Formation of Hirano Bodies after Inducible Expression of a Modified Form of an Actin-Cross-Linking Protein

Abstract

ABSTRACT Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 295) from the endogenous 34-kDa actin-binding protein of Dictyostelium discoideum leads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano body formation using an inducible promoter system ( rnrB ). By fusing the CT to a green fluorescent protein (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry, and Western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model.