MORPHOLOGICAL MECHANISMS OF ORGANELLE FUNCTION AND TRANSFORMATION IN CULTURE

培养物中细胞器功能和转化的形态机制

• 批准号: 4691814
• 负责人: M C WILLINGHAM
• 金额: --
• 依托单位: DIVISION OF CANCER BIOLOGY AND DIAGNOSIS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/4691814
• 关键词: Adenoviridae; Golgi apparatus; Pseudomonas; bacterial toxins; cell motility; cellular oncology; cytotoxicity; electron microscopy; epidermal growth factor; gel electrophoresis; image enhancement; immunochemistry; lysosomes; microinjections; molecular biology; monoclonal antibody; muscle proteins; neoplastic growth; neoplastic transformation; organelles; pinocytosis; receptor mediated endocytosis; tissue /cell culture; transforming virus; viral carcinogenesis

Neoplastic transformation produces many changes in cell physiology. Growth-promoting hormones produce some of these same changes. Endocytosis is a process that regulates both the interaction of cells with growth-promoting hormones and the entry of transforming viruses. In the last few years, our study of the pathway of endocytosis in cultured cells has revealed that epidermal growth factor (a growth promoting hormone)(EGF) and transferrin (a plasma iron-binding protein necessary for cell growth)(TF) are co-internalized in the same pathway into human carcinoma cells, but diverge from each other in the trans-reticular network of the Golgi system. Cytochemical experiments using electron microscopy have shown that the receptors for EGF and TF are also internalized with the ligands. However, EGF and its receptor have been found to be delivered to lysosomes and degraded, whereas TF and its receptor are recycled intact back to the cell surface. The morphologic divergence of these two ligand-receptor types appears to involve clathrin-coated pits of the Golgi system. We have now shown that cell fractionation using an anti-clathrin affinity absorbant technique revealed the same separation of TF and EGF seen morphologically. It was also shown that the entry of the EGF receptor could be at least partially induced by treatment of cells with phorbol esters in the absence of EGF. To evaluate the site of ligand-receptor sorting, we employed new markers for the Golgi and lysosomal systems, including: (1) Limax flavus lectin labeling of sialic acid residues in the trans-Golgi system, (2) LAMP-1 monoclonal antibody to a lysosomal membrane protein, (3) ABL-70 monoclonal antibody to a Golgi stack marker protein, (4) antibody to MEP as a marker of lysosomal matrix proteins, and (5) 2C6 monoclonal antibody as a marker for the alpha2-macroglobulin receptor. We examined the morphologic kinetics of delivery of ligand into the lysosomal system in double-label experiments using LAMP-1 as a marker for lysosomal membranes. We also devised a new method for preservation of membrane proteins for immunofluorescence detection.

肿瘤转化在细胞生理学中产生许多变化。 生长促进激素也会产生一些相同的变化。 内吞 是一个调节细胞与 生长促进激素和转化病毒的进入。 在 近年来，我们对培养细胞内吞途径的研究， 表皮生长因子（EGF）是一种生长促进激素。 和转铁蛋白（细胞必需的血浆铁结合蛋白） 生长因子）（TF）以相同的途径共同内化到人类癌中 细胞，但在细胞的跨网状网络中彼此分叉。 高尔基系统。 使用电子显微镜的细胞化学实验 EGF和TF的受体也被内化， 配体。 EGF及其受体被认为是 溶酶体并降解，而TF及其受体则完整回收 回到细胞表面。 这两个物种的形态差异 配体-受体类型似乎涉及高尔基体网格蛋白包被的小凹 系统 我们现在已经表明，使用抗网格蛋白的细胞分级分离 亲和吸附技术显示TF和EGF的分离效果相同 从形态上看。 研究还表明，表皮生长因子受体的进入 可以至少部分地通过用佛波醇处理细胞来诱导 在没有EGF的情况下。 评价配体-受体结合位点 分类时，我们采用了新的标记物用于高尔基体和溶酶体系统， 包括：（1）黄缘鲎凝集素标记唾液酸残基， trans-Golgi系统，（2）针对溶酶体膜的LAMP-1单克隆抗体 蛋白质，（3）针对高尔基体标志物蛋白质的ABL-70单克隆抗体， (4)作为溶酶体基质蛋白标志物的MEP抗体，和（5）2C 6 单克隆抗体作为α 2-巨球蛋白受体的标记物。 我们 检查了将配体递送到溶酶体中的形态学动力学 使用LAMP-1作为溶酶体标记物的双标记实验中的系统 膜。 我们还设计了一种新的膜保存方法 用于免疫荧光检测的蛋白质。