Mutant Actin Research Articles

Cryo-EM structures of cardiac muscle α-actin mutants M305L and A331P give insights into the structural mechanisms of hypertrophic cardiomyopathy

Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.

Actin cytoskeletal regulation of ciliogenesis in development and disease.

Primary cilia are antenna-like sensory organelles that are evolutionarily conserved in nearly all modern eukaryotes, from the single-celled green alga, Chlamydomonas reinhardtii, to vertebrates and mammals. Cilia are microtubule-based cellular projections that have adapted to perform a broad range of species-specific functions, from cell motility to detection of light and the transduction of extracellular mechanical and chemical signals. These functions render cilia essential for organismal development and survival. The high conservation of cilia has allowed for discoveries in C. reinhardtii to inform our understanding of the basic biology of mammalian primary cilia, and to provide insight into the genetic etiology of ciliopathies. Over the last two decades, a growing number of studies has revealed that multiple aspects of ciliary homeostasis are regulated by the actin cytoskeleton, including centrosome migration and positioning, vesicle transport to the basal body, ectocytosis, and ciliary-mediated signaling. Here, we review actin regulation of ciliary homeostasis, and highlight conserved and divergent mechanisms in C. reinhardtii and mammalian cells. Further, we compare the disease manifestations of patients with ciliopathies to those with mutations in actin and actin-associated genes, and propose that primary cilia defects caused by genetic alteration of the actin cytoskeleton may underlie certain birth defects.

Skeletal muscle actin mutation R256H causes cardiac hypocontractility by disrupting multiple actin functions demonstrated through multi-scale analysis

Skeletal muscle actin mutation R256H causes cardiac hypocontractility by disrupting multiple actin functions demonstrated through multi-scale analysis

A recurrent ACTA1 amino acid change in mosaic form causes milder asymmetric myopathy

We describe three patients with asymmetric congenital myopathy without definite nemaline bodies and one patient with severe nemaline myopathy. In all four patients, the phenotype had been caused by pathogenic missense variants in ACTA1 leading to the same amino acid change, p.(Gly247Arg). The three patients with milder myopathy were mosaic for their variants. In contrast, in the severely affected patient, the missense variant was present in a de novo, constitutional form. The grade of mosaicism in the three mosaic patients ranged between 20 % and 40 %. We speculate that the milder clinical and histological manifestations of the same ACTA1 variant in the patients with mosaicism reflect the lower abundance of mutant actin in their muscle tissue. Similarly, the asymmetry of body growth and muscle weakness may be a consequence of the affected cells being unevenly distributed. The partial improvement in muscle strength with age in patients with mosaicism might be due to an increased proportion over time of nuclei carrying and expressing two normal alleles.

Purification of modified mammalian actin isoforms for in vitro reconstitution assays

In vitro reconstitution assays using purified actin have greatly improved our understanding of cytoskeletal dynamics and their regulation by actin-binding proteins. However, early purification methods consisted of harsh conditions to obtain pure actin and often did not include correct maturation and obligate modification of the isolated actin monomers. Novel insights into the folding requirements and N-terminal processing of actin as well as a better understanding of the interaction of actin with monomer sequestering proteins such as DNaseI, profilin and gelsolin, led to the development of more gentle approaches to obtain pure recombinant actin isoforms with known obligate modifications. This review summarizes the approaches that can be employed to isolate natively folded endogenous and recombinant actin from tissues and cells. We further emphasize the use and limitations of each method and describe how these methods can be implemented to study actin PTMs, disease-related actin mutations and novel actin-like proteins.

The role of Trp79 in β-actin on histidine methyltransferase SETD3 catalysis.

Nτ-methylation of His73 in actin by histidine methyltransferase SETD3 plays an important role in stabilisation of actin filaments in eukaryotes. Mutations in actin and overexpression of SETD3 have been related to human diseases, including cancer. Herein, we investigated the importance of Trp79 in β-actin on productive human SETD3 catalysis. Substitution of Trp79 in β-actin peptides by its chemically diverse analogues reveals that the hydrophobic Trp79 binding pocket modulates the catalytic activity of SETD3, and that retaining a bulky and hydrophobic amino acid at the position 79 is important for efficient His73 methylation by SETD3. Molecular dynamics simulations show that the Trp79 binding pocket of SETD3 is ideally shaped to accommodate large and hydrophobic Trp79, contributing to the favourable release of water molecules upon binding. Our results demonstrate that the distant Trp79 binding site plays an important role in efficient SETD3 catalysis, contributing to the identification of new SETD3 substrates and development of chemical probes targeting the biomedically important SETD3.

