Transfection of Lens Cells with Phakinin Mutants

描述（由申请人提供）：拟议研究的长期目标是确定晶状体特异性珠状丝蛋白（BFP）细胞骨架如何有助于晶状体清晰度。我们的主要假设是，最近发现的与人类白内障形成相关的BF亚基phakinin突变中断了BF自组装或生化处理。特异性目的1将检验在体外条件下或在将BF亚基构建物与标记蛋白标签融合的培养细胞转染期间BFPs的原位生化特性不会改变的假设。这将通过共聚焦显微镜和免疫化学检测在培养的鸡晶状体环垫细胞中转染了与荧光蛋白（FPs）或HA表位融合的BF亚基构建物。特异性目的2将验证与人类白内障相关的phakinin突变在体外晶状体纤维分化过程中干扰BFPs的聚合和/或生化处理的假设。这将在转染与荧光蛋白或抗原决定因子融合的突变phakin亚单位的培养细胞中完成。用共聚焦显微镜和表达蛋白的免疫化学检测来监测BF聚合和生化过程。这些研究结果将有助于更好地了解珠丝功能和某些形式白内障的病因。

DESCRIPTION (provided by applicant): The long-term objectives of the proposed studies are to determine how the lens-specific beaded filament protein (BFP) cytoskeleton contributes to lenticular clarity. Our overlying hypothesis is that recently identified mutations in the BF subunit phakinin associated with human cataract formation interrupt BF self-assembly or biochemical processing. Specific Aim 1 will test the hypothesis that the in situ biochemical characteristics of BFPs are not altered under in vitro conditions or during the transfection of cultured cells with BF subunit constructs fused to marker protein tags. This will be accomplished with confocal microscopy and immunochemical detection in cultured chicken lens annular pad cells transfected with BF subunit constructs fused to fluorescent proteins (FPs) or the HA epitope. Specific Aim 2 will test the hypothesis that phakinin mutations associated with human cataracts interfere with the polymerization and/or biochemical processing of BFPs during in vitro lens fiber differentiation. This will be accomplished in cultured cells transfected with mutated phakinin subunits fused to fluorescent proteins or antigenic determinants. BF polymerization and biochemical processing will be monitored with confocal microscopy and immunochemical detection of the expressed proteins. The results of these studies will lead to a better understanding of beaded filament function and the etiology of some forms of cataract.