Characterization of Myosin in the Vertebrate Retina

本研究旨在研究青太阳鱼和斑马鱼视网膜中肌球蛋白的含量和亚型。这些硬骨鱼包含三种明确定义的细胞：视杆细胞、视锥细胞和视网膜色素上皮细胞(RPE)。所有这三种细胞类型都显示出明确的变化，对光、暗和某些化学物质做出反应。这些形状变化既发生在完整的视网膜中，也发生在分离的细胞中，称为视网膜运动。一些运动性改变已经被证明是肌动蛋白依赖的，所以很可能肌球蛋白也参与其中。肌球蛋白K/EDTA ATPase活性将用标准技术测量。肌球蛋白将使用针对三种非肌肉肌球蛋白的单抗或多克隆抗体进行免疫定位，还将制备针对视网膜肌球蛋白的单抗。肌球蛋白异构体的合成和沉积将在发育中的视网膜中确定；由于视网膜的发育方式，可以很容易地确定各种细胞类型的年龄和形态发生状态。鱼的视网膜对光、暗和其他刺激的反应比哺乳动物的视网膜变化更大。它们为研究肌球蛋白在细胞形状变化中的可能作用提供了一个方便的系统。已经在鱼类视网膜细胞中发现了肌动蛋白。在非肌肉组织中，至少有三种类型的肌球蛋白被鉴定出来，它们具有明显不同的性质。针对这些异构体的抗体已经制备出来。他们将被用来确定肌球蛋白在每种细胞类型中的存在形式，以及它被定位在哪里。如果视网膜肌球蛋白与已知类型不同，将制备针对它的单抗。由于细胞的位置在视网膜发育过程中有很好的特征，抗体将被用来观察亚型在这段时间内是否发生变化。由于基于肌球蛋白的细胞运动对许多生命过程都很重要，这一结果很可能也适用于其他系统。

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.