Studies of the glial fibrillary acidic protein: A model for intermediate filament assembly and its function in astrocyte process formation

All intermediate filament protein consists of an {dollar}alpha{dollar}-helical rod domain flanked by non-helical N-terminal head and C-terminal tail domains. The roles of these non-helical domains in the assembly and co-assembly of higher order filamentous structures have been studied by many groups but with quite contradictory results. Type III intermediate filaments are unique in that they can form homopolymers both in vitro and in vivo. The expression and assembly characteristics of carboxy- and amino-terminal deletion mutants of glial fibrillary acidic protein (GFAP), an astrocyte-specific type III intermediate filament protein, were examined by transient transfections of either vimentin-positive or vimentin-negative variants of human adrenocarcinoma-derived SW13 cell lines. Whereas complete deletion of the C-terminal tail domain of GFAP results in the formation of polymorphic aggregates, both intranuclear and cytoplasmic, in self-assembly experiments, efficient co-assembly of these tail-less GFAP mutants with vimentin can be achieved as long as the "KLLEGEE" sequence at the end of the {dollar}alpha{dollar}-helical rod domain is preserved. Only up to one-fifth of the C-terminal end of the tail domain can be deleted without affecting the capability of GFAP to self-assemble. A highly conserved "RDG"-containing motif in the tail domain may be necessary for self-assembly, but is not sufficient. The entire head domain seems to be required for self-assembly. All N-terminal deletion mutants of GFAP share the same phenotype of diffuse cytoplasmic staining when expressed in vimentin-negative SW13 cells. Although co-assembly with vimentin can still be achieved with completely head-less GFAP, preservation of some of the head domain greatly enhanced the efficiency.; Astroglial cells play an important role in orchestrating the migration and positioning of neurons during central nervous system development. Primary astroglia, as well as astrocytoma cells will extend long stable processes when co-cultured with granule neurons. In order to determine the function of the glial fibrillary acidic protein (GFAP), the major intermediate filament protein in astroglia and astrocytoma cells, we suppressed the expression of GFAP by stable transfection of an anti-sense GFAP construct in human astrocytoma U251MG cells. The resulting AS-U251 cells can no longer extend stable processes in the presence of granule neurons. To show that this effect is due specifically to the absence of GFAP, we reintroduced a fully encoding rat brain GFAP cDNA into these AS-U251 cells. The resulting rat GFAP appeared as a filamentous network and the reexpression of GFAP rescued the ability of these astrocytoma cells to form stable processes when co-cultured with neurons. Therefore, the glial specific intermediate filament protein, GFAP, is required for process extension of these astrocytoma cells in response to granule neurons.