Dietmar J. Manstein

Cellular processes such as cytokinesis, apoptosis and migration rely heavily on the myosin‐based contractility of the γ‐actin network in the submembrane cortex. Direct measurements of γ‐actin–myosin interactions through morphological and depletion investigations remain elusive. Here, we use a synthetic nanomachine, consisting of an array of myosin motors carried on a nanopositioner and brought to interact with an actin filament attached to a bead trapped in the focus of dual laser optical tweezers. The nanomachine is able to mimic the loading conditions of γ‐actin–myosin interactions in situ , allowing measurements of the maximum steady force ( F ₀ ) and of the shortening velocity against loads < F ₀ . Comparative measurements are conducted on wild‐type γ‐actin and γ‐actin carrying the E334Q mutation, associated with non‐muscle actinopathies. Our results show that the force of the single actin–myosin interaction is 2.5 pN for the wild‐type actin and is halved by the mutation. The kinetics of motor attachment–detachment, underpinning the rate of isometric force rise and the force–velocity relation, are also reduced by a factor of two, resulting in a reduction of the maximum nanomachine power to one‐fifth. The identification and quantitative definition of the loss of basic function caused by the E334Q γ‐actin mutation serve as a starting point for understanding the chain of remodelling events leading to the pathological phenotype and demonstrate the potential of the nanomachine for targeted therapeutic interventions. image Key points: Mutations in cytoskeletal actin cause rare pathologies classified as non‐muscle actinopathies (NMAs), including the Baraitser–Winter cerebrofrontofacial syndrome characterized by neural cortex abnormalities leading to facial dysmorphism, developmental delay and organ malformations. Cytoskeleton dynamics control cell morphology and migration and rely on the interaction of non‐muscle myosin II with cytoskeletal γ‐actin, but the system's mechanical performance and its blunting by NMA‐causing mutations in γ‐actin have still to be defined. Here, a synthetic nanomachine is used to record the relevant mechano‐kinetic parameters of the γ‐actin–myosin interaction under physiological loading conditions. Quantitative estimates of these parameters for wild‐type and E334Q mutant γ‐actin suggest that strong defects in the actin–myosin interaction mechanics may be one of the main molecular mechanisms leading to the pathological phenotype. This paper lays the groundwork for the quantitative definition of the basic function altered by NMA‐causing mutations and the evaluation of targeted therapies.

Histidine‐73 methylation of β‐actin by SETD3 modulates ATPase activity, filament assembly, and protein interactions. The pathogenic G74S mutation in cytoskeletal β‐actin, associated with Baraitser–Winter cerebrofrontofacial syndrome (BWCFF), alters the adjacent phosphate sensor loop, disrupting SETD3‐mediated methylation. Molecular docking indicates that SETD3 undergoes structural rearrangements to accommodate the mutant β‐actin, leading to reduced catalytic efficiency. Enzymatic assays confirm slower turnover of mutant actin peptides, while mass spectrometry reveals decreased histidine‐73 methylation in both recombinant mutant β‐actin and patient‐derived fibroblasts. This perturbance of SETD3‐mediated methylation likely generates β‐actin pools with distinct methylation states, varying across cell types and developmental stages, thereby impairing cytoskeletal dynamics and contributing to BWCFF pathology. Impact statement: This study reveals that the BWCFF‐linked G74S mutation in β‐actin disrupts SETD3‐mediated histidine‐73 methylation, impairing a critical post‐translational modification. It provides the first direct mechanistic link between a cytoskeletal actinopathy and altered methylation, highlighting potential targets for therapeutic intervention in β‐actin‐related developmental disorders.

