Assembly, disassembly, and exchange of glial fibrillary acidic protein.
The kinetics and dynamics of glial fibrillary acidic protein (GFAP) assembly was explored by a fluorescence energy transfer assay method. Purified GFAP was stoichiometrically labeled at a single cysteine residue with fluorescein-maleimide. Soluble labeled GFAP in a low ionic strength buffer was assembled into 10 nm filaments by rapidly increasing the ionic strength, and the kinetics of GFAP assembly was monitored by the reduction in fluorescence due to self-quenching of fluorescein. The extent of fluorescence quench correlated with both the formation of 10 nm filament morphology and the amount of protein pelleted at 12,000g. The assembly of GFAP is critically dependent upon both protein and magnesium ion concentration, and at the critical concentration for GFAP assembly is approximately 40 micrograms/ml. Disassembly of GFAP filaments was also observed as a relief of fluorescence quenching after dilution of labeled GFAP filaments. When labeled GFAP filaments were mixed with an excess of unlabeled filaments, a rapid increase of fluorescence was observed, which is due to an exchange of subunits between labeled and unlabeled GFAP filaments. These results indicate that GFAP filaments are dynamic structures and that a small pool of kinetically active unassembled GFAP subunits are in a dynamic equilibrium with assembled GFAP filaments. The ability of GFAP to assemble, disassemble, and undergo subunit exchange has important implications for the organization and dynamics of astroglia cell cytoskeleton during development and in response to injury.
Nakamura
Y
Y
Department of Neuropsychiatry, Osaka University Medical School, Japan.
Takeda
M
M
Angelides
K J
KJ
Tada
K
K
Hariguchi
S
S
Nishimura
T
T
D000818:Animals; D001253:Astrocytes / Q000378:metabolism; D002417:Cattle; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism*; D007382:Intermediate Filaments / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008274:Magnesium / Q000502:physiology
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005904:Glial Fibrillary Acidic Protein; C037041:fluorescein 5-maleimide; D008274:Magnesium