Abstract EC.06: Skeletal Muscle Actin Mutation R256H Disrupts Actin Polymerization And Cardiomyocyte Contractility: A Multi-scale Study Of Genetic Dilated Cardiomyopathy

Actin is conserved in all eukaryotic cells with six isoforms in humans. Skeletal muscle actin (ACTA1) is the primarily expressed isoform in skeletal muscle; however, it is also expressed at low levels in the heart. ACTA1 mutations are well known to cause skeletal myopathy, but mechanistic insights are incomplete due to difficulties with generating recombinant protein and limited in vivo modeling. The impact of ACTA1 mutations on cardiac function is controversial. We previously reported a novel missense heterozygous mutation in ACTA1, R256H, associated with dilated cardiomyopathy (DCM) without overt skeletal myopathy in patients. The objective of this study was to define the mechanistic basis by which ACTA1 R256H contributes to DCM. To establish the pathogenicity of ACTA1 R256H, we generated Acta1 R256H mice. Acta1 R256H/R256H mice display perinatal mortality. We also observed reduced survival and gross phenotypes in Acta1 R256H/+ mice over time. To further characterize ACTA1 R256H in human cardiomyocytes, we generated ACTA1 R256H/+ human induced pluripotent stem cells which we differentiated into cardiomyocytes. ACTA1 R256H/+ cardiomyocytes generated less force and displayed aberrant sarcomere structure compared to isogenic controls. To biochemically define the consequences of ACTA1 R256H protein, we developed a novel method to generate recombinant wildtype ACTA1 and ACTA1 R256H. Strikingly, ACTA1 R256H demonstrated severely reduced polymerization kinetics possibly accounting for the observed disruption in sarcomere structure. This finding was further supported by molecular dynamic simulations which identified impaired nucleotide binding in ACTA1 R256H compared to wildtype ACTA1 protein. Together, these findings reveal that a mutation in skeletal muscle actin results in increased mortality, cardiomyocyte hypocontractility, sarcomeric disruption, and impaired actin polymerization. Our work defines a cardiac function for skeletal muscle actin and establishes a multiscale approach to studying cardiomyopathy-associated actin mutations.

The intracellular and plasma membrane pools of phosphatidylinositol-4-monophosphate control megakaryocyte maturation and proplatelet formation

BackgroundMegakaryocytes (MKs) develop from hematopoietic stem cells after stimulation by the cytokine thrombopoietin. During megakaryopoiesis, MKs enlarge, undergo the process of endomitosis, and develop intracellular membranes (demarcation membrane system, DMS). During DMS formation, there is active transport of proteins, lipids, and membranes from the Golgi apparatus to the DMS. The most important phosphoinositide that controls anterograde transport from the Golgi apparatus to the plasma membrane (PM) is phosphatidylinositol-4-monophosphate (PI4P), whose levels are controlled by suppressor of actin mutations 1–like protein (Sac1) phosphatase at the Golgi and endoplasmic reticulum. ObjectivesHere we investigated the role of Sac1 and PI4P in megakaryopoiesis. MethodsWe analyzed Sac1 and PI4P localization in primary MKs derived from fetal liver or bone marrow and in the DAMI cell line by immunofluorescence. The intracellular and PM pools of PI4P in primary MKs were modulated by expression of Sac1 constructs from retroviral vector and inhibition of PI4 kinase IIIα, respectively. ResultsWe showed that in primary mouse MKs, PI4P is mostly found in the Golgi apparatus and the PM in immature MKs, while in mature MKs, it is found in the cell periphery and at the PM. The exogenous expression of wild-type but not C389S mutant (catalytically dead) Sac1 results in the perinuclear retention of the Golgi apparatus resembling immature MKs, with decreased ability to form proplatelets. The pharmacologic inhibition of PI4P production specifically at the PM also resulted in a significant decrease in MKs that form proplatelets. ConclusionThese results indicate that both intracellular and PM pools of PI4P mediate MK maturation and proplatelet formation.

Mutagenic analysis of actin reveals the mechanism of His161 flipping that triggers ATP hydrolysis.

