MEMBRANE SKELETON ASSEMBLY IN LENS DEVELOPMENT

位于脊椎动物眼睛纤维细胞的质膜下面 透镜是血影蛋白、肌动蛋白和肌动蛋白的高度交联的细胞骨架网络 丝和相关的蛋白质被称为膜 骷髅 本研究的长期目标是（1） 表征透镜纤维细胞膜的分子结构， （2）建立膜骨架组装机理， (3)以确定膜骨架在产生和 在透镜纤维细胞中维持专门的质膜区域。 在 本研究将着重于表达、定位和分子生物学方面的研究， 透镜原调节蛋白，一种新的原肌球蛋白结合和肌动蛋白的协会 细丝尖端的末端覆盖蛋白可能在 在调节肌动蛋白丝的组装和组织中的作用， 透镜纤维细胞膜骨架。 具体目标如下： (1)原调节蛋白、原肌球蛋白和血影蛋白的表达和组装 将在成年牛晶状体中作为透镜纤维细胞的函数进行检查 分化和成熟，使用免疫荧光染色， 冷冻切片与显微切割、亚细胞分离和 蛋白质印迹法。 此外，膜骨架的关联 在透镜纤维中具有专门质膜子域的组分 细胞（例如，粘连连接，球窝交错，间隙 连接）将通过免疫定位在超微结构 水平(2)为了确定可溶性未组装的原调节蛋白是否 与其他膜骨架成分在超分子 牛透镜胞质溶胶中的原调节蛋白复合物将经受 蔗糖梯度沉降、免疫共沉淀和超微结构 分析. 这个复合体有可能代表 未组装的透镜纤维细胞膜骨架的“亚单位”也将 接受检查。(3)为了检验可溶性原调节蛋白（和 其它膜骨架组分）是预定用于组装的前体 进入膜骨架，35 S-蛋氨酸，脉冲追踪标记 鸡胚透镜上皮细胞的原代培养细胞， 用Triton X-100提取并进行免疫沉淀。 (4)此外，鸡透镜原调节蛋白亚型的cDNA克隆和 原肌球蛋白将被分离和测序，和（5）表达和 原调节蛋白和原肌球蛋白的亚细胞分布， 将对这些信息进行审查。 我们的研究结果将 广泛的影响，膜骨架的作用， 质膜结构域在其他非红细胞，也将提供 未来研究膜作用的基本基础 骨骼在视觉调节和透镜纤维细胞发生中的作用 如白内障等疾病。

Underlying the plasma membrane of the fiber cells of the vertebrate eye lens is a highly cross-linked cytoskeletal network of spectrin, actin filaments and associated proteins that is referred to as the membrane skeleton. The long term objectives of this research are (1) to characterize the molecular organization of the lens fiber cell membrane skeleton, (2) to establish the mechanism of membrane skeleton assembly and (3) to determine the role of the membrane skeleton in generating and maintaining specialized plasma membrane domains in lens fiber cells. In this proposal, we will focus on the expression, localization and molecular associations of lens tropomodulin, a novel tropomyosin-binding and actin filament pointed end capping protein that is likely to play a critical role in regulating the assembly and organization of the actin filaments in the lens fiber cell membrane skeleton. The specific aims are as follows: (1) The expression and assembly of tropomodulin, tropomyosin and spectrin will be examined in adult bovine lenses as a function of lens fiber cell differentiation and maturation, using immunofluorescence staining of cryosections together with microdissection, subcellular fractionation and western blotting. In addition, the association of membrane skeleton components with specialized plasma membrane subdomains in the lens fiber cells (e.g., adherens junctions, ball-and-socket interdigitations, gap junctions) will be examined by immunolocalization at the ultrastructural level. (2) To determine whether soluble unassembled tropomodulin is associated with other membrane skeleton components in a supramolecular complex, tropomodulin from bovine lens cytosol will be subjected to sucrose gradient sedimentation, co-immunoprecipitation and ultrastructural analysis. The possibility that this complex could represent an unassembled "subunit" of the lens fiber cell membrane skeleton will also be examined. (3) To test the hypothesis that soluble tropomodulin (and other membrane skeleton components) are precursors destined for assembly into the membrane skeleton, 35S-methionine, pulse-chase labelling of primary culture cells of embryonic chick lens epithelial cells followed by extraction with Triton X-100 and immunoprecipitation will be performed. (4) In addition, cDNA clones for chick lens isoforms of tropomodulin and tropomyosin will be isolated and sequenced and (5) the expression and subcellular distribution of tropomodulin and tropomyosin proteins and these messages will be examined. The results of our studies will have broad implications for the role of the membrane skeleton in generating plasma membrane domains in other nonerythroid cells and will also provide the essential groundwork for future studies on the role of the membrane skeleton in visual accommodation and in the biogenesis of lens fiber cell pathologies such as cataracts.