FUNCTIONAL STUDIES OF INTERMEDIATE FILAMENTS IN GLIA

我们最近发现，星形胶质细胞需要胶质细胞特异性的 中间丝蛋白GFAP，以形成稳定的突起 原始性表达反义GFAP基因对神经元的反应 U251胶质瘤细胞系。这些实验首次证明了 中间纤维具有组织特有的功能以及 特定于组织的分布。尽管我们现在已经证明了 胶质细胞特有的中间丝是形成胶质细胞所必需的 稳定的星形细胞突起对神经元的反应，它们显然不是 足够了。为了研究神经胶质细胞突起的机制 ，我们提出了一系列实验，旨在研究 这种蛋白质影响细胞形态变化的机制(S) 当神经元被加入时，神经胶质细胞。对于这些研究，我们将使用 用分子生物学方法确定N-端头或 GFAP的C末端杆状区是该分子的功能结构域。我们 最近分离出了完全编码GFAP和Vimentin的cDNA， 非特异性中间丝蛋白。为了确定 GFAP分子的特定结构域，我们建议完成这些测序 克隆于真核表达载体中的GFAP cDNAs的构建及表达 转化成成纤维细胞。这些研究将使我们能够确定 蛋白质将与内源性波形蛋白中间体共同组装 细丝。我们还将把这些cDNA导入到先前制备的 反义GFAP细胞株，表明我们可以拯救这些能力 细胞通过形成星形细胞突起对神经元做出反应。然而，为了 要准确定位GFAP的功能域，我们将不得不做好准备 额外的反义GFAP细胞系仅转导5‘ 或cDNA的3‘端，制备vientin-GFAP杂化构建体，以及 确定哪种杂交分子可以恢复神经胶质细胞的能力 通过形成稳定的星形细胞突起对神经元作出反应。我们 期望能够识别出GFAP中的重要序列(S) 分子的缺失和诱变研究。最终，我们希望我们 可以描绘出发生的一系列事件，导致 当神经元被加入到神经胶质细胞中时，星形细胞过程以及在 发育中的神经系统。

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.