The dynamic assembly of actin is controlled by the hydrolysis of ATP, bound to the center of the molecule. Upon polymerization, actin undergoes a conformational change from the monomeric G-form to the fibrous F-form, which is associated with the flipping of the side chain of His161 toward ATP. His161 flipping from the gauche-minus to gauche-plus conformation leads to a rearrangement of the active site water molecules, including ATP attacking water (W1), into an orientation capable of hydrolysis. We previously showed that by using a human cardiac muscle α-actin expression system, mutations in the Pro-rich loop residues (A108G and P109A) and in a residue that was hydrogen-bonded to W1 (Q137A) affect the rate of polymerization and ATP hydrolysis. Here, we report the crystal structures of the three mutant actins bound to AMPPNP or ADP-Pi determined at a resolution of 1.35-1.55Å, which are stabilized in the F-form conformation with the aid of the fragmin F1 domain. In A108G, His161 remained non-flipped despite the global actin conformation adopting the F-form, demonstrating that the side chain of His161 is flipped to avoid a steric clash with the methyl group of A108. Because of the non-flipped His161, W1 was located away from ATP, similar to G-actin, which was accompanied by incomplete hydrolysis. In P109A, the absence of the bulky proline ring allowed His161 to be positioned near the Pro-rich loop, with a minor influence on ATPase activity. In Q137A, two water molecules replaced the side-chain oxygen and nitrogen of Gln137 almost exactly at their positions; consequently, the active site structure, including the W1 position, is essentially conserved. This seemingly contradictory observation to the reported low ATPase activity of the Q137A filament could be attributed to a high fluctuation of the active site water. Together, our results suggest that the elaborate structural design of the active site residues ensures the precise control of the ATPase activity of actin.

Integrating comparative modeling and accelerated simulations reveals conformational and energetic basis of actomyosin force generation

Muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin. Direct characterization of how small changes in the myosin-actin complex impact its force production remains challenging. Molecular dynamics (MD) simulations, although capable of studying protein structure-function relationships, are limited owing to the slow timescale of the myosin cycle as well as a lack of various intermediate structures for the actomyosin complex. Here, employing comparative modeling and enhanced sampling MD simulations, we show how the human cardiac myosin generates force during the mechanochemical cycle. Initial conformational ensembles for different myosin-actin states are learned from multiple structural templates with Rosetta. This enables us to efficiently sample the energy landscape of the system using Gaussian accelerated MD. Key myosin loop residues, whose substitutions are related to cardiomyopathy, are identified to form stable or metastable interactions with the actin surface. We find that the actin-binding cleft closure is allosterically coupled to the myosin motor core transitions and ATP-hydrolysis product release from the active site. Furthermore, a gate between switch I and switch II is suggested to control phosphate release at the prepowerstroke state. Our approach demonstrates the ability to link sequence and structural information to motor functions.

ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy

Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.

A solution to the long-standing problem of actin expression and purification

Actin is the most abundant protein in the cytoplasm of eukaryotic cells and interacts with hundreds of proteins to perform essential functions, including cell motility and cytokinesis. Numerous diseases are caused by mutations in actin, but studying the biochemistry of actin mutants is difficult without a reliable method to obtain recombinant actin. Moreover, biochemical studies have typically used tissue-purified α-actin, whereas humans express six isoforms that are nearly identical but perform specialized functions and are difficult to obtain in isolation from natural sources. Here, we describe a solution to the problem of actin expression and purification. We obtain high yields of actin isoforms in human Expi293F cells. Experiments along the multistep purification protocol demonstrate the removal of endogenous actin and the functional integrity of recombinant actin isoforms. Proteomics analysis of endogenous vs. recombinant actin isoforms confirms the presence of native posttranslational modifications, including N-terminal acetylation achieved after affinity-tag removal using the actin-specific enzyme Naa80. The method described facilitates studies of actin under fully native conditions to determine differences among isoforms and the effects of disease-causing mutations that occur in all six isoforms.

Abstract P1059: Establishing The Mechanistic Basis Of Dilated Cardiomyopathy Associated With The Skeletal Muscle Actin Mutation R256H

