Characterization of Myosin in the Vertebrate Retina

本研究拟对猪肌肉中肌球蛋白的含量和亚型进行研究， 绿色翻车鱼和斑马鱼的视网膜。这些硬骨鱼 包含三种明确的细胞类型：视杆细胞、视锥细胞和RPE细胞 （视网膜色素上皮细胞）。三种细胞类型都显示 明确定义的变化，以响应光，黑暗，和某些 化学品这些形状变化发生在完整的视网膜， 在孤立的细胞中，被称为视网膜运动。一些 运动性的变化已被证明是肌动蛋白依赖性的，所以它 肌球蛋白可能也参与其中。肌球蛋白K/EDTA ATP酶 将通过标准技术测量活性。肌球蛋白将是 使用针对以下的单克隆或多克隆抗体进行免疫定位： 三种类型的非肌肉肌球蛋白，和单克隆抗体 还将制备视网膜肌球蛋白。肌球蛋白异构体合成和 沉积将在发育中的视网膜中确定;由于 视网膜发育的方式，年龄和形态发生的状态， 可以容易地确定各种细胞类型。 %%% 鱼视网膜对光线、黑暗和其他环境的反应更大。 比哺乳动物的视网膜更刺激。他们提供了一个方便的系统 为研究肌球蛋白在细胞凋亡中的作用， 形状肌动蛋白已经在鱼类视网膜细胞中被发现。在 在非肌肉组织中，至少有三种类型的肌球蛋白被 它们具有明显不同的属性。抗体 已经制备了这些同种型。它们将被用来确定 肌球蛋白的哪种形式存在于每种细胞类型中，以及它在哪里。 本地化。如果视网膜肌球蛋白不同于已知类型， 将制备针对它的单克隆抗体。由于 细胞的位置在视网膜发育过程中被很好地表征， 这些抗体将被用来观察在细胞周期中同种型是否发生变化。 这次由于肌球蛋白为基础的细胞运动是重要的， 生命过程，其结果很可能将适用于其他 系统也是。

This proposal is to study the content and isoforms of myosin in the retina of green sunfish and zebrafish. These teleost fish contain three well-defined types of cells: rods, cones, and RPEs (retinal pigmented epithelial cells). All three cell types show well-defined changes in response to light, darkness, and certain chemicals. These shape changes occur both in the intact retina and in isolated cells, and are known as retinomotor movements. Some of the motility changes have been shown to be actin-dependent, so it is likely that myosin is also involved. Myosin K/EDTA ATPase activity will be measured by standard techniques. Myosin will be immunolocalized using monoclonal or polyclonal antibodies against three types of non-muscle myosin, and monoclonal antibodies against retinal myosin will also be prepared. Myosin isoform synthesis and deposition will be determined in the developing retina; because of the way the retina develops, the age and state of morphogenesis of the various cell types can be easily determined. %%% Fish retinas change more in response to light, darkness, and other stimuli than mammalian retinas do. They provide a convenient system for studying the possible role of myosin in the changes in cell shape. Actin has already been identified in fish retinal cells. In non-muscle tissues, at least three types of myosin have been identified, which have markedly different properties. Antibodies to these isoforms have been prepared. They will be used to determine which form of myosin is present in each cell type, and where it is localized. If retinal myosin is different from the known types, monoclonal antibodies will be prepared against it. Since the location of cells is well-characterized during retinal development, the antibodies will be used to see whether isoforms change during this time. Since myosin-based cell motility is important for many life processes, it is likely that the results will apply to other systems as well.