Dynamic remodeling of the neuronal cytoskeleton by mutations in beta- and gamma-actin isoforms leading to Non-Muscle Actinopathies (NMAs)

This thesis investigates the role of non-muscle actin isoforms in the structural dynamics of the membrane-cytoskeleton system in neurons during maturation, neural progenitor cells (NPCs) and differentiated neurons (NCs). The study employs advanced technologies to compare mechanical, biochemical, and morphological parameters in WT neurons and neurons with two actin mutations linked to Baraitser-Winter Cerebrofrontofacial syndrome (BWCFF): β-actin R196H and γ-actin T203M. The mechanical analysis uses dual laser optical tweezers (DLOT), enabling precise quantification of two key responses that define cellular mechanics: membrane indentation and tether formation. Membrane indentation assesses resistance to deformation, while tether formation evaluates membrane-cytoskeleton adhesion and viscoelastic behavior during tether dynamics. These responses reveal interactions among actin filaments, actin-binding proteins, and the plasma membrane. Complementary biochemical and immunocytochemical analyses, via Western blot and immunofluorescence, quantify and localize actin isoforms (β-, γ-, and α-smooth muscle actin) and assess filament organization. Both β-R196H and γ-T203M mutations impact early neural differentiation, each with distinct mechanical, morphological, and biochemical signatures. β-R196H NPCs show reduced size and altered mechanical properties, lowering resistance to stress. However, when mechanical parameters are normalized for size, the reduced cell dimensions appear to compensate for high membrane tension. Upon differentiation, β-R196H NCs resemble WT NCs, displaying enhanced membrane-cytoskeleton plasticity. Conversely, γ-T203M NPCs, similar in size to WT, exhibit unambiguous mechanical deficits, including reductions in both damped and undamped elastic coefficients, matching those seen in WT neurons treated with Latrunculin A (an F-actin disruptor). This impaired plasticity persists at the NC stage, indicating disrupted actin polymerization and filament stability. The mutation also causes overexpression of all actin isoforms, including α-smooth muscle actin (typically absent in neurons), likely as a failed compensatory attempt to restore cytoskeletal integrity. Mechanical, biochemical, and morphological data from the NPC stage highlight two distinct compensatory mechanisms addressing membrane-cytoskeleton weakening. β-R196H NPCs recover rigidity through cell size reduction and γ-actin–mediated stabilization, whereas γ-T203M NPCs increase actin expression but fail to restore function due to defective polymerization. These differences point to isoform-specific roles: in WT neurons, β-actin is enriched at the cell periphery—supporting dynamic remodeling tasks like synaptic plasticity, while γ-actin is more cytoplasmically distributed, contributing to structural stability, especially in axons. Thus, in β-R196H NPCs, γ-actin stabilizes the cytoskeleton in a reduced-size context, enabling compensation. In contrast, γ-T203M NPCs cannot achieve similar stabilization, due to impaired γ-actin polymerization. Overall, the study shows that mutations in actin genes profoundly alter isoform expression, cytoskeletal dynamics, and mechanical properties, compromising neural cell integrity and offering a mechanistic understanding of BWCFF pathology. Control experiments in fibroblasts show no significant mechanical differences between WT and mutants, indicating neuron-specific effects. This highlights the specialized role of actin in neuronal functions like axon elongation and synaptic remodeling, which are more vulnerable to cytoskeletal disruption than in non-neuronal cells.