Actin, which is expressed in all eukaryotic cells, is an essential component of the cardiac and skeletal muscle sarcomere thin filament. Mutations in actin have been associated with a wide range of diseases, including skeletal myopathies and cardiomyopathies. Humans express six actin isoforms with two that are thought to be highly enriched in sarcomeres and differ by only four amino acids: skeletal muscle actin (ACTA1) and cardiac actin (ACTC1). While ACTC1 is exclusively expressed in the adult heart, ACTA1 is predominantly expressed in skeletal muscle with lower levels detected in cardiac muscle. As such, ACTA1 mutations are well known to cause skeletal myopathy with minimal effects on cardiac function. However, I previously reported a family carrying a novel heterozygous mutation in ACTA1 (ACTA1 R256H) with dilated cardiomyopathy (DCM) and no clinical evidence of skeletal myopathy. The objective of this study is to elucidate the molecular mechanism(s) by which ACTA1 R256H may cause heart failure. I generated ACTA1 R256H/+ human pluripotent stem cells (hPSCs) using Cas9/CRISPR gene editing. Using traction force microscopy, I observed that ACTA1 R256H/+ hPSC-derived cardiomyocytes display reduced contractility. To define relevant mechanisms, I devised a novel approach to purify milligram quantities of recombinant human ACTA1, which has served as a remarkable challenge for the field. Importantly, this purification technique circumvents contamination from endogenous actins and produces functional protein capable of polymerization. Moreover, in vitro motility assays demonstrated that reconstituted human thin filaments containing recombinant ACTA1 are functional and activated in a calcium-regulated manner. Intriguingly, purified ACTA1 R256H appears to have a polymerization defect, suggesting a possible mechanism by which this mutation leads to a defect in contractility. Together, these studies suggest for the first time that a mutation in skeletal muscle actin, ACTA1 R256H, causes cardiomyocyte hypocontractility while also suggesting a potential molecular mechanism. Finally, this study also introduces a new purification method essential for biochemical and biophysical analysis of human ACTA1 mutations.

Implications of the Actin Cytoskeleton on the Multi-Step Process of [PSI+] Prion Formation.

Yeast prions are self-perpetuating misfolded proteins that are infectious. In yeast, [PSI+] is the prion form of the Sup35 protein. While the study of [PSI+] has revealed important cellular mechanisms that contribute to prion propagation, the underlying cellular factors that influence prion formation are not well understood. Prion formation has been described as a multi-step process involving both the initial nucleation and growth of aggregates, followed by the subsequent transmission of prion particles to daughter cells. Prior evidence suggests that actin plays a role in this multi-step process, but actin’s precise role is unclear. Here, we investigate how actin influences the cell’s ability to manage newly formed visible aggregates and how actin influences the transmission of newly formed aggregates to future generations. At early steps, using 3D time-lapse microscopy, several actin mutants, and Markov modeling, we find that the movement of newly formed aggregates is random and actin independent. At later steps, our prion induction studies provide evidence that the transmission of newly formed prion particles to daughter cells is limited by the actin cytoskeletal network. We suspect that this limitation is because actin is used to possibly retain prion particles in the mother cell.

Actin isovariant ACT7 controls root meristem development in Arabidopsis through modulating auxin and ethylene responses.

The meristem is the most functionally dynamic part in a plant. The shaping of the meristem requires constant cell division and elongation, which are influenced by hormones and the cytoskeletal component, actin. Although the roles of hormones in modulating meristem development have been extensively studied, the role of actin in this process is still elusive. Using the single and double mutants of the vegetative class actin, we demonstrate that actin isovariant ACT7 plays an important role in root meristem development. In the absence of ACT7, but not ACT8 and ACT2, depolymerization of actin was observed. Consistently, the act7 mutant showed reduced cell division, cell elongation, and meristem length. Intracellular distribution and trafficking of auxin transport proteins in the actin mutants revealed that ACT7 specifically functions in the root meristem to facilitate the trafficking of auxin efflux carriers PIN1 and PIN2, and consequently the transport of auxin. Compared with act7, the act7act8 double mutant exhibited slightly enhanced phenotypic response and altered intracellular trafficking. The altered distribution of auxin in act7 and act7act8 affects the response of the roots to ethylene, but not to cytokinin. Collectively, our results suggest that ACT7-dependent auxin-ethylene response plays a key role in controlling Arabidopsis root meristem development.

Cyclic-AMP Increases Nuclear Actin Monomer Which Promotes Proteasomal Degradation of RelA/p65 Leading to Anti-Inflammatory Effects.

