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

基本信息

批准号：
2133742
负责人：
Diana Gilligan
金额：
$ 9.17万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1992
资助国家：
美国
起止时间：
1992-01-15 至 1997-09-29
项目状态：
已结题

项目摘要

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.

该项目的远距离目标是对多样化的理解膜骨骼蛋白的功能，添加剂。最初确定在红细胞中，几乎在所有细胞中表达添加剂。这个建议将重点介绍两种细胞类型的添加素功能：1）培养的上皮细胞，添加剂位于细胞细胞边界，而2）培养单核细胞系，添加剂局部与吞噬体。膜骨骼蛋白对于维持上皮至关重要细胞骨架组织的完整性和破坏是癌。膜骨骼蛋白对于宿主也很重要防御;白细胞的趋化性，依从性和吞噬作用是取决于膜骨骼蛋白功能。克隆编码Adducin的α和β亚基的红细胞cDNA（请参阅初步数据）将允许使用分子和细胞生物学技术研究上皮素和单核细胞。具体目的包括：I。）生理分析 adducin的功能，a。）上皮细胞系：1。中源性内源性通过转染上皮细胞系的附加素功能表达向量编码alpha和beta adducin的截短形式或反义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。确定添加剂磷酸化是否为细胞周期依赖性。 b。）单核细胞系：1。表征通过对培养的巨噬细胞的显微注射，加多丁素在吞噬作用带有抗adducin抗体或附加素的荧光标记结构域。 2。单核细胞的显微注射（或转染）带有附加素表达向量。 ii。）表征组织特异性添加剂同工型，a。）其他βadducin mRNA的cDNA克隆：1。放大使用基于PCR引物在克隆的大鼠脾脏cDNA上。 2。从人脑中分离克隆使用红细胞βadducin cDNA探针的cDNA文库。 b。）相关具有细胞功能的Adducin同工型表达1。使用同工型特异性抗体的组织特异性蛋白表达免疫印迹和免疫荧光显微镜。 2。扩展电流对附加素定位的了解，包括神经组织和心脏肌肉。 iii。）鉴定下真核生物中的adducin同源物一个。确定是否在果蝇中表达adducin同源物。通过免疫印迹全细胞或胚胎。 b。分离的cDNA克隆编码Adducin同源物。 c。创造带有附加素突变的菌株，以进一步分析 adducin。