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，以形成稳定的过程， 通过反义GFAP mRNA的组成性表达， U251胶质瘤细胞系。这些实验提供了第一个证据， 中间丝具有组织特异性功能， 组织特异性分布。尽管我们现在已经表明， 神经胶质特异性中间丝是形成 稳定的星形胶质细胞过程对神经元的反应，它们显然不是 足够了。为了研究神经胶质细胞的机制， 形成，我们提出了一系列的实验，旨在调查 这种蛋白质可以影响形状变化的机制 当神经元加入时，神经胶质细胞。对于这些研究，我们将使用 分子生物学方法来确定是否N-末端头部或 GFAP的C-末端杆状区域是该分子的功能结构域。我们 最近分离出了GFAP和波形蛋白的完全编码cDNA， 非特异性中间丝蛋白。为了确定 具体结构域的GFAP分子，我们建议完成测序这些 克隆到真核表达载体中的GFAP cDNA 转化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组装 细丝。我们还将这些cDNA插入到先前制备的 反义GFAP细胞系，以表明我们可以拯救这些细胞的能力， 细胞通过形成星形胶质细胞突起对神经元作出反应。但要 为了能够精确定位GFAP的功能域，我们必须准备 仅用5 ′-寡核苷酸转染的另外的反义GFAP细胞系 或3’末端，制备波形蛋白-GFAP杂合构建体，和 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胶质细胞突起对神经元作出反应。我们 期望能够鉴定GFAP中的重要序列 分子通过缺失和诱变研究。最终，我们希望 可以描述一系列事件的发生，导致形成 当神经元加入胶质细胞时，星形胶质细胞的过程，以及 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.