The second messenger, cAMP has potent immunosuppressive and anti-inflammatory actions. These have been attributed, in part, to the ability of cAMP-induced signals to interfere with the function of the proinflammatory transcription factor Nuclear Factor-kappa B (NF-κB). However, the mechanisms underlying the modulation of NF-κB activity by cAMP remain unclear. Here we demonstrate an important role for cAMP-mediated increase in nuclear actin monomer levels in inhibiting NF-κB activity. Elevated cAMP or forced expression of a nuclear localised polymerisation defective actin mutant (NLS-ActinR62D) inhibited basal and TNFα induced mRNA levels of NF-κB-dependent genes and NF-κB-dependent reporter gene activity. Elevated cAMP or NLS-ActinR62D did not affect NF-κB nuclear translocation but did reduce total cellular and nuclear RelA/p65 levels. Preventing the cAMP-induced increase in nuclear actin monomer, either by expressing a nuclear localised active mutant of the actin polymerising protein mDIA, silencing components of the nuclear actin import complex IPO9 and CFL1 or overexpressing the nuclear export complex XPO6, rescued RelA/p65 levels and NF-κB reporter gene activity in forskolin-stimulated cells. Elevated cAMP or NLS-ActinR62D reduced the half-life of RelA/p65, which was reversed by the proteasome inhibitor MG132. Accordingly, forskolin stimulated association of RelA/p65 with ubiquitin affinity beads, indicating increased ubiquitination of RelA/p65 or associated proteins. Taken together, our data demonstrate a novel mechanism underlying the anti-inflammatory effects of cAMP and highlight the important role played by nuclear actin in the regulation of inflammation.

Functional Characterization of Cardiac Actin Mutants Causing Hypertrophic (p.A295S) and Dilated Cardiomyopathy (p.R312H and p.E361G).

Human wild type (wt) cardiac α-actin and its mutants p.A295S or p.R312H and p.E361G correlated with hypertrophic or dilated cardiomyopathy, respectively, were expressed by using the baculovirus/Sf21 insect cell system. The c-actin variants inhibited DNase I, indicating maintenance of their native state. Electron microscopy showed the formation of normal appearing actin filaments though they showed mutant specific differences in length and straightness correlating with their polymerization rates. TRITC-phalloidin staining showed that p.A295S and p.R312H exhibited reduced and the p.E361G mutant increased lengths of their formed filaments. Decoration of c-actins with cardiac tropomyosin (cTm) and troponin (cTn) conveyed Ca2+-sensitivity of the myosin-S1 ATPase stimulation, which was higher for the HCM p.A295S mutant and lower for the DCM p.R312H and p.E361G mutants than for wt c-actin. The lower Ca2+-sensitivity of myosin-S1 stimulation by both DCM actin mutants was corrected by the addition of levosimendan. Ca2+-dependency of the movement of pyrene-labeled cTm along polymerized c-actin variants decorated with cTn corresponded to the relations observed for the myosin-S1 ATPase stimulation though shifted to lower Ca2+-concentrations. The N-terminal C0C2 domain of cardiac myosin-binding protein-C increased the Ca2+-sensitivity of the pyrene-cTM movement of bovine, recombinant wt, p.A295S, and p.E361G c-actins, but not of the p.R312H mutant, suggesting decreased affinity to cTm.

Bound nucleotide can control the dynamic architecture of monomeric actin

Polymerization of actin into cytoskeletal filaments is coupled to its bound adenine nucleotides. The mechanism by which nucleotide modulates actin functions has not been evident from analyses of ATP- and ADP-bound crystal structures of the actin monomer. We report that NMR chemical shift differences between the two forms are globally distributed. Furthermore, microsecond–millisecond motions are spread throughout the molecule in the ATP form, but largely confined to subdomains 1 and 2, and the nucleotide binding site in the ADP form. Through these motions, the ATP- and ADP-bound forms sample different high-energy conformations. A deafness-causing, fast-nucleating actin mutant populates the high-energy conformer of ATP-actin more than the wild-type protein, suggesting that this conformer may be on the pathway to nucleation. Together, the data suggest a model in which differential sampling of a nucleation-compatible form of the actin monomer may contribute to control of actin filament dynamics by nucleotide.

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.

Mutation E334Q in ACTG1 leads to perturbed interactions with cofilin and cytoskeletal myosin isoforms

Mutation E334Q in γ-cytoskeletal actin causes symptoms diverging from previously described actinopathies with a distinct clinical phenotype. The patients suffer from a mild speech disorder, frontal dysgyria, and severe hypotonia in some cases. Wild–type γ–actin and p.E334Q were produced as actin–thymosin β4–His–tag fusion proteins using the Baculovirus Sf9-insect cell system. Purification via NiNTA-affinity chromatography and digestion with chymotrypsin yielded homogeneous wild–type and mutant actin with N–terminal acetylation and without any additional sequence modifications, as verified by MS/MS analysis.