Background and Aims: The present study analysed the expression patterns of class-5 myosin motor proteins (MYO5a, b, and c) in the heart with a specific focus on the role of MYO5b. Methods: RNA-sequencing, quantitative real-time polymerase chain reaction, immunohistochemistry, Western blot, immunoprecipitation, and proteomics were performed in mice and human tissues. Functional analyses were performed in mice with a cardiac-specific knockout (KO) of MYO5b (αMHC-Cretg/−; MYO5bflox/flox), wild-type (WT) (MYO5bflox/flox), and αMHC-Cretg/− mice and in isolated adult cardiomyocytes. Next-generation sequencing screened for MYO5B gene variants in a cohort of sudden cardiac death in the young/sudden infant death syndrome patients. Results: The expression of MYO5b, but not MYO5a or c, increased during postnatal cardiomyocyte maturation. Myosin-5b was reduced in end-stage failing human hearts and infarcted murine hearts. Heterozygous rare and likely pathogenic missense MYO5B gene variants (n = 6) were identified in three patients of a cohort of young patients (n = 95) who died of sudden cardiac death in the young/sudden infant death syndrome. MYO5b-KO mice revealed impaired electric conductance and metabolism, developed sarcomeric disarrangement, heart failure and death with altered mRNA levels for genes involved in sarcomere organization, fatty acid and glucose metabolism, ion channel sub-units, and Ca2+-homeostasis prior to heart failure. In cardiomyocytes, myosin-5b is associated with mitochondrial and ribosomal proteins. Myosin-5b-associated ribonucleoprotein particles (RNPs) contained mRNAs of sarcomeric, metabolic, cytoskeletal, and ion channel proteins. Conclusions: MYO5b is the major MYO5 gene expressed in postnatal cardiomyocytes where it transports vesicles, proteins, and multi-protein complexes. Among these are mRNA/RNP complexes affecting electric conductance, sarcomere homeostasis, cell metabolism, and cytoskeletal organization. Impairment in MYO5b expression and function promotes cardiac dysfunction, heart failure, and death.

Baraitser–Winter cerebrofrontofacial syndrome (BWCFF) is the most common and best‐defined clinical entity associated with heterozygous single‐point missense mutations in cytoskeletal β‐actin. Patients present with distinct craniofacial anomalies and neurodevelopmental disabilities of variable severity. To date, the most frequently observed variants affect residue R196 of cytoskeletal β‐actin. Patients carrying the p.R196 variants are likely to suffer from pachygyria, probably due to neuronal migration defects contributing to the development of abnormal convolutions of the cerebral cortex. Here, we report on the recombinant production, purification and characterization of the BWCFF hotspot variants p.R196H, p.R196C and p.R196S. Our findings reveal that the stability of the monomeric variants remains unaffected, suggesting that the disease mechanism involves the incorporation of these variants into actin filaments. This incorporation alters F‐actin stability and polymerization dynamics to varying degrees, depending on the specific variant. These effects are consistent with the positioning of residue R196 near the helical filament axis. Observed changes include an increased critical concentration for polymerization, reduced elongation rates and accelerated filament depolymerization. Within the actin‐related protein 2/3 (Arp2/3)‐generated branch junction complex, which is critical for processes such as cell migration and endocytosis, residue R196 is located at the interface between the first protomer of the nucleated filament and the Arp2 subunit. Variant p.R196H specifically results in reduced branching efficiency and impaired branch stability. Future research will seek to elucidate the impact of these actin filament defects on cellular processes and their contribution to the multifaceted pathophysiology of BWCFF, with a particular emphasis on cortical development.

Myosin 5c (Myo5c) is a motor protein that is produced in epithelial and glandular tissues, where it plays an important role in secretory processes. Myo5c is composed of two heavy chains, each containing a generic motor domain, an elongated neck domain consisting of a single α–helix with six IQ motifs, each of which binds to a calmodulin (CaM) or a myosin light chain from the EF–hand protein family, a coiled–coil dimer–forming region and a carboxyl–terminal globular tail domain. Although Myo5c is a low duty cycle motor, when two or more Myo5c–heavy meromyosin (HMM) molecules are linked together, they move processively along actin filaments. We describe the purification and functional characterization of human Myo5c–HMM co–produced either with CaM alone or with CaM and the essential and regulatory light chains Myl6 and Myl12b. We describe the extent to which cofilaments of actin and Tpm1.6, Tpm1.8 or Tpm3.1 alter the maximum actin–activated ATPase and motile activity of the recombinant Myo5c constructs. The small allosteric effector pentabromopseudilin (PBP), which is predicted to bind in a groove close to the actin and nucleotide binding site with a calculated ΔG of −18.44 kcal/mol, inhibits the motor function of Myo5c with a half–maximal concentration of 280 nM. Using immunohistochemical staining, we determined the distribution and exact localization of Myo5c in endothelial and endocrine cells from rat and human tissue. Particular high levels of Myo5c were observed in insulin–producing β–cells located within the pancreatic islets of Langerhans.

