LAMININ-5 AND HEMIDESMOSOMES IN ORAL EPITHELIAL CELLS

半桥粒，细胞基质连接器装置，用于口服 上皮细胞也附着在牙龈的结缔组织上 如牙齿表面。 在口腔上皮细胞的细胞质中 半桥粒也是细胞骨架锚定到 细胞表面在牙周病的过程中， 必要性，调节半桥粒完整性作为上皮细胞 细胞沿着牙齿迁移形成“长连接 上皮”。 在我们研究半桥粒在 牙周病的发病机制我们必须有一个基本的 了解参与这两个分子的组成部分， 组装和结构维护 形态实体 为此，在本计划的项目1中， 项目申请，我们计划把我们的努力集中在功能 的粘附性细胞外糖蛋白组分的分析 层粘连蛋白-5的半桥粒及其对口腔上皮细胞的影响 细胞 在目标I中，我们解决了以下结构域的识别： 层粘连蛋白-5异源三聚体参与半桥粒成核 组装件. 这将涉及重组层粘连蛋白-5的制备 蛋白质以及层粘连蛋白-5肽。 在Aim 2中， 将测定重组蛋白和肽的能力 a）诱导口腔上皮细胞的快速粘附，和B） 半桥粒的核装配。 作为替代策略 为了鉴定层粘连蛋白-5的功能性重要结构域， 目的2我们将使用肽竞争的粘附性能， 完整层粘连蛋白-5。 在目标3中，我们希望确定底物 层粘连蛋白-5的结合位点。 为此，蛋白水解片段的 将测定层粘连蛋白-5的a）它们粘附于表面的能力 和/或B）与天然层粘连蛋白-5竞争底物结合。 功能活性片段将进一步表征为： 微测序 在目标4中，细胞与层粘连蛋白结合的影响- 将对半桥粒组分进行生化测定。 我们特别感兴趣的是层粘连蛋白5 通过磷酸化事件在基质细胞信号传导中起作用。 最后，在目标5中，我们希望确定 层粘连蛋白-5。这种联系对于稳定 牙齿表面的口腔上皮。

Hemidesmosomes, cell-matrix connector devices, are used by oral epithelial to attach to the connective tissue of the gingiva as well as the tooth surface. In the cytoplasm of an oral epithelial cell hemidesmosomes are also sites of cytoskeleton anchorage to the cell surface. During the course of periodontal disease, there is, of necessity, modulation in hemidesmosome integrity as epithelial cells migrate along the tooth to form the "long junctional epithelium". Before we can study the role of hemidesmosomes in the pathogenesis of periodontal disease we must have a basic understanding of the molecular components involved in both the assembly and structural maintenance of this complex morphological entity. To this end, in Project 1 of this program project application, we plan to focus our efforts on functional analyses of an adhesive extracellular glycoprotein component of hemidesmosomes called laminin-5 and its impact on oral epithelial cells. In Aim I we address the identification of domains of the laminin-5 heterotrimer involved in nucleation of hemidesmosome assembly. This will involve preparation of recombinant laminin-5 proteins as well as laminin-5 peptides. In Aim 2, these recombinant proteins and peptides will be assayed for their ability a) to induce rapid adhesion of oral epithelial cells and b) to nucleate assembly of hemidesmosomes. As alternative strategies for identification of functional important domains of laminin-5, in Aim 2 we will use peptides to compete the adhesive properties of intact laminin-5. In Aim 3, we wish to identify the substrate binding site(s) of laminin-5. To this end, proteolytic fragments of laminin-5 will be assayed for a) their ability to adhere to surfaces and/or b) to compete with native laminin-5 for substrate binding. Functionally active fragments will be further characterized by microsequencing. In Aim 4, the impact of cell binding to laminin- 5 on hemidesmosome components will be assayed biochemically. We are particularly interested in the possibility that laminin-5 plays a role in matrix-cell signaling via phosphorylation events. Finally, in Aim 5, we wish to identify membrane interactions of laminin-5. This linkage is necessary for the stabilization of the oral epithelium on the tooth surface.