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: 8126431
• 负责人: ANDREW G MASELLI
• 金额: $ 10.03万
• 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/8126431
• 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; 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; Work; base; cell fixing; cell motility; cell type; comparative; crosslink; light microscopy; plastin; 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.

随着人口老龄化，神经退行性疾病成为主要的健康问题。一个最小的身上 研究神经退行性疾病的结构表现是平野小体。平野小体是肌动蛋白 基于内含物，这已经在具有广泛的 神经退行性疾病，包括阿尔茨海默病。虽然超微结构和蛋白质 平野小体的组成是众所周知的，导致平野小体形成的事件级联 仍然未知。确定导致组装的事件对于理解 病理学中的平野小体。我们的假设是平野体的形成是由于不受控制的肌动蛋白 这种不受控制的交联，与细胞的自然交联 能力，生成一个稳定的平野身体结构.平野身体形成的主要原因之一是 缺乏活细胞模型系统。我们已经证明，截断的表达式 具有不受控制的肌动蛋白交联活性的肌动蛋白结合蛋白可以诱导Hirano小体的形成， 真核生物模式系统盘基网柄藻（Dictyosteelium discoideum）（Maselli，2002; Maselli，2003）。模特Hirano 在网骨藻中形成的体具有与在人脑中发现的平野体相似的特征 （Maselli，2002年）。通过表达截短的肌动蛋白结合蛋白（t-abp）绿色荧光蛋白（GFP） 与诱导型表达系统融合，我们将能够观察到细胞中平野小体的形成。通过 将t-abp探针与丝状肌动蛋白的探针结合，我们可以观察到发生在 平野身体形成的最早阶段。扩展我们的假设，人类细胞中t-abp的一个可能来源 是细胞自身肌动蛋白结合蛋白的蛋白水解切割片段。我们打算验证我们的假设 通过在细胞中表达可能的切割片段，并观察细胞的内含物形成。的 平野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，并使用免疫电镜和FIAsH标记技术，以关联我们的 从光镜观察到超微结构。更好地理解事件的级联和 平野身体形成的可能触发因素将进一步加深我们对平野潜在作用的理解 神经退行性疾病的身体。