Mutant Actin Research Articles

P6 - Redox Modification of the Cytoskeleton During Cell Adhesion and Migration

Actin cytoskeleton assembly and organization are modulated by reactive oxygen species and the cellular redox environment. Focal adhesions link the cytoskeleton to the extracellular matrix and play an important role in growth and migration of vascular smooth muscle cells. We have previously shown that NADPH oxidase Nox4, a H2O2-producing enzyme, and its binding partner, polymerase delta interacting protein 2 (Poldip2), regulate turnover of focal adhesions. Downstream targets of Nox4 include Hic-5 and Hsp27, as well as the small GTPase, Rho A. Activation of Smad signaling by TGFp, which is known to strengthen focal adhesions, is Nox4-dependent, as is upregulation of Hsp27 and Hic-5 by TGFp. Similarly, overexpression of Poldip2 activates RhoA to prevent focal adhesion dissolution, while dominant-negative RhoA reverses the effects of Nox4/Poldip2 on focal adhesions. More recently, we discovered that H2O2 levels increase during integrin-mediated cell attachment due to activation of NOX4. Attachment induces F-actin oxidation by sulfenylation, and depletion of Poldip2 or NOX4 using siRNA, or scavenging of endogenous H2O2 with catalase, inhibits adhesion-induced F-actin oxidation. Of importance, the binding of F-actin to vinculin, a protein present in focal adhesion complexes that regulates focal adhesion maturation, is inhibited after transfection of an oxidation-resistant actin mutant. Silencing of Poldip2 or NOX4 also impairs actin-vinculin interaction. Taken together, our work has identified a major role for Poldip2/Nox4 in cytoskeletal organization, focal adhesion turnover and smooth muscle cell migration.

Isogenic Pairs of hiPSC-CMs with Hypertrophic Cardiomyopathy/LVNC-Associated ACTC1 E99K Mutation Unveil Differential Functional Deficits.

SummaryHypertrophic cardiomyopathy (HCM) is a primary disorder of contractility in heart muscle. To gain mechanistic insight and guide pharmacological rescue, this study models HCM using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying the E99K-ACTC1 cardiac actin mutation. In both 3D engineered heart tissues and 2D monolayers, arrhythmogenesis was evident in all E99K-ACTC1 hiPSC-CMs. Aberrant phenotypes were most common in hiPSC-CMs produced from the heterozygote father. Unexpectedly, pathological phenotypes were less evident in E99K-expressing hiPSC-CMs from the two sons. Mechanistic insight from Ca2+ handling expression studies prompted pharmacological rescue experiments, wherein dual dantroline/ranolazine treatment was most effective. Our data are consistent with E99K mutant protein being a central cause of HCM but the three-way interaction between the primary genetic lesion, background (epi)genetics, and donor patient age may influence the pathogenic phenotype. This illustrates the value of isogenic hiPSC-CMs in genotype-phenotype correlations.

Production of Human β-Actin Using a Bacterial Expression System with a Cold Shock Vector.

Actin is one of the most abundant proteins in the cytoplasm of eukaryotic cells and plays important roles in a variety of cellular functions. However, it has been difficult to produce actin in substantial amounts using bacterial expression systems. In this article, a new method is described for the production of recombinant actin in bacterial cells. Human β-actin (His-tagged) can be expressed using a cold shock vector, pCold, in a bacterial expression system and then separated with a Ni-chelating resin, followed by a polymerization/depolymerization cycle or column chromatography with the Ni-chelating resin. The purified recombinant β-actin shows normal polymerization ability compared with commercially available β-actin purified from human platelets. This article also describes the preparation of mutant actin(G168R). This purified mutant exhibits impaired polymerization ability. The system and procedures described here will provide a useful method for the production of actin isoforms and their mutants. © 2018 by John Wiley & Sons, Inc.

The Molecular Mechanisms of Mutations in Actin and Myosin that Cause Inherited Myopathy.

The discovery that mutations in myosin and actin genes, together with mutations in the other components of the muscle sarcomere, are responsible for a range of inherited muscle diseases (myopathies) has revolutionized the study of muscle, converting it from a subject of basic science to a relevant subject for clinical study and has been responsible for a great increase of interest in muscle studies. Myopathies are linked to mutations in five of the myosin heavy chain genes, three of the myosin light chain genes, and three of the actin genes. This review aims to determine to what extent we can explain disease phenotype from the mutant genotype. To optimise our chances of finding the right mechanism we must study a myopathy where there are a large number of different mutations that cause a common phenotype and so are likely to have a common mechanism: a corollary to this criterion is that if any mutation causes the disease phenotype but does not correspond to the proposed mechanism, then the whole mechanism is suspect. Using these criteria, we consider two cases where plausible genotype-phenotype mechanisms have been proposed: the actin “A-triad” and the myosin “mesa/IHD” models.

