CYTOSKELETAL REGULATION BY EXTRACELLULAR MATRIX ADHESION

内皮细胞在许多生理和病理过程中发挥作用， 如炎症、血栓形成、伤口修复和新生血管。 这些过程都涉及内皮细胞与其他细胞的粘连。 以及细胞外基质。有能力经历的先决条件 在迁移和管道形成过程中的形状变化是一种灵活的 细胞骨架。免疫荧光研究表明， 迁移和黏附过程中的细胞骨架组织。然而， 支配细胞骨架转变的分子事件有 目前还不能很好地理解。我们有初步数据表明 细胞骨架重排的一种机制是通过改变 蛋白质4.1亚型。蛋白4.1是一种细胞骨架蛋白，具有 广泛的组织分布。它的红系功能很好- 特征包括与幽灵蛋白和肌动蛋白的结合以及 跨膜糖蛋白和脂类。它被表示为一系列 通过选择性剪接信使核糖核酸产生的异构体。这项提议将 重点阐明其分布和类型的变化。 与不同细胞外黏附时产生的蛋白质4.1亚型 二维和三维细胞培养中的基质成分 暴露于转化生长因子-β。蛋白质4.1亚型的分布和类型 将首先建立用于融合的静息内皮细胞 文化。这将涉及蛋白质和信使核糖核酸分析，包括 免疫印迹、免疫荧光、Northern印迹、核酸酶保护、 代谢标记和聚合酶链式反应。血管内皮细胞 然后在相同的条件下用同样的技术进行检查 促进细胞外基质的迁移和改变 组成。更长的(170kD)蛋白4.1 异构体是通过对尚未确定的区域进行选择性剪接而产生的 将使用诸如随机等技术来处理核苷酸序列 CDNA端的扩增。在蛋白质4.1亚型的模式具有 这些多肽的功能被发现是显著的 将会被检查。这些多肽与其他细胞骨架蛋白的结合 通过亲和层析对膜受体进行分析， 固相分析、沉淀法、竞争分析和定点定位 诱变。最终，这些实验可能会导致未来 粘着产生的跨膜信号的研究 细胞外基质及其调控机制 信使核糖核酸选择性剪接。这项工作应该进一步阐明 对继发性血管损伤等过程的分子反应 血管成形术和动脉内膜切除术，血栓形成，新生血管， 炎症和伤口修复。

The endothelium functions in many physiological and pathological processes, such as inflammation, thrombosis, wound repair, and neovascularization. These processes all involve adhesion of the endothelial cell to other cells and to the extracellular matrix. A prerequisite for the ability to undergo shape changes during migration and tube formation is a flexible cytoskeleton. Immunofluorescence studies have demonstrated alterations in cytoskeletal organization during migration and adhesion. However, the molecular events which govern the transformations of the cytoskeleton are currently not well understood. We have preliminary data which suggest that one mechanism for cytoskeletal rearrangement would be via changes in protein 4.1 isoforms. Protein 4.1 is a cytoskeletal protein with widespread tissue distribution. Its erythroid functions are well- characterized and include binding to spectrin and actin as well as transmembrane glycoproteins and lipids. It is expressed as a series of isoforms which arise by alternative splicing of mRNA. This proposal will focus on the elucidation of the changes in the distribution and types of protein 4.1 isoforms which occur upon adhesion to different extracellular matrix components in two and three-dimensional cell cultures and with exposure to TGF-beta. The distribution and types of protein 4.1 isoforms will first be established for the resting endothelial cell in confluent cultures. This will involve both protein and mRNA analysis, including immunoblotting, immunofluorescence, Northern blotting, nuclease protection, metabolic labeling and polymerase chain reaction. The endothelial cells will then by examined with the same techniques under conditions that promote migration and with alteration in the extracellular matrix composition. The question of whether the longer (170 kD) protein 4.1 isoform arise by alternative splicing of as yet unidentified regions of nucleotide sequence will be addressed with techniques such as random amplification of cDNA ends. After the pattern of protein 4.1 isoforms has been identified, the functions of those peptides found to be significant will be examined. Binding of these peptides to other cytoskeletal proteins and to membrane receptors will be analyzed by affinity chromatography, solid phase assay, sedimentation, competition assay, and site directed mutagenesis. Eventually, these experiments could lead to future investigations of the transmembrane signal generated by adhesion to extracellular matrix and the mechanism by which the cell regulates alternative splicing of mRNA. This work should further elucidate the molecular response to processes such as vascular injury secondary to angioplasty and endarterectomy, thrombosis, neovascularization, inflammation and wound repair.