STUDIES OF CELL JUNCTIONS & CELL MEMBRANES IN THE LENS

我们建议扩展对连接复合物和细胞膜的研究 在脊椎动物的眼透镜中。 本申请的长期目标是获得更好的 了解结构特征、分布和功能 各种类型的细胞连接在透镜中的作用。 细胞膜是 Al细胞连接的主要成分。 据信， 连接和细胞膜在维持正常的 结构生理学和透镜的透明度。 我们扩展我们 研究目标是深入了解细胞膜的作用 参与有用大分子的内吞作用和囊泡运输 进入透镜。 研究计划的具体目标如下：（1）直接 透镜中自然间隙结连接子结构分析与比较 上皮细胞和纤维细胞，使用快速冷冻技术;（2）研究 缝隙连接与肌动蛋白束和肌动蛋白相关 组分，使用细胞松弛素-D处理，以及改进的固定 含戊二醛、赖氨酸和单宁酸;（3）免疫细胞化学 间隙连接蛋白（MP 70和/或MP26）、肌动蛋白和肌动蛋白的定位- 成束蛋白;（4）细胞超微结构和免疫细胞化学分析 鸡透镜形态发生过程中粘附连接的形成;（5） 形态学和免疫细胞化学研究的发生，结构 以及桥粒和相关细胞骨架成分在透镜中的性质 不同种属的上皮细胞;（6）超微结构和免疫细胞化学 研究了与细胞凋亡相关的独特膜结构域（网格蛋白样）， 纤维细胞中球窝连接的形成;（7） 包被囊泡和非包被囊泡的结构和分布 不同物种的透镜，使用改进的固定和蛋白质示踪剂 方法：（8）研究生长因子等因素对 透镜中的液相和受体介导的内吞作用;（9） 免疫细胞化学测定可能涉及的一些生长 利用其特异性抗体作为探针研究内吞作用中的因子。 使用的方法包括（1）透射电子显微镜， 本实验室开发的改良固定方法;（2）快速冷冻 在没有预先化学固定的液氦温度下， 结合自由置换、冷冻断裂和深蚀刻 技术;（3）常规冷冻断裂TEM;（4）免疫荧光 免疫胶体金标记技术;（5）蛋白质示踪和细胞化学 方法.

We propose to extend the studies of junctional complexes and cell membranes in the ocular lens of vertebrates. The long-term objectives of this application are to gain a better understanding in structural characteristics, distributions and functional roles of various types of cell junctions in the lens. Cell membranes are the major component of al cell junctions. It is believed that cell junctions and cell membranes play important roles in maintaining normal architecture physiology, and transparency of the lens. We extend our research goals to gain some insights into the roles of cell membranes involved in endocytosis and vesicular transport of useful macromolecules into the lens. The specific aims of the research plan are as follows: (1) Direct structural analysis and comparison of native gap-junction connexons in lens epithelium and fiber cells, using rapid-freezing techniques; (2) Study association of gap junctions with actin bundles and actin-associated components, using cytochalasin-D treatments, and an improved fixation containing glutaraldehyde, lysine and tannic acid; (3) Immunocytochemical localization of gap junction proteins (MP70 and/or MP26), actin and actin- bundling proteins; (4) Ultrastructural and immunocytochemical analyses of formation of adherens junctions during chick lens morphogenesis; (5) Morphological and immunocytochemical studies of the occurrence, structure and nature of desmosomes and associated cytoskeletal components in lens epithelium of various species; (6) Ultrastructural and immunocytochemical investigations of unique membrane domains (clathrin-like) associated with formation of ball-and-socket junctions in fiber cells; (7) Visualization of structure and distribution of coated vesicles and non-coated vesicles in the lens of various species, using improved fixation and protein tracer methods; (8) Study the effects of growth factors and other factors on fluid-phase and receptor-mediated endocytosis in the lens; (9) Immunocytochemical determination of possible involvements of some growth factors in endocytosis using their specific antibodies as probes. Methodology used incudes (1) transmission electron microscopy using the improved fixation method developed in this laboratory; (2) rapid-freezing in liquid helium temperature without prior chemical fixation, and in conjunction with free-substitution, freeze-fracture and deep-etching techniques; (3) conventional freeze-fracture TEM; (4) immunofluorescence and immunogold labeling techniques, and (5) protein tracer and cytochemical methods.