MEMBRANE SKELETON ASSEMBLY IN LENS DEVELOPMENT

脊椎动物眼睛纤维细胞的质膜下面 晶状体是由血影蛋白、肌动蛋白组成的高度交联的细胞骨架网络。 细丝和相关蛋白质，称为膜 骷髅。这项研究的长期目标是(1) 表征晶状体纤维细胞膜的分子结构 骨架，(2)建立膜骨架组装机制， (3)确定膜骨架在生成和释放过程中的作用 维持晶状体纤维细胞中特殊的质膜结构域。在……里面 本提案中，我们将重点介绍基因的表达、定位和分子 新型原肌球蛋白结合蛋白--晶状体原调节蛋白与肌动蛋白的关系 丝状末端封端蛋白很可能发挥关键作用 在调节肌动蛋白细丝的组装和组织中的作用 晶状体纤维细胞膜骨架。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晶状体纤维细胞的功能进行检查 分化和成熟，使用免疫荧光染色 冷冻切片与显微解剖、亚细胞分离和 Western blotting。此外，膜骨架的缔合 晶状体纤维中含有特殊质膜亚域的成分 细胞(例如，粘连连接、球窝交错、间隙 连接)将通过超微结构的免疫定位进行检查 水平。(2)测定可溶性未组装原调节蛋白是否 与超分子中的其他膜骨架成分相关联 复合体，从牛晶状体胞浆中提取的原调节蛋白将受到 蔗糖梯度沉淀、免疫共沉淀和超微结构 分析。这个建筑群可能代表着 未组装的晶状体纤维细胞膜骨架的亚单位也会 接受检查。(3)检验可溶原调节蛋白(和 其他膜骨架组件)是用于组装的前体 进入膜骨架，35S-蛋氨酸，脉冲追逐标记 鸡胚晶状体上皮细胞的原代培养 将进行Triton X-100提取和免疫沉淀。 (4)鸡晶状体原调节蛋白和原调节蛋白亚型的cDNA克隆 原肌球蛋白将被分离和测序和(5)表达和 原肌球蛋白和原肌球蛋白的亚细胞分布 我们将检查这些消息。我们的研究结果将会有 膜骨架在生成过程中的作用的广泛含义 在其他非红系细胞中的质膜结构域，也将提供 为未来研究膜的作用奠定必要的基础 视觉调节中的骨骼和晶状体纤维细胞的生物发生 白内障等病理性疾病。

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.