MOLECULAR ULTRASTRUCTURE UNDERLYING CELL ADHESION

肿瘤细胞的转移潜能是最重要的决定因素 用于肿瘤疾病的预后。 在分子步骤中 成功转移所需的是几个离散事件， 涉及肿瘤细胞通过结缔组织屏障的侵袭。 细胞侵袭力涉及粘附和蛋白水解的细胞外基质， 基质和基底膜，以及细胞运动。 这项建议 主要研究质膜相关蛋白酶和粘附分子 人类恶性黑色素瘤细胞，这是重要的介导， 这些细胞的侵袭性和转移潜力。 最近的工作从 本实验室从人类中鉴定出几种新的膜蛋白酶， 恶性黑色素瘤细胞，鸡胚细胞，鸡转化 细胞和鸡胚。 独特的细胞表面结构， 侵袭伪足存在于表达恶性表型的细胞上， 已经被证明与细胞的侵袭性有关。 的 侵袭伪足是活性酪氨酸磷酸化、定位 β 1整合素，以及细胞外基质的主动降解 通过膜蛋白酶。 本提案的目标是（1）确定 膜蛋白酶的生物化学性质 蛋白酶活性测定，单克隆抗体细胞定位 生产、亚细胞分级、抑制剂和抗体亲和力 蛋白质生物化学，包括微测序，以及 分子克隆，（2）确定膜蛋白酶如何与 其他蛋白酶激活降解，以及它们在表达中的作用 （3）探讨细胞侵袭力的调控机制 入侵，特别是侧重于直接或间接的相互作用 具有假定的运动因子（包括层粘连蛋白）的膜蛋白酶 A链肽，自分泌运动因子，酸性成纤维细胞生长 因子和白细胞介素-I在侵袭性伪足诱导过程中的作用， 在稳定过程中定位于侵袭伪足的人整合素 的侵袭相关膜，和（4）以确定表达和 蛋白酶和粘附分子在实验性侵袭中的定位 和转移模型和人类肿瘤的扫描共聚焦显微镜 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.