LYSOSOME BIOGENESIS IN NORMAL AND TUMOR CELLS

正常细胞和肿瘤细胞中的溶酶体生物发生

• 批准号: 2181288
• 负责人: PHILIP D STAHL
• 金额: $ 29.89万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181288
• 关键词: ADP ribosylation; G protein; Xenopus oocyte; endocytosis; guanine nucleotide binding protein; intracellular transport; lysosomes; membrane biogenesis; membrane fusion; monoclonal antibody; neoplastic cell; phospholipase A2; tissue /cell culture; transfection /expression vector; vesicle /vacuole; western blottings

DESCRIPTION: Receptor-mediated endocytosis is a fundamental process carried out by all cells and is characterized by the internalization of plasma membrane-derived coated vesicles and a series of ordered and specific fusion events among coated vesicles, endosomes, and lysosomes. During the last grant period, in vitro assays were developed to reconstituted early fusion events. Heterotrimeric G proteins and a variety of other factors emerged as important regulators of endocytic vesicle fusion. These assays will now be used to identify key GTP binding proteins and other novel regulators such as phospholipase A2 and characterize their mechanisms of action. Dr. Stahl plans four major specific aims. (1) To use density shift and/or immunoisolation techniques to prepare enriched preparations of endosomes for biochemical and morphological analysis. These fractions will be used to identify cytosolic and membrane proteins associated with endosomes that have been "primed" for recognition and fusion. Ultrastructural characterization of putative fusion pores will be continued. Finally, monoclonal antibodies will be prepared that inhibit fusion. (2) To elucidate the role of heterotrimeric G proteins and ADPribosylation factor (ARF) in fusion, G proteins present in the enriched vesicle fraction will be identified by western blot and mutant or wild type G protein subunits and different ARF family members overexpressed in intact cells using the Sindbis virus expression system. Effects on in vitro fusion or endocytosis in intact cells will be sought. (3) Since G proteins are likely to be activated by upstream effectors, such effectors will be sought. Given that clathrin adaptor proteins have an effect on the in vitro fusion assay, the possibility that adaptins themselves serve this effector function will be evaluated. The role of downstream effectors, such as rab5, NSF and phospholipase A2 will also be evaluated. (4) Endosome-lysosome fusion will be reconstituted in permeabilized cell systems or by using Xenopus oocytes injected with mRNA encoding molecules of interest.

描述：受体介导的内吞作用是一个基本过程 由所有细胞执行，以此为特征 质膜衍生的涂层囊泡和一系列有序的囊泡和 涂层囊泡，内体和溶酶体之间的特定融合事件。 在最后一个赠款期间，开发了体外测定 重组的早期融合事件。 异三聚体G蛋白和A 出现的各种其他因素是内吞的重要调节剂 囊泡融合。 这些测定现在将用于识别密钥GTP 结合蛋白和其他新型调节剂，例如磷脂酶A2和 表征他们的作用机制。 Stahl博士计划四个专业 具体目标。 （1）使用密度转移和/或免疫分散技术准备 生化和形态学的内体富集制剂 分析。 这些分数将用于识别胞质和 与内体相关的膜蛋白已被“启动” 识别和融合。推定的超微结构特征 融合孔将继续。 最后，单克隆抗体将是 准备抑制融合。 （2）阐明异三聚体G蛋白和 融合中的adpribosylation因子（ARF），G蛋白存在于G蛋白中 富集的囊泡分数将通过蛋白质印迹和突变体鉴定 或野生型G蛋白亚基和不同的ARF家族成员 使用信德氏病毒表达系统在完整细胞中过表达。 对完整细胞中体外融合或内吞作用的影响将是 寻求。 （3）由于G蛋白可能会被上游效应子激活，所以 将寻求此类效应子。鉴于网格蛋白衔接蛋白具有 对体外融合测定的影响，适应蛋白的可能性 本身将评估此效应函数。 的作用 下游效应子，例如RAB5，NSF和磷脂酶A2也将 进行评估。 （4）内体 - 散糖体融合将在透化细胞中重组 系统或使用注入mRNA编码分子的爪蟾卵母细胞 感兴趣的。