a nuclear localization signal (NLS). Raising nuclear abundance of a NLS wild-type actin, we observed phalloidin- and LifeAct-positive actin bundles forming a nuclear cytoskeletal network consisting of curved F-actin. In contrast, a polymer-stabilizing actin mutant (NLS-G15S-actin) deficient in interacting with the actin-binding protein cofilin generated a nuclear actin network reminiscent of straight stress fiber-like microfilaments in the cytoplasm. We provide a first electron microscopic description of such nuclear actin polymers suggesting bundling of actin filaments. Employing different cell types from various species including neurons, we show that the morphology of and potential to generate nuclear actin are conserved. Finally, we demonstrate that nuclear actin affects cell function including morphology, serum response factor-mediated gene expression, and herpes simplex virus infection. Our data suggest that actin is able to form filamentous structures inside the nucleus, which share architectural and functional similarities with the cytoplasmic F-actin.

A tumorigenic actin mutant alters fibroblast morphology and multicellular assembly properties

Tumor initiation and progression are accompanied by complex changes in the cytoarchitecture that at the cellular level involve remodeling of the cytoskeleton. We report on the impact of a mutant β-actin (G245D-actin) on cell structure and multicellular assembly properties. To appreciate the effects of the Gly245Asp substitution on the organization of the actin cytoskeleton, we examined the polymerization properties of G245D-actin in vitro by pyrene polymerization assays and total internal reflection fluorescence microscopy (TIRF). The mutant actin on its own has a significantly reduced polymerization efficiency compared to native actin but also modifies the polymerization of actin in copolymerization experiments. Comparison of the structure of Rat-2 fibroblasts and a stably transfected derivate called Rat-2-sm9 revealed the effects of G245D-actin in a cellular environment. The overall actin levels in Rat-2-sm9 show a 1.6-fold increase with similar amounts of mutant and wild-type actin. G245D-actin expression renders Rat-2-sm9 cells highly tumorigenic in nude mice. In Rat-2-sm9 monolayers, G245D-actin triggers the formation of extensive membrane ruffles, which is a characteristic feature of many transformed cells. To approximate complex cell-cell and cell-matrix interactions that occur in tumors and might modulate the effects of G245D-actin, we extended our studies to scaffold-free 3D spheroid cultures. Bright field and scanning electron microscopy (SEM) show that Rat-2-sm9 and Rat-2 cells share essential features of spheroid formation and compaction. However, the resulting spheroids exhibit distinct phenotypes that differ mainly in surface structure and size. The systematic comparison of transformed and normal spheroids by SEM provides new insights into scaffold-free fibroblast spheroid formation.

Rapid Nucleotide Exchange Renders Asp-11 Mutant Actins Resistant to Depolymerizing Activity of Cofilin, Leading to Dominant Toxicity in Vivo

Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.

Covering the Cover

Covering the Cover

Thoracic Aortic Aneurysm (TAAD)-causing Mutation in Actin Affects Formin Regulation of Polymerization

More than 30 mutations in ACTA2, which encodes α-smooth muscle actin, have been identified to cause autosomal dominant thoracic aortic aneurysm and dissection. The mutation R256H is of particular interest because it also causes patent ductus arteriosus and moyamoya disease. R256H is one of the more prevalent mutations and, based on its molecular location near the strand-strand interface in the actin filament, may affect F-actin stability. To understand the molecular ramifications of the R256H mutation, we generated Saccharomyces cerevisiae yeast cells expressing only R256H yeast actin as a model system. These cells displayed abnormal cytoskeletal morphology and increased sensitivity to latrunculin A. After cable disassembly induced by transient exposure to latrunculin A, mutant cells were delayed in reestablishing the actin cytoskeleton. In vitro, mutant actin exhibited a higher than normal critical concentration and a delayed nucleation. Consequently, we investigated regulation of mutant actin by formin, a potent facilitator of nucleation and a protein needed for normal vascular smooth muscle cell development. Mutant actin polymerization was inhibited by the FH1-FH2 fragment of the yeast formin, Bni1. This fragment strongly capped the filament rather than facilitating polymerization. Interestingly, phalloidin or the presence of wild type actin reversed the strong capping behavior of Bni1. Together, the data suggest that the R256H actin mutation alters filament conformation resulting in filament instability and misregulation by formin. These biochemical effects may contribute to abnormal histology identified in diseased arterial samples from affected patients.

G146V Mutation at the Hinge Region of Actin Reveals a Myosin Class-specific Requirement of Actin Conformations for Motility

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac β-myosin ATPase to only 50% of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

Does Uncoupling of Troponin I Phosphorylation from Changes in Myofibrillar Ca2+-Sensitivity Play a Role in the Pathogenesis of Cardiomyopathy?

In normal heart, troponin I (TnI) phosphorylation alters myofibrillar Ca2+-sensitivity and increases the speed of Ca2+-dissociation (lusitropic effect). We have discovered several cases in human heart muscle (DCM mutations, HCM mutations, septal myectomy) where Ca2+-sensitivity is independent of TnI phosphorylation.The ACTC E361G DCM mutation had no effect upon Ca2+-sensitivity at all when tested in thin filaments containing donor heart troponin. However if troponin was dephosphorylated, the wild-type actin containing thin filaments had a higher Ca2+-sensitivity than ACTC E361G containing thin filaments. Thus, Ca2+-sensitivity is uncoupled from TnI phosphorylation. This was also observed with another 6 DCM mutations (TnT R141W, TnI K36Q, TnC G159D and Tm E40K and E54K).The ACTC E99K HCM mutation causes an increase in Ca2+-sensitivity, but uncoupling also occurs, as there is no difference in the Ca2+-sensitivity of ACTC E99K containing thin filaments with phosphorylated and dephosphorylated troponin. The uncoupling must be a direct effect of the mutation, which influences TnI properties allosterically.We have been studying myectomy samples as a source of human HCM tissue. The Ca2+-sensitivity of thin filaments with HOCM troponin was the same as donor heart troponin even though the TnI phosphorylation levels were very different. This uncoupling is independent of the mutation causing HCM so one of the troponin subunits is functionally abnormal. When HOCM troponin was exchanged with recombinant TnT there was a 1.9-fold higher Ca2+-sensitivity as was expected with the low level of phosphorylation. Therefore a change in TnT is responsible for the observed disruption of the Ca2+-sensitivity-phosphorylation relationship in human myectomy samples.Thus, the ‘uncoupling’ phenomenon seems to be a widespread consequence of cardiomyopathy and plays a key role in the pathogenesis of familial cardiomyopathy.