MORPHOLOGICAL MECHANISMS OF ORGANELLE FUNCTION AND TRANSFORMATION IN CULTURE

肿瘤转化会导致细胞生理学上的许多变化。 促进生长的荷尔蒙会产生一些同样的变化。内吞作用 是一个调节细胞与细胞相互作用的过程 促进生长的激素和转化病毒的进入。在 近年来，我们对培养细胞的内吞作用途径进行了研究 已发现表皮生长因子(一种促生长激素)(EGF) 和转铁蛋白(一种细胞必需的血浆铁结合蛋白 生长)(Tf)以相同的途径共同内化到人类肿瘤中 细胞，但在跨网状网络中彼此分叉 高尔基体系。使用电子显微镜进行的细胞化学实验 研究表明，EGF和TF的受体也内化于 配基。然而，已经发现EGF及其受体被递送到 溶酶体降解，而转铁蛋白及其受体原封不动地循环 回到细胞表面。两者在形态上的差异 配体-受体类型似乎涉及高尔基体网蛋白包裹的凹陷 系统。我们现在已经证明了使用反络合蛋白进行细胞分离 亲和吸附技术显示组织因子和表皮生长因子的分离相同 从形态上看。研究还表明，EGF受体的进入 可能至少部分是通过佛波醇处理细胞而诱导的 酯类在没有EGF的情况下。评估配体-受体的位置 分类时，我们为高尔基体和溶酶体系统使用了新的标记， 主要内容包括：(1)黄毛藻凝集素标记唾液酸残基 反式高尔基体系统，(2)抗溶酶体膜的LAMP-1单抗 蛋白，(3)抗高尔基堆叠标记蛋白的ABL-70单抗， (4)抗MEP抗体作为溶酶体基质蛋白的标志物；(5)2C6 作为α2-巨球蛋白受体标志物的单抗。我们 研究了配基进入溶酶体的形态动力学 LAMP-1作为溶酶体标记物的双标记实验系统 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。

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.