Profilin 1 with the amyotrophic lateral sclerosis associated mutation T109M displays unaltered actin binding and does not affect the actin cytoskeleton

Axel Freischmidt, Marcel Schöpflin, Marisa S. Feiler, Ann-Katrin Fleck, Albert C. Ludolph, Jochen H. Weishaupt

The recent identification of several mutations in PFN1, a protein involved in actin dynamics, strengthens the hypothesis that pathology of amyotrophic lateral sclerosis is linked to cytoskeletal defects. Impaired actin binding is a common denominator of several PFN1 mutations associated with amyotrophic lateral sclerosis, although further mechanisms may also contribute to the death of motor neurons. In this study we examine the actin binding properties of PFN1 carrying the causal T109M mutation and its effects on the actin cytoskeleton. Actin binding of PFN1 T109M was examined by co-immunoprecipitation experiments, a split luciferase complementation assay and a pulldown assay with recombinant PFN1. The actin cytoskeleton was investigated by fluorescence microscopy and by ultracentrifuge separation of globular and filamentous actin fractions followed by Western blotting. Using different technical approaches we show that PFN1 T109M displays unaltered actin binding. Furthermore we show that the actin cytoskeleton is not affected by PFN1 carrying the T109M mutation. Our data suggest that actin independent mechanisms contribute to the pathogenicity of PFN1 T109M and possibly other PFN1 mutations.