Sodium fluoride as a nucleating factor for Mg-actin polymerization

Dynamic instability of actin filaments can be inhibited by Pi analogs beryllium fluoride and aluminium fluoride that mimic the intermediate ADP-Pi state and stabilize actin filaments. On the other hand, the phosphoryl transfer enzymes can be activated in the absence of aluminium by magnesium fluoride if magnesium ions and sodium fluoride (NaF) were present in the solution. Whether magnesium fluoride promotes functional activities of actin is not known. Here we show, for the first time, that sodium fluoride strongly accelerates polymerization of highly dynamic Mg-F-actin assembled from the monomers proteolytically cleaved between Gly42 and Val43 within the D-loop with actin-specific protease protealysin (Pln-actin), apparently due to stabilization of nuclei formed at the initial step of actin polymerization. Thereby, NaF did not inhibit the ATPase activity (subunit exchange) on Pln-F-actin, did not increase the amount of Pln-F-actin sedimented by ultracentrifugation, and did not stabilize the inter-strand contacts of Pln-F-actin. On the other hand, NaF diminished accessibility of the nucleotide binding cleft of Mg-G-actin to trypsin, pointing to an additional cleft closure, and additionally protected the D-loop from the protealysin cleavage in Mg-F-actin, thus indicating that the longitudinal contacts are stabilized. We also demonstrate that in cultured cells NaF can directly promote assembly of F-actin structures under conditions when the corresponding activity of the RhoA pathway is inhibited. These data suggest that the NaF-induced assembly of actin filaments is promoted by magnesium fluoride that can be formed by the NaF-originating fluoride and the actin tightly bound magnesium. - Highlights: • NaF is found to rescue nucleation of instable proteolytically cleaved Mg-F-actin. • The NaF-induced tightening of the longitudinal contacts within Mg-F-actin is shown. • NaF promotes assembly of F-actin structures when the RhoA pathway is inhibited. • Magnesium fluoride may be formed from NaF and the actin tightly bound Mg²⁺.