Mechanics of blunting of actin-myosin interaction dynamics by the actinopathy-causing mutation E334Q in cytoskeletal γ-actin.

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 (F0) and of the shortening velocity against loads < F0. 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. 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.