Formation, Stability, and Structure of Hirano Bodies Using a Live Cell System

使用活细胞系统的平野体的形成、稳定性和结构

• 批准号: 7897946
• 负责人: ANDREW G MASELLI
• 金额: $ 17.14万
• 依托单位: CHICAGO STATE UNIVERSITY
• 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/7897946
• 关键词: Actin-Binding Protein; Actins; Age; Alzheimer' s Disease; Antibodies; Antigens; Binding; Binding Sites; Biological Assay; Biological Models; Biological Preservation; Brain; Caspase; Cell model; Cell physiology; Cells; Characteristics; Chemicals; Chicago; Crosslinker; Cytoskeleton; DNA Sequence Rearrangement; Data; Dictyostelium; Dictyostelium discoideum; Event; F-Actin; Fimbrin; Freezing; Funding; Green Fluorescent Proteins; Health; Homologous Gene; Human; Image; Immersion Investigative Technique; Immunoelectron Microscopy; Individual; Kinetics; Lead; Libraries; Life; Light Microscope; Literature; Methodology; Methods; Modeling; Neurodegenerative Disorders; Pathology; Peptide Hydrolases; Phagocytosis; Pinocytosis; Population; Process; Protein Fragment; Protein Isoforms; Proteins; Role; Sepharose; Site; Source; Staging; Structure; System; T-plastin; Technology; Testing; Time; Universities; Work; base; cell fixing; cell motility; cell type; comparative; crosslink; light microscopy; plastin; programs; promoter; sample fixation

Neurodegenerative diseases become a major health concern as the population ages. One of the least studied structural manifestations of neurodegenerative disease are Hirano bodies. Hirano bodies are actin based inclusions, which have been identified in the brains of individuals with a broad range of neurodegenerative disorders including Alzheimer's disease. Although the ultrastructure and protein composition of Hirano bodies is well known the cascade of events that leads to Hirano body formation remains unknown. Determining the events that lead to assembly is important to understanding the role of Hirano bodies in pathology. Our hypothesis is that Hirano bodies form as the result of uncontrolled actin cross-linker activity and that this uncontrolled cross-linking, in concert with the cell's natural cross-linking ability, generate a stable Hirano body structure. One of the major reasons that Hirano body formation has not been studied is the lack of a live cell model system. We have shown that the expression of a truncated actin binding protein with uncontrolled actin cross-linking activity can induce the formation of Hirano bodies in the eukaryotic model system Dictyostelium discoideum (Maselli, 2002 ;Maselli, 2003). The model Hirano bodies formed in Dictyostelium have similar characteristics to the Hirano bodies found in the human brain (Maselli, 2002). By expressing a truncated actin binding protein (t-abp) Green Fluorescent Protein (GFP) fusion with an inducible expression system we will be able to observe Hirano body formation in cells. By combining the t-abp probe with a probe for filamentous actin we can observe changes that take place at the earliest stages of Hirano Body formation. Extending our hypothesis, a likely source of t-abp in human cells are proteolytic cleavage fragments of the cells own actin biding proteins. We propose to test our hypothesis by expressing the likely cleavage fragments in cells, and observing the cells for inclusion formation. The ultrastructure of Hirano Bodies is key to understanding both their formation and stability. We will determine the optimal fixation method and used immuno electron microscopy and FIAsH tag technology to correlate our observations from light microscopy to the ultrastructure. Better understanding the cascade of events and the possible triggers for Hirano Body formation will further our understanding of the potential roles of Hirano Bodies in neurodegenerative disease.

随着人口年龄的增长，神经退行性疾病成为主要健康问题。最少之一 研究的神经退行性疾病的结构表现是Hirano身体。海兰尸体是肌动蛋白 基于包含物，这些包含物已在具有广泛范围的个体的大脑中识别 神经退行性疾病，包括阿尔茨海默氏病。虽然超微结构和蛋白质 众所周知的是众所周知的事件级联 仍然未知。确定导致集会的事件对于理解的作用很重要 病理学中的植物。我们的假设是，由于不受控制的肌动蛋白而形成了平民体 交联活动以及这种不受控制的交联，与单元的自然交联一致 能力，产生稳定的Hirano身体结构。 Hirano身体形成的主要原因之一 没有研究的是缺乏现场电池模型系统。我们已经证明了截短的表达 肌动蛋白结合蛋白具有不受控制的肌动蛋白交联活性可以诱导静脉体的形成 真核模型系统迪斯特尔迪斯特尔（Maselli，2002; Maselli，2003）。模型Hirano 在Dictyostelium中形成的物体具有与人脑中发现的Hirano体的相似特征 （Maselli，2002）。通过表达截短的肌动蛋白结合蛋白（T-ABP）绿色荧光蛋白（GFP） 与诱导表达系统的融合，我们将能够观察细胞中的Hirano身体形成。经过 将T-ABP探针与探针结合了丝状肌动蛋白，我们可以观察到发生的变化 Hirano身体形成的最早阶段。扩展我们的假设，这是人类细胞中T-ABP的可能来源 是细胞自己肌动蛋白b的蛋白水解裂解片段。我们建议检验我们的假设 通过表达细胞中可能的切割片段，并观察细胞的包含形成。这 广场体的超微结构是了解其形成和稳定性的关键。我们将确定 最佳固定方法和使用免疫电子显微镜和FIASH标签技术来关联我们 从光学显微镜到超微结构的观察结果。更好地了解一系列事件和 可能的触发触发器形成将进一步理解我们对Hirano的潜在作用的理解 神经退行性疾病的身体。