MOLECULAR ULTRASTRUCTURE UNDERLYING CELL ADHESION

肿瘤细胞的转移潜能是最重要的决定因素 以判断肿瘤疾病的预后。在分子步骤中 成功转移所需的几个离散事件 参与肿瘤细胞通过结缔组织屏障的侵袭。 细胞侵袭性与细胞外的黏附和蛋白分解有关 基质和基底膜，以及细胞运动性。这项建议 重点介绍质膜相关的蛋白水解酶和黏附分子 在人类恶性黑色素瘤细胞中发挥重要作用 这些细胞的侵袭性和转移潜能。最近的工作来自 本实验室鉴定了几种新的人膜蛋白水解酶。 恶性黑色素瘤细胞、鸡胚胎细胞、转化鸡 细胞和鸡胚。独特的细胞表面结构称为 在表达恶性表型的细胞上存在不定形 已被证明与细胞的侵袭性有关。这个 跨足类是活性酪氨酸磷酸化的部位，定位 β1整合素和细胞外基质的主动降解 通过膜蛋白水解酶。这项建议的目标是(1)确定 膜蛋白水解酶的生化性质研究 蛋白水解酶活性测定、单抗细胞定位 生产、亚细胞分级、抑制物和抗体亲和力 层析，蛋白质生物化学，包括微测序，以及 分子克隆，(2)确定膜蛋白水解酶如何与 激活降解的其他蛋白酶及其在表达中的作用 细胞侵袭性的研究：(3)研究细胞侵袭的调控机制 入侵，特别是侧重于直接或间接的 含有包括层粘连蛋白在内的假定运动因子的膜蛋白 A链肽、自分泌运动因子、酸性成纤维细胞生长 因子和白介素I在诱导性蹄内翻足和 人类整合素在稳定过程中定位于不定足类 侵袭相关膜，以及(4)确定其表达和 实验性侵袭过程中蛋白水解酶和黏附分子的定位 用扫描共聚焦显微镜观察转移模型和人类肿瘤 和免疫电子显微镜。

The metastatic potential of tumor cells is the most important determinant for the prognosis of neoplastic disease. Among the molecular steps required for successful metastasis are several discrete events that involve the invasion of tumor cells through connective tissue barriers. Cell invasiveness concerns adhesion to and proteolysis of extracellular matrices and basement membranes, as well as cell motility. This proposal focuses on plasma membrane-associated proteases and adhesion molecules of human malignant melanoma cells that are important in mediating the invasiveness and metastatic potential of these cells. Recent work from this laboratory identifies several novel membrane proteases from human malignant melanoma cells, chicken embryonic cells, chicken transformed cells, and chicken embryos. Unique cell surface structures called invadopodia are present on cells expressing the malignant phenotype and have been shown to be involved in the invasiveness of cells. The invadopodia are sites of active tyrosine-phosphorylation, localization of beta1 integrins, and active degradation of the extracellular matrix by membrane proteases. The goals of this proposal are (1) to determine the biochemical nature of membrane proteases by the combined approaches of protease activity assays, cellular localization by monoclonal antibody production, sub-cellular fractionation, inhibitor- and antibody-affinity chromatography, protein biochemistry including microsequencing, and molecular cloning, (2) to determine how membrane proteases interact with other proteases to activate degradation, and their role in the expression of cell invasiveness, (3) to investigate the control mechanisms of cell invasion, particularly focusing on the direct or indirect interaction of membrane proteases with putative motility factors including the laminin A chain peptide, autocrine motility factor, acidic fibroblast growth factor, and interleukin-I during the induction of invadopodia and with the human integrins that localize to invadopodia during the stabilization of invasion-associated membranes, and (4) to determine the expression and localization of protease and adhesion molecules in experimental invasion and metastasis models and human tumors by scanning confocal microscopy and immuno-electron microscopy.