Non-muscle actinopathy-associated loss-of-function actin variants modulate cytoskeletal reorganization.

Variants in ACTB gene encoding for cytoplasmic β-actin result in a group of rare disorders called non-muscle actinopathies (NMA). A subgroup of NMA patients with ACTB pLoF (predicted loss-of-function) disorder have various congenital malformations, such as microcephaly and intellectual disability. We investigated the cellular effects of an ACTB pLoF missense variant, G302A, and a four-amino-acid deletion, S338-I341 in cytoskeletal β-actin, in patient-derived fibroblast cells. We found that neither of the mutations affected the organization of actin or the width of the actin-filament bundles, while the mutation G302A reduced the stiffness of the cells as measured by using atomic force microscopy. The latter effect might be associated with the misorganization of tubulin and with the increased size and number of focal adhesions. When we challenged the cells by monolayer stretching and followed the reorganization of the actin cytoskeleton, we found that G302A mutant cells showed denser actin filament bundles within the cells compared to wild type cells. At the same time, the extent of cofilin reorganization from the cell periphery was increased upon stretch, and this correlated with an increased cofilin phosphorylation. In the case of deletion, while the extent of cofilin phosphorylation increased, the extent of reorganization was unaltered; rather, the phosphorylation of myosin light chain, important in counteracting external force, was reduced by 75%. We could partially rescue this effect by overexpressing the active form of the formin mDia1. Our findings open the possibility to verify the cellular phenotype in the most affected patients' cells, in neurons.