RBC ADDUCIN--MOLECULAR/CELL BIOLOGY OF TISSUE ISOFORMS

红细胞内收蛋白——组织亚型的分子/细胞生物学

• 批准号: 3081004
• 负责人: Diana Gilligan
• 金额: $ 8.81万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-15 至 1994-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3081004
• 关键词: Caenorhabditis elegans; Dictyostelium; Drosophilidae; Paramecium; antisense nucleic acid; brain; chemotaxis; complementary DNA; electron microscopy; epithelium; erythrocytes; gene expression; genetic library; heart; immunofluorescence technique; intercellular connection; laboratory rabbit; macrophage; membrane proteins; messenger RNA; microinjections; molecular cloning; monocyte; peripheral nervous system; phagocytosis; phosphorylation; polymerase chain reaction; protein biosynthesis; protein isoforms; protein sequence; protein structure function; site directed mutagenesis; spinal cord; tissue /cell culture; transfection; western blottings

The long range goal of this project is an understanding of the diverse functions of a membrane skeletal protein, adducin. Initially identified in erythrocytes, adducin is expressed in nearly all cells. This proposal will focus on adducin function in two cell types: 1) cultured epithelial cells, where adducin is localized to cell-cell borders, and 2) cultured monocytic cell lines, where adducin is localized to phagosomes. Membrane skeletal proteins are essential for maintenance of epithelial integrity and disruption of cytoskeletal organization is a feature of carcinomas. Membrane skeletal proteins are also important for host defense; chemotaxis, adherence, and phagocytosis by leukocytes are dependent upon membrane skeletal protein function. The cloning of erythrocytes cDNAs encoding both alpha and beta subunits of adducin (see preliminary data) will allow a combined approach using molecular and cell biology techniques to study adducin function in epithelia and monocytes. The specific aims include: I.) Analysis of physiological functions of adducin, a.) Epithelial cell lines: 1. Interrupt endogenous adducin function by transfection of epithelial cell lines with expression vectors encoding truncated forms of alpha and beta adducin or antisense mRNA. 2. Analyze effects of transfection on junction formation using immunofluorescence, electron microscopy and in vivo phosphorylation. 3. Determine whether adducin phosphorylation is cell-cycle dependent. b.) Monocytic cell lines: 1. Characterize adducin's role in phagocytosis by microinjection of cultured macrophages with anti-adducin antibodies or fluorescently tagged domains of adducin. 2. Microinjection (or transfection) of monocytes with adducin expression vectors. II.) Characterization of tissue-specific adducin isoforms, a.) cDNA cloning of additional beta adducin mRNAs: 1. Amplify the alternative 3' end of erythrocyte beta adducin using PCR primers based upon the cloned rat spleen cDNA. 2. Isolate clones from a human brain cDNA library using erythrocyte beta adducin cDNA probes. b.) Correlation of adducin isoform expression with cellular functions 1. Analyze tissue-specific protein expression using isoform-specific antibodies for immunoblotting and immunofluorescence microscopy. 2. Expand current knowledge of adducin localization to include nervous tissue and cardiac muscle. III.) Identification of adducin homologues in lower eukaryotes a. Determine whether adducin homologues are expressed in Drosophila, C. elegans, Dictyostelium, or Paramecium by immunoblotting whole cells or embryos. b. Isolate cDNA clones encoding adducin homologues. c. Create strains with adducin mutations in order to further analyze functions of adducin.

该项目的长期目标是了解多元化 膜骨架蛋白内收蛋白的功能。 初步确定 在红细胞中，内收蛋白几乎在所有细胞中表达。这个提议 将重点关注两种细胞类型中的内收蛋白功能：1) 培养的上皮细胞 细胞，其中内收蛋白定位于细胞-细胞边界，并且 2) 培养 单核细胞系，其中内收蛋白定位于吞噬体。 膜骨骼蛋白对于维持上皮细胞的功能至关重要 细胞骨架组织的完整性和破坏是 癌。 膜骨骼蛋白对宿主也很重要 防御;白细胞的趋化性、粘附性和吞噬作用是 依赖于膜骨架蛋白的功能。 的克隆 编码内收蛋白 α 和 β 亚基的红细胞 cDNA（参见 初步数据）将允许使用分子和 研究上皮内收蛋白功能的细胞生物学技术和 单核细胞。 具体目标包括： I.) 生理分析 内收蛋白的功能，a.) 上皮细胞系： 1. 中断内源性 通过转染上皮细胞系来发挥内收蛋白功能 编码α和β内收蛋白的截短形式的表达载体或 反义mRNA。 2. 分析转染对连接形成的影响 使用免疫荧光、电子显微镜和体内 磷酸化。 3. 确定内收蛋白是否磷酸化 细胞周期依赖性。 b.) 单核细胞系： 1. 表征 内收蛋白在培养巨噬细胞显微注射吞噬作用中的作用 具有抗内收蛋白抗体或荧光标记的内收蛋白结构域。 2. 内收蛋白表达的单核细胞显微​​注射（或转染） 向量。 II.) 组织特异性内收蛋白亚型的表征，a.) 其他 β 内收蛋白 mRNA 的 cDNA 克隆： 1. 扩增 使用基于 PCR 引物的红细胞 β 内收蛋白的替代 3' 末端 克隆的大鼠脾脏 cDNA。 2. 从人脑中分离克隆 使用红细胞β内收蛋白cDNA探针的cDNA文库。 b.) 相关性 内收蛋白亚型表达与细胞功能的关系 1. 分析 使用异构体特异性抗体进行组织特异性蛋白质表达 免疫印迹和免疫荧光显微镜。 2. 扩大电流 内收蛋白定位知识，包括神经组织和心脏 肌肉。 III.) 低等真核生物中内收蛋白同源物的鉴定 一个。确定内收蛋白同源物是否在果蝇、C. 线虫、盘基网柄菌或草履虫，通过免疫印迹全细胞或 胚胎。 b.分离编码内收蛋白同源物的 cDNA 克隆。 c. 创造 具有内收蛋白突变的菌株，以进一步分析其功能 内收蛋白。