DESMOSOMES--DYNAMICS AND ASSEMBLY

细胞-细胞粘附结构，桥粒，密切参与 上皮组织的形态发生。跨膜桥粒 桥粒胶蛋白（Desmocollin，Dsc）和桥粒芯糖蛋白（Desmoglein，Dsg）是一种多糖蛋白， cadherins基因家族，并在桥粒功能中发挥重要作用。 桥粒钙粘蛋白的细胞内区域整合在 细胞质斑块，作为中间 细丝。关于桥粒组装的机制和 功能没有关于桥粒动力学的数据报道。 我们的初步结果表明，直接钙依赖的相互作用 在Dsg和Dsc的胞外区之间。我们假设 这些相互作用不仅介导细胞-细胞粘附， Dsg/Dsc复合物依赖于斑珠蛋白和桥粒斑蛋白组装成 桥粒本提案的总体目标是评估这一点 假说.为此，我们将定义氨基酸残基内 桥粒钙粘蛋白的胞外区参与Dsg-Dsc 相互作用，并确定这种相互作用对桥粒的影响 组装件.我们还将研究桥粒钙粘蛋白是否能够形成 这种相互作用对桥粒组装的影响。我们将研究桥粒是否 钙粘蛋白能够形成同二聚体。结合此 调查中，我们将检查斑珠蛋白的功能， 桥粒虽然我们之前的实验证明了 这种蛋白在桥粒装配中的作用还不清楚。 最近，一种新的桥粒重建模型， HIT-1080细胞是在我实验室建立的。这种模式为我们提供了 有机会利用野生型基因研究斑珠蛋白的功能， HT-1080细胞中表达的蛋白质的类型和突变形式。最终 这项建议的一部分集中在阐明桥粒动力学， 尤其是上皮细胞在迁移过程中。完成 为此，我们构建了PgGFP嵌合蛋白， 斑珠蛋白和绿色荧光蛋白。我们已经证明这种嵌合体 被适当地整合到桥粒中， 通过延时荧光显微镜观察细胞。利用这个奇美拉， 研究桥粒组装和拆卸的动力学， 对上皮片层损伤和不同细胞的反应 文化环境。本提案中描述的实验将 为理解分子机制提供了不可或缺的数据 负责上皮组织在发育和创伤中的重塑 治愈

The cell-cell adhesion structures, desmosomes, are intimately involved in the morphogenesis of epithelial tissues. Transmembrane desmosomal glycoproteins, desmocollin (Dsc) and desmoglein (Dsg), belong to a multi- gene family of cadherins and play an important role in desmosome function. The intracellular regions of desmosomal cadherins are integrated in the cytoplasmic plaque, serving as an anchorage site for intermediate filaments. Little is known about the mechanisms of desmosome assembly and function. No data has been reported concerning desmosomal dynamics. Our preliminary results indicate a direct calcium-dependent interaction between the extracellular regions of Dsg and Dsc. We hypothesize that these interactions not only mediate cell-cell adhesion, but also trigger plakoglobin and desmoplakin dependent assembly of Dsg/Dsc complexes into desmosomes. The broad goal of the present proposal is to evaluate this hypothesis. To this end we will define amino acid residues within extracellular regions of the desmosomal cadherins involved in Dsg-Dsc interactions and determine the effect of this interaction on desmosome assembly. We will also study whether desmosomal cadherins are able to form this interaction on desmosome assembly. We will study whether desmosomal cadherins are able to form homodimers. In conjunction with this investigation we will examine the function of the plakoglobin in desmosomes. While our previous experiments demonstrated a critical role of this protein in desmosome assembly, its function is not well understood. Recently a novel model of desmosome reconstitution in plakoglobin-negative HIT-1080 cells was established in my laboratory. This model provides us with an opportunity to investigate the function of plakoglobin using wild type and mutant forms of the protein expressed in HT-1080 cells. The final part of this proposal is focused on elucidating desmosome dynamics in epithelial cells, in particular, during their migration. To accomplish this, we have constructed a PgGFP chimeric protein consisting of intact plakoglobin and Green Fluorescent protein. We have shown that this chimera is integrated properly into desmosomes that can be visualized in living cells by time-lapse fluorescence microscopy. Using this chimera we will investigate the dynamics of assembly and disassembly of desmosomes in response to injury of an epithelial sheet and to different cellular environments in culture. The experiments described in this proposal will provide data indispensable for understanding the molecular mechanisms responsible for remodeling of epithelial tissue in development and wound healing.