MOLECULAR ANALYSIS OF OCULAR LENS BEADED FILAMENTS

描述：珠状丝是结构独特的细胞骨架 元件，由两种蛋白质组成，仅在纤维细胞中表达。 目镜透镜（CP 49和CP 115）。 由于珠丝蛋白的表达 当它被打开时，其他细胞骨架蛋白的表达被关闭。 这最终使珠状细丝成为主导，也许是唯一的 成熟纤维细胞中的细胞骨架结构。 在此前的融资中， 在此期间，PI建立了很多关于序列和结构的信息。 珠丝蛋白。 该信息为以下方面提供了依据 提出旨在：确定珠状细丝功能的问题 在透镜中;确定珠状灯丝对透镜的重要性 生物学;确定珠丝蛋白的分子机制 大会;建立如何，作为中间灯丝的极端成员 家庭，串珠灯丝已利用独特的修改，主要 序列和二级结构，以适应透镜的特定功能。 的 PI建议三个主要的调查途径：I。什么是/是 珠状纤维在透镜纤维细胞生物学中的作用？ 他 将采用超微结构，生物化学和遗传学方法，包括 种系“敲除/敲入”策略。 二. 什么是分子 两种珠丝蛋白之间的相互作用 他们怎么 他们的主要任务是什么？他们的主要任务是什么？ 序列和二级结构 他将使用表达的蛋白质，酵母二号 混合分析，组装研究，交联，分析色谱， 和突变分析。 三. 使用什么机制来管理 透镜纤维细胞的细胞质中珠状纤维蛋白的水平？ 这些蛋白质的化学计量是如何保持平衡的， 珠丝蛋白磷酸化的作用？ 他会用生殖细胞 敲除、蛋白质和mRNA水平分析、转染和组装 问题研究

DESCRIPTION: The beaded filament is a structurally unique cytoskeletal element, composed of two proteins expressed only in the fiber cell of the ocular lens (CP49 and CP115). As the expression of Beaded Filament proteins is turned on, the expression of other cytoskeletal proteins is turned off. This ultimately leaves the Beaded Filament as the dominant, and perhaps sole cytoskeletal structure in the mature fiber cell. In the previous funding period the PI established much about the sequence and structure of the Beaded Filament proteins. This information provides a rationale basis for asking questions designed to: establish the function of the Beaded Filament in the lens; determine the importance of the Beaded Filament to lens biology; determine the molecular mechanisms of Beaded Filament protein assembly; establish how, as extreme members of the Intermediate Filament family, the Beaded Filament has utilized unique modifications to primary sequence and secondary structure to adapt to lens-specific functions. The PI proposes three major avenues of investigation: I. What is/are the role(s) of the Beaded Filament in the biology of the lens fiber cell? He will employ ultrastructural, biochemical, and genetic approaches, including germline "knock out/knock in" strategies. II. What are the molecular interactions between the two Beaded Filament proteins? How do they assemble, and what is the impact of the unique changes seen in their primary sequence and secondary structure? He will use expressed proteins, Yeast Two Hybrid analysis, assembly studies, cross-linking, analytical chromatography, and mutational analysis. III. What mechanisms are used to regulate the levels of Beaded Filament proteins in the cytoplasm of the lens fiber cell? How is the stoichiometry of these proteins kept in balance, and what is the role of Beaded Filament protein phosphorylation? He will use germline knockouts, analysis of protein and mRNA levels, transfection, and assembly studies.