Baraitser–Winter cerebrofrontofacial syndrome (BWCFF) is the most common and best–defined clinical entity associated with heterozygous single–point missense mutations in cytoskeletal β–actin. Patients present with distinct craniofacial anomalies and neurodevelopmental disabilities of variable severity. To date, the most frequently observed variants affect residue R196 of cytoskeletal β–actin, with the variant p.R196H being the most common. Patients carrying the p.R196H variant are likely to suffer from pachygyria, probably due to neuronal migration defects contributing to the development of abnormally thick convolutions of the cerebral cortex. Here, we describe the recombinant production, purification and biochemical characterization of the BWCFF hotspot variant p.R196H. The stability and nucleotide interaction of monomeric p.R196H are unaffected, indicating a disease mechanism involving incorporation of p.R196H protomers into actin filaments. Incorporation of the variant strongly affects F–actin stability and polymerization dynamics, consistent with the position of residue R196 close to the helical axis of the actin filament and an important interstrand contact. The changes observed include an increased critical concentration of polymerization, a reduced elongation rate and an increase in the rate of filament depolymerization. In the Arp2/3–generated branch junction complex, which is essential for cell migration and endocytosis, R196 is located at the interface between the first protomer of the nucleated daughter filament and the Arp2 subunit of the Arp2/3 complex. Assays probing the interaction of p.R196H filaments with the Arp2/3 complex show a reduced efficiency of branch generation. Branch stability is impaired, as evidenced by a reduction in the number of branches and spontaneous debranching events. Furthermore, in their interaction with different types of cytoskeletal myosin motors, p.R196H filaments show isoform–specific differences. While p.R196H filaments move WT–like on lawns of surface–immobilized non–muscle myosin–2A, motility on myosin–5A is 30 % faster.

Familial hypertrophic cardiomyopathy (HCM) affects .2% of the world's population and is inherited in an autosomal dominant manner. Mutations in cardiac α‐actin are the cause in 1%–5% of all observed cases. Here, we describe the recombinant production, purification, and characterization of the HCM‐linked cardiac α‐actin variants p.A21V and p.D26N. Mass spectrometric analysis of the initially purified recombinant cardiac α‐actin variants and wild‐type protein revealed improper N‐terminal processing in the Spodoptera frugiperda (Sf‐9) insect cell system, compromising the labeling of the protein with fluorescent probes for biochemical studies. Therefore, we produced N‐terminal deletion mutants lacking the N‐terminal cysteine (ΔC2). The ΔC2 wild‐type construct behaved similar to porcine cardiac α‐actin purified from native Sus scrofa heart tissue and all ΔC2 constructs showed improved fluorescent labeling. Further analysis of untruncated and ΔC2 constructs showed that while neither the A21V nor the D26N mutation affects nucleotide binding, they cause a similar slowing of the rate of filament formation as well as a reduction in the thermal stability of monomeric and filamentous cardiac α‐actin. In vitro motility assays and transient‐kinetic studies probing the interaction of the actin variants with cardiac β‐myosin revealed perturbed actomyosin interactions and a reduced motile activity for the p.D26N variant. Addition of the small molecule effector EMD 57033, which targets cardiac β‐myosin, rescued the approximately 40% drop in velocity observed with the p.D26N constructs and activated the motile activity of wild‐type and p.D26N to the same level of 1100 nm s⁻¹.

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser–Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.