MYOFIBRILLOGENESIS IN LIVING CARDIAC MUSCLE CELLS

拟议实验的长期目标是了解 控制胚胎心肌肌原纤维组装的参数 细胞和调控肌原纤维的维持和附着的细胞 成年动物的心肌细胞。实验的主要重点是 方法是通过分析活细胞内的肌原纤维 荧光标记细胞骨架蛋白的显微注射及其应用 定量光学技术。第一个具体目标是分析 在活体分离的胚胎心肌细胞中肌原纤维形成 视频和3D共聚焦显微镜来测试这一假说 肌原纤维是一种缩写形式，前肌原纤维， 通过掺入肌原纤维蛋白而延长。第二个目标 将研究非肌肉肌球蛋白IIB在 肌原纤维形成来验证这个分子负责的假说 对于前肌原纤维的初始短肌节单位的排列 这将成为成熟的肌原纤维的更长的肌节。这个 第三个目的是检验这样一种假设，即在Z中发现的一种蛋白质Zeugmatin- 带，负责融合前和前的Z-小体 新生的肌原纤维形成成熟的肌原纤维的Z带。第四个目标 是为了确定肌原纤维如何在蛋白质过程中保持其完整性 通过显微方法分析肌动蛋白进入细胞内的位置 成年心肌细胞的肌节。第五个具体目标是分析 表面Z带与细胞膜的功能性连接 一种在可变形橡胶表面生长细胞的新方法。五花八门 微注射探针将被用来检验这些Z-带的假设 通过整合素-纽蛋白-α-肌动蛋白系统偶联到表面 分子。提出了研究离体心的先进光学方法 肌肉细胞允许关于肌萎缩侧索硬化的形成和修复机制的假说 肌原纤维直接在活的心肌细胞中进行测试。一个 将使用集成的微观方法跟踪个人 心肌细胞从光镜到电子显微镜水平。这些 方法应该产生关于心肌细胞基本过程的新事实 从胚胎和成人心脏中分离出来。

The long-term goals of the proposed experiments are to understand the parameters governing the assembly of myofibrils in embryonic cardiac muscle cells and those governing the maintenance and attachment of myofibrils in cardiomyocytes of adult animals. The major emphasis of the experimental approaches is on analyzing myofibrils inside living cells via the microinjection of fluorescently labeled cytoskeletal proteins and the use of quantitative optical techniques. The first specific aim is to analyze myofibrillogenesis in living isolated embryonic cardiac myocytes using video and 3D-confocal microscopy to test the hypothesis that the future myofibril is laid down in a shortened version, the premyofibril, that lengthens by the incorporation of myofibrillar proteins. The second aim will investigate the role of non-muscle myosin IIB during myofibrillogenesis to test the hypothesis that this molecule is responsible for the alignment of the initial short sarcomeric units of the premyofibril which will become the longer sarcomeres of the mature myofibrils. The third aim is to test the hypothesis that zeugmatin, a protein found in Z- Bands, is responsible for the fusion of the Z-Bodies of the pre- and nascent myofibrils into Z-Bands of the mature myofibrils. The fourth aim is to determine how myofibrils maintain their integrity during protein turnover by analyzing with microscopic methods where actin enters the sarcomere of adult cardiomyocytes. The fifth specific aim is to analyze the functional attachment of the surface Z-Bands to the cell membrane using a novel method of growing cells on a deformable rubber surface. Various microinjected probes will be used to test the hypothesis that these Z-bands are coupled to the surface via a system of integrin-vinculin-alpha-actin molecules. The advanced optical methods proposed to study isolated cardiac muscle cells allow hypotheses about the formation and repair mechanisms of myofibrils to be tested directly in the living cardiac myocyte. An integrated microscopic approach will be used to follow individual cardiomyocytes from the light to the electron microscopic level. These approaches should yield new facts about basic processes in cardiac myocytes isolated from embryonic and adult hearts.