FUNCTIONAL STUDIES OF INTERMEDIATE FILAMENTS IN GLIA

我们最近发现星形胶质细胞需要胶质特异性

We have recently shown that astrocytes require the glial-specific intermediate filament protein GFAP, in order to form stable processes in response to neurons by constitutive expression of anti-sense GFAP mRNA in the U251 glioma cell line. These experiments provided the first proof that intermediate filaments have tissue-specific functions as well as tissue-specific distributions. Although we have now shown that glial-specific intermediate filaments are necessary for the formation of stable astrocytic processes in response to neurons, they are clearly not sufficient. In order to study the mechanism by which glial processes are formed, we propose a series of experiments that are designed to investigate the mechanism(s) by which this protein can affect the shape changes of glial cells when neurons are added. For these studies, we will use molecular biological methods to determine whether the N-terminal head or C-terminal rod region of GFAP is the functional domain of the molecule. We have recently isolated fully encoding cDNAs for both GFAP and vimentin, the non-specific intermediate filament protein. In order to determine the specific domain of the GFAP molecule, we propose to finish sequencing these cDNAs and transfect the GFAP cDNAs cloned in eukaryotic expression vectors into fibroblasts. These studies will allow us to determine whether the protein will co-assemble with the endogenous vimentin intermediate filaments. We will also transfect these cDNAs into the previously prepared anti-sense GFAP cell lines, to show that we can rescue the ability of these cells to respond to neurons by forming astrocytic processes. However, to be able to pinpoint the functional domain of GFAP, we will have to prepare additional anti-sense GFAP cell lines transfected only with either the 5' or 3' end of the cDNAs, prepare vimentin-GFAP hybrid constructs, and determine which hybrid molecule can restore the ability of the glial cells to respond to neurons by the formation of stable astrocytic processes. We expect to be able to identity the important sequence(s) in the GFAP molecule by deletion and mutagenesis studies. Ultimately, we hope that we can delineate the series of events that occur to cause the formation of astrocytic processes when neurons are added to glial cells, as well as in the developing nervous system.