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 细胞和那些管理维持和附着的肌原纤维， 成年动物的心肌细胞。 实验的重点是 方法是通过分析活细胞内的肌原纤维， 荧光标记的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 用离体胚胎心肌细胞的肌原纤维发生 视频和3D共聚焦显微镜来测试未来的假设， 肌原纤维以缩短的形式，即前肌原纤维， 通过肌原纤维蛋白的掺入而延长。 第二个目的 将研究非肌肉肌球蛋白IIB在 肌原纤维形成来检验这种分子负责 对于前肌原纤维的初始短肌节单位的排列 其将成为成熟肌原纤维的较长的肌节。 的 第三个目的是检验一种在Z- 带，负责融合的Z-机构的前和 新生肌原纤维进入成熟肌原纤维的Z带。 第四个目标 是为了确定肌原纤维在蛋白质分解过程中如何保持其完整性， 通过显微镜方法分析肌动蛋白进入细胞的位置， 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上生长细胞的新方法。 各种 显微注射探针将被用来测试假设，这些Z带 通过整联蛋白-黏着斑蛋白-α-肌动蛋白系统与表面偶联 分子。 研究离体心脏的先进光学方法 肌肉细胞允许关于形成和修复机制的假设， 在活心肌细胞中直接测试肌原纤维。 一个 将使用综合显微镜方法跟踪个体 心肌细胞从光镜水平到电镜水平。 这些 这些方法应该会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.