Pasquale Bianco

Neuronal maturation is governed by the integration of intrinsic programs, such as gene regulation and cellular metabolism, with external mechanical cues. The conversion of mechanical forces into developmental responses (mechanotransduction) requires mechanical coupling of neurons to the extracellular matrix and neighboring cells. This coupling is mediated by the actin cytoskeleton and associated transmembrane protein complexes. However, the specific role of the β- and γ-actin isoforms in mechanotransduction, and the functional consequences of their pathogenic mutations, remain poorly understood. To address this question, we combined optical tweezers mechanics with immunofluorescence in human iPSC-derived neural progenitor cells (NPCs) and postmitotic neurons (NCs), both wild-type (WT) and carrying pathogenic mutations in β-actin (R196H) or γ-actin (T203M) associated with Baraitser-Winter cerebrofrontofacial (BWCFF) syndrome. Using membrane tether elongation as a proxy for early protrusion formation, we found that γ-T203M NPCs require a fourfold lower force than WT NPCs and, upon differentiation, fail to develop the neurite-filopodia architecture retaining abundant immature protrusions In contrast, β-R196H NPCs exhibit only reduction of cell surface tension associated with increased neuron fragility, without pronounced protrusion abnormalities. These results reveal non-redundant roles of β- and γ-actin isoforms in neuronal mechanotransduction and provide mechanistic insight into the pathogenesis of BWCFF syndrome.

Variants in cytoskeletal actin encoding genes are associated with a broad spectrum of disorders, called non‐muscle actinopathies. Among them, the Baraitser–Winter cerebrofrontofacial syndrome (BWCFF) displays the most severe symptoms, such as intellectual disability and epilepsy. We found that the BWCFF‐associated R196H mutation results in reduced proliferation and migration of patient‐derived fibroblast cells, and the latter is likely related to decreased fibronectin expression. The mutation causes a 50% drop in filamentous (F‐) actin content, which is correlated with an approximately fourfold reduction in the stiffness of patient‐derived cells probed with atomic force microscopy (AFM). We observed no significant defects either in the organization of the cellular actin cytoskeleton, analyzed by superresolution (STED) microscopy, or in the structure of purified filaments, explored with AFM. On the other hand, the more parallel orientation of the mutant actin bundles might be caused by the perturbed interaction of actin with the Arp2/3 complex. Manipulating the cells by mechanical forces through the application of the dual laser optical tweezers (DLOT) technique suggests that the mutation weakens the attachment of cytoskeletal actin to the plasma membrane. Inducing dynamic reorganization of actin by uniaxial stretching revealed that the interaction of cofilin with actin is also weakened by the mutation. Thus, the impaired function of actin to form filaments and interact with either cofilin or the Arp2/3 complex may result in the malfunction of dendritic spines, and the reduced cellular proliferation and migration might account for the lissencephaly phenotype of patients.

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.

A data fitting procedure is devised and thoroughly tested to provide self-consistent estimates of the relevant mechanokinetic parameters involved in a plausible scheme underpinning the output of an ensemble of myosin II molecular motors mimicking the contraction of skeletal muscle. The method builds on a stochastic model accounting for the force exerted by the motor ensemble operated both in the low and high force-generating regimes corresponding to different temperature ranges. The proposed interpretative framework is successfully challenged against simulated data, meant to mimic the experimental output of a one-dimensional synthetic nanomachine powered by pure muscle myosin isoforms.

In maximally Ca²⁺-activated demembranated fibres from the mammalian skeletal muscle, the depression of the force by lowering the temperature below the physiological level (~35 °C) is explained by the reduction of force in the myosin motor. Instead, cooling is reported to not affect the force per motor in Ca²⁺-activated cardiac trabeculae from the rat ventricle. Here, the mechanism of the cardiac performance depression by cooling is reinvestigated with fast sarcomere-level mechanics. We determine the changes in the half-sarcomere compliance of maximally Ca²⁺-activated demembranated rat trabeculae in the range of temperatures of 10–30 °C and analyse the data in terms of a simplified mechanical model of the half-sarcomere to extract the contribution of myofilaments and myosin motors. We find that the changes in the ensemble force are due to changes in the force per motor, while the fraction of actin-attached motors remains constant independent of temperature. The results demonstrate that in the cardiac myosin, as in the skeletal muscle myosin, the force-generating transition is endothermic. The underlying large heat absorption indicates the interaction of extended hydrophobic surfaces within the myosin motor, like those suggested by the crystallographic model of the working stroke.

