BIOSYNTHESIS OF SCHWANN CELL EXTRACELLULAR MATRIX

该项目的长期目标是了解 细胞外基质(ECM)与神经系统的发育。这个 雪旺细胞的终末分化，这涉及到在 轴突膜和髓鞘的形成严格依赖于接触 使用ECM。在体内和原代细胞培养中，这种细胞外基质是基础 雪旺细胞在手术前和手术过程中合成的膜 髓鞘形成时期。本提案涉及以下三个相关方面 总体目标；即表达的结构、功能和调控 雪旺细胞表面硫酸乙酰肝素蛋白多糖(HSPGs)的表达。 我们实验室以前的研究表明，雪旺细胞 合成两种不同的细胞表面HSPG，它们的模式不同 与细胞表面的结合：脂质锚定的HSPG和 与先前发现的上皮细胞跨膜相似或相同 HSPG Syndecan。我们实验室的其他实验表明，这些 热休克蛋白G参与雪旺细胞与基底细胞的相互作用 膜蛋白层粘连蛋白。我们建议用免疫细胞化学的方法来研究 方法将这些HSPG在雪旺细胞中进行亚细胞定位。 文化分化的不同阶段。我们还将确定 无论是神经细胞还是基底膜接触，这都是已知的 强烈影响雪旺细胞的行为，影响合成， 分布，或由雪旺细胞处理这些HSPG。这些 实验将利用生化、免疫学和 分子技术。为了研究细胞表面热休克蛋白G的功能，我们 将在雪旺细胞和神经细胞的培养中添加特定的抗体 并观察它们对雪旺细胞组装能力的影响 基底膜新生及对外源性基底膜的反应 扩散、包裹轴突和髓鞘形成轴突。我们还将尝试 脂质锚定雪旺细胞核心蛋白编码基因的克隆 HSPG。在这些实验中，雪旺细胞的c DNA表达文库 用抗体或寡核苷酸进行筛选。核心蛋白质序列将 被亚克隆到适当的载体中进行进一步分析，如DNA 序列确定。

The long range goal of this project is to understand the role of extracellular matrix (ECM) in the development of the nervous system. The terminal differentiation of Schwann cells, which involves spreading on axonal membranes and myelin formation is strictly dependent upon contact with ECM. In vivo and in primary cell cultures this ECM is the basement membrane that is synthesized by the Schwann cells prior to and during the period of myelination. This proposal addresses 3 related aspects of this overall goal; namely, the structure, function and regulation of expression of cell surface heparan sulfate proteoglycans (HSPGs) of Schwann cells. Previous studies from our laboratory have shown that Schwann cells synthesize 2 distinct cell surface HSPGs that differ in their mode of association with the cell surface: a lipid anchored HSPG and an HSPG that is similar or identical to a previously identified epithelial transmembrane HSPG syndecan. Other experiments from our laboratory suggest that these HSPGs are involved in the interaction of Schwann cell with the basement membrane protein laminin. We propose to investigate by immunocytochemical methods the subcellular localization of these HSPGs in Schwann cells at various stages of differentiation in culture. We will also determine whether nerve cell or basement membrane contact, which are known to strongly influence Schwann cell behavior, affect the synthesis, distribution, or processing these HSPGs by Schwann cells. These experiments will utilize a combination of biochemical, immunological and molecular techniques. To examine the function of the cell surface HSPGs we will add specific antibodies to cultures of Schwann cells and nerve cells and observe their effects on the ability of the Schwann cells to assembly basement membrane de novo and to respond to exogenous basement membrane be spreading on and ensheathing and myelinating axons. We will also attempt to clone cDNA encoding the core protein of the lipid anchored Schwann cell HSPG. For these experiment a Schwann cell cDNA expression library will be screened with antibodies or oligonucleotides. Core protein sequences will be subcloned into the appropriate vectors for further analysis, such as DNA sequence determination.