Classifying Cardiac Actin Mutations Associated With Hypertrophic Cardiomyopathy.

Mutations in the cardiac actin gene (ACTC1) are associated with the development of hypertrophic cardiomyopathy (HCM). To date, 12 different ACTC1 mutations have been discovered in patients with HCM. Given the high degree of sequence conservation of actin proteins and the range of protein–protein interactions actin participates in, mutations in cardiac actin leading to HCM are particularly interesting. Here, we suggest the classification of ACTC1 mutations based on the location of the resulting amino acid change in actin into three main groups: (1) those affecting only the binding site of the myosin molecular motor, termed M-class mutations, (2) those affecting only the binding site of the tropomyosin (Tm) regulatory protein, designated T-class mutations, and (3) those affecting both the myosin- and Tm-binding sites, called MT-class mutations. To understand the precise pathogenesis of cardiac actin mutations and develop treatments specific to the molecular cause of disease, we need to integrate rapidly growing structural information with studies of regulated actomyosin systems.

Genetic compensation triggered by actin mutation prevents the muscle damage caused by loss of actin protein

The lack of a mutant phenotype in homozygous mutant individuals’ due to compensatory gene expression triggered upstream of protein function has been identified as genetic compensation. Whilst this intriguing process has been recognized in zebrafish, the presence of homozygous loss of function mutations in healthy human individuals suggests that compensation may not be restricted to this model. Loss of skeletal α-actin results in nemaline myopathy and we have previously shown that the pathological symptoms of the disease and reduction in muscle performance are recapitulated in a zebrafish antisense morpholino knockdown model. Here we reveal that a genetic actc1b mutant exhibits mild muscle defects and is unaffected by injection of the actc1b targeting morpholino. We further show that the milder phenotype results from a compensatory transcriptional upregulation of an actin paralogue providing a novel approach to be explored for the treatment of actin myopathy. Our findings provide further evidence that genetic compensation may influence the penetrance of disease-causing mutations.

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.

Profilin Directly Promotes Microtubule Growth through Residues Mutated in Amyotrophic Lateral Sclerosis

Profilin is an abundant actin monomer-binding protein with critical actin regulatory roles invivo [1, 2]. However, profilin also influences microtubule dynamics in cells, which may be mediated in part through its interactions with formins that in turn bind microtubules [3, 4]. Specific residues on human profilin-1 (PFN1) are mutated in patients with amyotrophic lateral sclerosis (ALS) [5, 6]. However, the observation that some ALS-linked PFN1 mutants fail to alter cellular actin organization or dynamics [5-8] or invitro actin-monomer affinity [9] has been perplexing, given that profilin is best understood as an actin regulator. Here, we investigated direct effects of profilin on microtubule dynamics and whether ALS-linked mutations in PFN1 disrupt such functions. We found that human, fly, and yeast profilin homologs all directly enhance microtubule growth rate by several-fold invitro. Microtubule stimulatory effects were unaffected by mutations in the canonical actin- or poly-proline-binding sites of profilin. Instead, microtubule activities depended on specific surface residues on profilin mutated in ALS patients. Furthermore, microtubule effects were attenuated by increasing concentrations of actin monomers, suggesting competition between actin and microtubules for binding profilin. Consistent with these biochemical observations, a 2-fold increase in the expression level of wild-type PFN1, but not the ALS-linked PFN1 mutants, increased microtubule growth rates in cells. Together, these results demonstrate that profilin directly enhances the growth rate of microtubules. They further suggest that ALS-linked mutations in PFN1 may perturb cellular microtubule dynamics and/or the coordination between the actin and microtubule cytoskeletons, leading to motor neuron degeneration.

D292V Actin Mutation Stabilizes Tropomyosin in the Off-State of the Thin Filament

A cluster of basic residues on the surface of actin (K326, K328 and R147) are needed for F-actin to bind to acidic residues on tropomyosin in the blocked- and closed-states of the muscle thin filament (Li et al., 2011; Lehman et al., 2013; Orzechowski et al., 2013; Fischer et al., 2016). However, the binding of tropomyosin to actin is necessarily weak so that regulatory transitions across actin can proceed at low-energy cost. We propose that negatively charged actin residue D292 lying adjacent to residues K326, K328 and R147 is required to temper the binding of actin to tropomyosin allowing regulatory-switching.

Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins.

The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins.

Mutations in actin used for structural studies partially disrupt β-thymosin/WH2 domains interaction.

Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model β-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both β-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains.

