Transfection of Lens Cells with Phakinin Mutants

描述(申请人提供)：拟议研究的长期目标是确定晶状体特有的珠状丝状蛋白(BFP)细胞骨架如何有助于晶状体清晰度。我们的假设是，最近发现的与人类白内障形成相关的BF亚单位phakinin的突变中断了BF的自我组装或生化过程。特定目的1将验证这样一种假设，即BFP的原位生化特性在体外条件下或在将BF亚单位构建融合到标记蛋白质标签的培养细胞中时不会改变。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完成，该细胞携带融合到荧光蛋白(FP)或HA表位的BF亚单位构建物。特定目标2将测试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BFP的聚合和/或生化处理的假设。这将在将突变的phakinin亚单位与荧光蛋白或抗原决定簇融合在一起的培养细胞中完成。BF聚合和生化过程将通过共聚焦显微镜和表达蛋白的免疫化学检测进行监测。这些研究的结果将有助于更好地了解珠状纤维的功能和某些形式的白内障的病因。

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.