The distinct role of actin isoforms on mechanosensing-based maturation of hiPSC-derived neurons unveiled by isoform-specific mutations

Abstract 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.