The Baraitser-Winter Cerebrofrontofacial Syndrome recurrent R196H variant in cytoplasmic β-actin impairs its cellular polymerization and stability

Variants in cytoskeletal actin encoding genes are associated with a broad spectrum of disorders, called non-muscle actinopathies. Among them, Baraitser-Winter cerebrofrontofacial syndrome (BWCFF) displays the most severe symptoms, such as intellectual disability and epilepsy. The exact consequences of the mutation on actins properties, however, are not fully understood. Here we explored the cellular effects of the R196H mutation in patient-derived fibroblasts. We show that the heterozygous mutation causes an actin polymerization defect in cells, leading to a fifty percent decrease in filamentous (F-) actin content. This effect can be rescued by the addition of the actin-polymerizing and stabilizing drug, jasplakinolide. 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 stabilized with phalloidin, explored with atomic force microscopy (AFM). The reduced F-actin content correlated with an approximately fourfold reduction in the stiffness of patient-derived cells probed with AFM. 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 dynamical reorganization of actin by uniaxial stretching revealed that the interaction of cofilin with actin is also weakened by the mutation. Based on the existing cofilin-actin structures, the binding of cofilin may weaken the interaction of the neighboring residue E195 with K113, one of the lateral contacts stabilizing the filament. Thus, the mutation possibly exerts its effect through the destabilization of the interfilament interactions, potentially interfering allosterically with cofilin binding during actin depolymerization.