The regulation of heart function is attributed to a dual filament mechanism: i) the Ca ²⁺ -dependent structural changes in the regulatory proteins of the thin, actin-containing filament making actin available for myosin motor attachment, and ii) the release of motors from their folded (OFF) state on the surface of the thick filament allowing them to attach and pull the actin filament. Thick filament mechanosensing is thought to control the number of motors switching ON in relation to the systolic performance, but its molecular basis is still controversial. Here, we use high spatial resolution X-ray diffraction data from electrically paced rat trabeculae and papillary muscles to provide a molecular explanation of the modulation of heart performance that calls for a revision of the mechanosensing hypothesis. We find that upon stimulation, titin-mediated structural changes in the thick filament switch motors ON throughout the filament within ~½ the maximum systolic force. These structural changes also drive Myosin Binding Protein-C (MyBP-C) to promote first motor attachments to actin from the central 1/3 of the half-thick filament. Progression of attachments toward the periphery of half-thick filament with increase in systolic force is carried on by near-neighbor cooperative thin filament activation by attached motors. The identification of the roles of MyBP-C, titin, thin and thick filaments in heart regulation enables their targeting for potential therapeutic interventions.

Myosin II is the muscle molecular motor that works in two bipolar arrays in each thick filament of the striated (skeletal and cardiac) muscle, converting the chemical energy into steady force and shortening by cyclic ATP–driven interactions with the nearby actin filaments. Different isoforms of the myosin motor in the skeletal muscles account for the different functional requirements of the slow muscles (primarily responsible for the posture) and fast muscles (responsible for voluntary movements). To clarify the molecular basis of the differences, here the isoform–dependent mechanokinetic parameters underpinning the force of slow and fast muscles are defined with a unidimensional synthetic nanomachine powered by pure myosin isoforms from either slow or fast rabbit skeletal muscle. Data fitting with a stochastic model provides a self–consistent estimate of all the mechanokinetic properties of the motor ensemble including the motor force, the fraction of actin–attached motors and the rate of transition through the attachment–detachment cycle. The achievements in this paper set the stage for any future study on the emergent mechanokinetic properties of an ensemble of myosin molecules either engineered or purified from mutant animal models or human biopsies.

The mechanical performances of the vertebrate skeletal muscle during isometric and isotonic contractions are interfaced with the corresponding energy consumptions to define the coupling between mechanical and biochemical steps in the myosin–actin energy transduction cycle. The analysis is extended to a simplified synthetic nanomachine in which eight HMM molecules purified from fast mammalian skeletal muscle are brought to interact with an actin filament in the presence of 2 mM ATP, to assess the emergent properties of a minimum number of motors working in ensemble without the effects of both the higher hierarchical levels of striated muscle organization and other sarcomeric, regulatory and cytoskeleton proteins. A three-state model of myosin–actin interaction is able to predict the known relationships between energetics and transient and steady-state mechanical properties of fast skeletal muscle either in vivo or in vitro only under the assumption that during shortening a myosin motor can interact with two actin sites during one ATP hydrolysis cycle. Implementation of the molecular details of the model should be achieved by exploiting kinetic and structural constraints present in the transients elicited by stepwise perturbations in length or force superimposed on the isometric contraction.

Titin is a molecular spring in parallel with myosin motors in each muscle half-sarcomere, responsible for passive force development at sarcomere length (SL) above the physiological range (>2.7 μm). The role of titin at physiological SL is unclear and is investigated here in single intact muscle cells of the frog ( Rana esculenta ), by combining half-sarcomere mechanics and synchrotron X-ray diffraction in the presence of 20 μM para-nitro-blebbistatin, which abolishes the activity of myosin motors and maintains them in the resting state even during activation of the cell by electrical stimulation. We show that, during cell activation at physiological SL, titin in the I-band switches from an SL-dependent extensible spring (OFF-state) to an SL-independent rectifier (ON-state) that allows free shortening while resisting stretch with an effective stiffness of ~3 pN nm ⁻¹ per half-thick filament. In this way, I-band titin efficiently transmits any load increase to the myosin filament in the A-band. Small-angle X-ray diffraction signals reveal that, with I-band titin ON, the periodic interactions of A-band titin with myosin motors alter their resting disposition in a load-dependent manner, biasing the azimuthal orientation of the motors toward actin. This work sets the stage for future investigations on scaffold and mechanosensing-based signaling functions of titin in health and disease.

Contraction of striated muscle is regulated by a dual mechanism involving both thin, actin-containing filament and thick, myosin-containing filament. Thin filament is activated by Ca²⁺ binding to troponin, leading to tropomyosin displacement that exposes actin sites for interaction with myosin motors, extending from the neighbouring stress-activated thick filaments. Motor attachment to actin contributes to spreading activation along the thin filament, through a cooperative mechanism, still unclear, that determines the slope of the sigmoidal relation between isometric force and pCa (−log[Ca²⁺]), estimated by Hill coefficient n_H. We use sarcomere-level mechanics in demembranated fibres of rabbit skeletal muscle activated by Ca²⁺ at different temperatures (12–35 °C) to show that n_H depends on the motor force at constant number of attached motors. The definition of the role of motor force provides fundamental constraints for modelling the dynamics of thin filament activation and defining the action of small molecules as possible therapeutic tools.