IMMUNOCYTOCHEMISTRY OF MEMEBRANE BIOSYNTHESIS IN EYE

膜蛋白的极化分布是MOST的一个特征 分化的细胞，尤其在视网膜中表达 光感受器。我们试图确定细胞机制的分类和 膜蛋白从合成部位到合成部位的矢量分布 功能场所。以往的研究主要集中在视蛋白的生物合成上。 并展示了高尔基体和高尔基体后囊泡在视蛋白中的作用 输送到杆件内段的顶端。这些研究已经 在EM采用高分辨率免疫细胞化学技术 水平。抗原既存在于细胞内部，也存在于细胞内。 在薄片上使用多克隆或鼠单抗的表面 视网膜包埋在Lowicryl K4M、白蛋白或冷冻蔗糖中。这个 新技术正在探索细胞表面抗原的分布 使用薄(2 Nm)的超高分辨率扫描电子显微镜 Nb或Ta涂层。结合的抗体由 使用生物素抗体的链霉亲和素-生物素桥技术和 链霉亲和素-金或使用链霉亲和素-生物素-白蛋白-金 或生物素-铁蛋白结合物。标记密度通过以下方式进行量化 半自动形态测量法。抗视蛋白抗体将用于研究视杆细胞 外节段视盘形态发生与视蛋白运输的终末阶段 从睫状脊复合体到外段 连接纤毛质膜。抗蟾蜍视网膜和脑的抗体 将生成(Na+，K+)ATPase，并比较其特异性和交叉 与现有多克隆和单抗的反应性 对抗蟾酥肾ATPase。将产生新的单克隆抗体来 其他光感受器和视网膜神经元质膜作为 高尔基体中膜蛋白的分选。的专一性 抗体将通过结合到电泳法进行进一步评估 视网膜蛋白和纯化的ATPase及其蛋白水解酶的转印 碎片。使用几种类型的抗体对抗几种不同的 光感受器和其他视网膜神经元中的抗原应该可以让我们 确定我们关于特定分类的调查结果在多大程度上 视蛋白可以推广到其他视网膜膜蛋白。双标签 技术应直接评估高尔基体在分选中的作用 这些蛋白质。

Polarized distribution of membrane proteins is a characteristic of most differentiated cells and is especially expressed in retinal photoreceptors. We seek to determine to cellular mechanisms of sorting and vectorial distribution of membrane proteins from sites of synthesis to sites of function. Prior studies have concentrated on opsin biosynthesis and demonstrated the role of the Golgi and post-Golgi vesicles in opsin transport to the apex of the rod inner segment. These studies have employed high resolution techniques of immunocytochemistry at the EM level. Antigens are localized in the interior of cells as well as on cell surfaces using polyclonal or mouse monoclonal antibodies on thin sections of retinas embedded in Lowicryl K4M, albumin or frozen sucrose. The distribution of cell surface antigens is being explored by new techniques of ultrahigh resolution scanning electron microscopy using thin (2nm) niobium or tantalum coatings. Bound antibodies are detected by the streptavidin-biotin-bridge technique using biotinyl antibodies and streptavidin-gold or using streptavidin followed by biotinyl-albumin-gold or biotinyl-ferritin conjugates. Labeling densities are quantitated by semiautomated mophometry. Antiopsin antibodies will be used to study rod outer segment disk morphogenesis and terminal stages of opsin transport from the periciliary ridge complex to the outer segment along the connecting cilium plasma membrane. Antibodies to toad retina and brain (Na+, K+) ATPase will be generated and compared for specificity and cross reactivity with presently available polyclonal and monoclonal antibodies against toad kidney ATPase. New monoclonal antibodies will be generated to other photoreceptor and retinal neuron plasma membranes as probes of the sorting of membrane proteins in the Golgi apparatus. Specificity of the antibodies will be further evaluated by binding to electrophoretic transblots of retinal proteins and purified ATPase and its proteolytic fragments. The use of several types of antibodies to several different antigens in photoreceptors and other retinal neurons should allow us to determine the degree to which our findings regarding specific sorting of opsin can be generalized to other retinal membrane proteins. Double label techniques should directly evaluate the role of the Golgi in sorting of these proteins.