Molecular Dynamics Simulation Reveal the Mechanism of Resistance of Mutant Actins to Latrunculin A – Insight into Specific Modifications to Design Novel Drugs to Overcome Resistance

Mutant actins D157E and R183A-D184A are reported to resist the anticancer drug Latrunculin A (LAT); though identified, the mechanism of resistance is not clearly understood. To design better molecules that can overcome the resistance caused by mutations it is important to define precise pharmacophoric regions in LAT based on the mechanism of resistance on the mutant actin -LAT interactions. To address this we have conducted 20 nano seconds (ns) simulation of mutant actins - LAT complex and compared it with the 20ns simulation of wild actin - LAT complex. Functions as the binding free energy, distance between LAT and binding site residues, LAT and actin domains, dihedral angle analysis, motional correlation were studied of these simulations. Grounded on these studies, four sites in LAT are identified to be crucial for modification. Bulkier ring moieties containing nitrogen in place of the double bonded oxygen in the macrocyclic lactone ring may be considered to establish interactions with Glu214. The nitrogen in 2-thiazolidinone moiety can be substituted with a hydrophobic ring to stabilise the interaction with the Asp157Glu and the oxygen in the cyclohexane of LAT with hydrophilic groups to strengthen their interaction with Tyr69. The nitrogen of the 2-thiazolidinone moiety can be replaced with nitrogen containing rings to improve inhibition of the actin polymerisation. Apart from this chemical groups on the sulphur of 2-thiazolidinone moiety to improve the hydrophobic interaction with actin is also identified for modification. Based on this a combinatorial library of 46 LAT analogs was generated and docked with the wild and mutant actins to identify potent leads to become anti-actin anticancer drugs.

Two Deafness-Causing Actin Mutations (DFNA20/26) Have Allosteric Effects on the Actin Structure

Point mutations in γ-cytoplasmic actin have been shown to result in autosomal-dominant, nonsyndromic, early-onset deafness. Two mutations at the same site, K118M and K118N, provide a unique opportunity to compare the effects of two dissimilar amino acid substitutions that produce a similar phenotype in humans. K118 resides in a helix that runs from K113 to T126, and mutations that alter the position, dynamics, and/or biochemistry of this helix can result in a wide range of pathologies. Using a combination of computational and experimental studies, both employing yeast actin, we find that these mutations at K118 result in changes in the structure and dynamics of the DNase-I loop, alterations in the structure of the H73 loop as well as the side-chain orientations of W79 and W86, changes in nucleotide exchange rates, and significant shifts in the twist of the actin monomer. Interestingly, in the case of K118N, the twist of the monomer is nearly identical to that of the F-actin protomer, and in vitro polymerization assays show that this mutation results in faster polymerization. Taken together, these results indicate that mutations at this site give rise to a series of small changes that can be tolerated in vivo but result in misregulation of actin assembly and dynamics.

The N-terminal tropomyosin- and actin-binding sites are important for leiomodin 2's function.

Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2-knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43-90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124-201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin poly-merization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.

Actin Tyrosine-53-Phosphorylation in Neuronal Maturation and Synaptic Plasticity.

Rapid reorganization and stabilization of the actin cytoskeleton in dendritic spines enables cellular processes underlying learning, such as long-term potentiation (LTP). Dendritic spines are enriched in exceptionally short and dynamic actin filaments, but the studies so far have not revealed the molecular mechanisms underlying the high actin dynamics in dendritic spines. Here, we show that actin in dendritic spines is dynamically phosphorylated at tyrosine-53 (Y53) in rat hippocampal and cortical neurons. Our findings show that actin phosphorylation increases the turnover rate of actin filaments and promotes the short-term dynamics of dendritic spines. During neuronal maturation, actin phosphorylation peaks at the first weeks of morphogenesis, when dendritic spines form, and the amount of Y53-phosphorylated actin decreases when spines mature and stabilize. Induction of LTP transiently increases the amount of phosphorylated actin and LTP induction is deficient in neurons expressing mutant actin that mimics phosphorylation. Actin phosphorylation provides a molecular mechanism to maintain the high actin dynamics in dendritic spines during neuronal development and to induce fast reorganization of the actin cytoskeleton in synaptic plasticity. In turn, dephosphorylation of actin is required for the stabilization of actin filaments that is necessary for proper dendritic spine maturation and LTP maintenance. Dendritic spines are small protrusions from neuronal dendrites where the postsynaptic components of most excitatory synapses reside. Precise control of dendritic spine morphology and density is critical for normal brain function. Accordingly, aberrant spine morphology is linked to many neurological diseases. The actin cytoskeleton is a structural element underlying the proper morphology of dendritic spines. Therefore, defects in the regulation of the actin cytoskeleton in neurons have been implicated in neurological diseases. Here, we revealed a novel mechanism for regulating neuronal actin cytoskeleton that explains the specific organization and dynamics of actin in spines. The better we understand the regulation of the dendritic spine morphology, the better we understand what goes wrong in neurological diseases.

Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.

Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly.

RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions.

Molecular Effects of Deafness Mutations in Actin

Point mutations in γ-cytoplasmic actin have been shown to cause autosomal-dominant non-syndromic early onset deafness. Of the eleven known actin mutations, two are unique in that they occur in the same residue. The mutations, K118M in one family and K118N in a second family, provide a unique opportunity to compare the effects of two dissimilar amino acid substitutions that produce a similar phenotype in humans. K118 resides in a helix that runs from from K113 to T126, and mutations that alter the position, dynamics and/or biochemistry of this helix can result in a wide range of pathologies. Using a combination of computational and experimental studies, both using yeast actin, we find that these mutations at K118 result in changes in the structure and dynamics of the DNase-I loop, alterations in the structure of the H73 loop as well as the sidechain orientations of W79 and W86, changes in nucleotide exchange rates, and significant shifts in the twist of the actin monomer. Interestingly for the case of K118N, the twist of the monomer is nearly identical to that for the F-actin protomer, and in vitro polymerization assays show that this mutation actually results in faster polymerization. Taken together, it is evident that mutations at this site give rise to a series of smaller changes that can be tolerated in vivo, but result in misregulation of actin assembly and dynamics.

The Actin F352S Nemaline Myopathy Mutation Disrupts Indirect Flight Muscle Structure and Function in Drosophila

Nemaline myopathy is a congenital human muscle disorder characterized by masticatory, limb and respiratory weakness. Dominant negative disease-causing mutations have been identified in ten genes that notably encode sarcomeric thin filament proteins including α actin (ACTA1). While most of these mutations cause a loss of muscle function, the F352S actin mutation elevates myosin cross-bridge strain and steady-state isometric force production and, thereby, apparently increases contractile function in humans. To investigate the F352S-induced myopathic cascade from the level of single molecules through the level of whole muscle we generated transgenic Drosophila that express the mutation in Act88F, the indirect flight muscle (IFM) actin gene. Mutant heterozygotes were flightless. Fluorescent and electron microscopy revealed breaks in mutant IFM fibers and extensive myofibrillar disarray. For example, the highly ordered double hexagonal thin/thick filament lattice, at the periphery of myofibrils, was distorted with myofilaments occasionally losing longitudinal orientation. Z-lines showed streaming and formed “zebra bodies”. The microscopic alterations are consistent with inappropriate acto-myosin interaction, unevenly distributed force, and destructive hypercontraction. Interestingly, in vitro sliding velocities of purified IFM F-actin from wild-type (3.17±0.12 μm/s) vs. F352S mutant heterozygous (2.88±0.09 μm/s, mean±SEM) flies did not significantly differ. Similarly, no differences in maximum calcium-activated velocity (3.88±0.11 vs. 3.77±0.10 μm/s) or in cooperativity (nH = 2.13±0.27 vs. 2.62±0.46) were observed for F-actin in the presence of tropomyosin and troponin. However, F352S heterozygous filaments showed a significant (p < 0.01) increase in calcium sensitivity relative to control (pCa50 = 6.11 +/- 0.03 vs. 5.99 +/- 0.03), consistent with mutation-induced gain in molecular function. Going forward, our novel Drosophila model will be used to screen for in vivo modifiers of mutant muscle performance.Supported by NIH 2PO1 AR052354 (P.D. Allen PI; CFA Core D).

Assembly and Maintenance of Myofibrils in Striated Muscle.

In this chapter, we present the current knowledge on de novo assembly, growth, and dynamics of striated myofibrils, the functional architectural elements developed in skeletal and cardiac muscle. The data were obtained in studies of myofibrils formed in cultures of mouse skeletal and quail myotubes, in the somites of living zebrafish embryos, and in mouse neonatal and quail embryonic cardiac cells. The comparative view obtained revealed that the assembly of striated myofibrils is a three-step process progressing from premyofibrils to nascent myofibrils to mature myofibrils. This process is specified by the addition of new structural proteins, the arrangement of myofibrillar components like actin and myosin filaments with their companions into so-called sarcomeres, and in their precise alignment. Accompanying the formation of mature myofibrils is a decrease in the dynamic behavior of the assembling proteins. Proteins are most dynamic in the premyofibrils during the early phase and least dynamic in mature myofibrils in the final stage of myofibrillogenesis. This is probably due to increased interactions between proteins during the maturation process. The dynamic properties of myofibrillar proteins provide a mechanism for the exchange of older proteins or a change in isoforms to take place without disassembling the structural integrity needed for myofibril function. An important aspect of myofibril assembly is the role of actin-nucleating proteins in the formation, maintenance, and sarcomeric arrangement of the myofibrillar actin filaments. This is a very active field of research. We also report on several actin mutations that result in human muscle diseases.