Baraitser–Winter Syndrome Hotspot Mutation R196H in Cytoskeletal β–actin Reduces F–actin Stability and Perturbs Interaction with the Arp2/3 Complex

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Abstract
Baraitser–Winter cerebrofrontofacial syndrome (BWCFF) is the most common and best–defined clinical entity associated with heterozygous single–point missense mutations in cytoskeletal β–actin. Patients present with distinct craniofacial anomalies and neurodevelopmental disabilities of variable severity. To date, the most frequently observed variants affect residue R196 of cytoskeletal β–actin, with the variant p.R196H being the most common. Patients carrying the p.R196H variant are likely to suffer from pachygyria, probably due to neuronal migration defects contributing to the development of abnormally thick convolutions of the cerebral cortex. Here, we describe the recombinant production, purification and biochemical characterization of the BWCFF hotspot variant p.R196H. The stability and nucleotide interaction of monomeric p.R196H are unaffected, indicating a disease mechanism involving incorporation of p.R196H protomers into actin filaments. Incorporation of the variant strongly affects F–actin stability and polymerization dynamics, consistent with the position of residue R196 close to the helical axis of the actin filament and an important interstrand contact. The changes observed include an increased critical concentration of polymerization, a reduced elongation rate and an increase in the rate of filament depolymerization. In the Arp2/3–generated branch junction complex, which is essential for cell migration and endocytosis, R196 is located at the interface between the first protomer of the nucleated daughter filament and the Arp2 subunit of the Arp2/3 complex. Assays probing the interaction of p.R196H filaments with the Arp2/3 complex show a reduced efficiency of branch generation. Branch stability is impaired, as evidenced by a reduction in the number of branches and spontaneous debranching events. Furthermore, in their interaction with different types of cytoskeletal myosin motors, p.R196H filaments show isoform–specific differences. While p.R196H filaments move WT–like on lawns of surface–immobilized non–muscle myosin–2A, motility on myosin–5A is 30 % faster.

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