Abstract
Glial fibrillary acidic protein (GFAP) is classified as a type III intermediate filament protein predominantly expressed in mature astrocytes. It has the ability to self-assemble into 10 nm filaments in vitro, making it particularly valuable for elucidating the sequences essential for filament assembly. In this study, we created a series of deletion mutants targeting sequences in the N-terminal, C-terminal, and central rod domains to explore the sequences critical for the assembly of GFAP into 10 nm filaments. The impact of these deletions on filament formation was evaluated through in vitro assembly studies and transduction assays conducted with primary astrocytes. Our data revealed that deletions at the carboxy end resulted in abnormalities in either filament diameter calibration or lateral association, whereas deletions at the amino-terminal end significantly disrupted the filament assembly process, particularly restricting filament elongation. Furthermore, we discovered that the filament-forming sequences within the rod domain varied in their contributions to filament assembly and network formation. These findings enhance our understanding of the GFAP assembly process in vitro and provide a detailed mapping of the essential regions required for GFAP assembly. These insights hold significant implications for Alexander disease arising from deletion mutations in GFAP.