Requirements for Hirano body formation.

Abstract

Hirano bodies are paracrystalline F-actin-rich structures associated with diverse conditions, including neurodegeneration and aging. Generation of model Hirano bodies using altered forms of Dictyostelium 34-kDa actin-bundling protein allows studies of their physiological function and mechanism of formation. We describe a novel 34-kDa protein mutant, E60K, with a point mutation within the inhibitory domain of the 34-kDa protein. Expression of E60K in Dictyostelium induces the formation of model Hirano bodies. The E60K protein has activated actin binding and is calcium regulated, unlike other forms of the 34-kDa protein that induce Hirano bodies and that have activated actin binding but lack calcium regulation. Actin filaments in the presence of E60K in vitro show enhanced resistance to disassembly induced by latrunculin B. Actin filaments in model Hirano bodies are also protected from latrunculin-induced depolymerization. We used nocodazole and blebbistatin to probe the role of the microtubules and myosin II, respectively, in the formation of model Hirano bodies. In the presence of these inhibitors, model Hirano bodies can form but are smaller than controls at early times of formation. The ultrastructure of model Hirano bodies did not reveal any major difference in structure and organization in the presence of inhibitors. In summary, these results support the conclusion that formation of model Hirano bodies is promoted by gain-of-function actin filament bundling, which enhances actin filament stabilization. Microtubules and myosin II contribute to but are not required for formation of model Hirano bodies.