Myofibrillogenesis in Living Cardiac Muscle Cells

描述（由申请人提供）：肌原纤维的关键亚基，Z带，串联连接肌节收缩单位，也作为中间丝、中肋和膜的附着位点。这种肌原纤维复合体是如何组装的尚不清楚。第一个具体的目的是分析组装的Z-带活细胞在两个禽文化系统：心前中胚层文化，其中第一肌原纤维的形式，和10天的胚胎心肌细胞培养，其中新的肌原纤维组装在细胞与预先存在的肌原纤维。第二个具体目的是测试突变的Z -带肌联蛋白片段的表达（对应于报道参与肥厚性心肌病的突变）将改变Z-带稳定性、干扰α-辅肌动蛋白交联肌动蛋白丝的能力和/或诱导转染的心肌细胞中的肌原纤维紊乱的想法。第三个具体的目的是分析一个新的Z带蛋白，NspI-1，在Z体的成熟肌原纤维的Z带的过渡和Z带结构的维护中的作用。Nspl-1可能是连接Z带、中链、中间丝和膜的潜在重要分子。实验方法包括使用编码连接到肌节蛋白和突变结构域的绿色荧光蛋白组成员的质粒转染心肌细胞。活细胞的图像将通过光学技术进行分析，例如光漂白后荧光恢复（FRAP）、共聚焦和去卷积显微镜。这些先进的光学方法与分子和生物化学技术相结合，将用于测试有关肌原纤维形成的假设，以及突变Z带肌联蛋白片段对与它们相互作用的蛋白质动力学的影响。这些方法应该产生新的见解的作用，Z-带在心肌细胞中的肌原纤维组装的基本和病理过程。

DESCRIPTION (provided by applicant): A key subunit of myofibrils, the Z-band, links the sarcomeric contractile units in series and also serves as attachment sites for the intermediate filaments, the costameres and membranes. How this myofibrillar complex is assembled is unknown. The first specific aim is to analyze the assembly of Z-bands in live cells in two avian culture systems: precardiac mesoderm cultures in which the first myofibrils form, and 10-day embryonic cardiomyocyte cultures in which new myofibrils assemble in cells with pre-existing myofibrils. The second specific aim is to test the idea that expression of a mutated Z -band titin fragment, corresponding to a mutation reported to be involved in Hypertrophic Cardiomyopathy, will alter Z-band stability, interfere with the ability of alpha-actinin to cross-link actin filaments, and/or induce myofibril disarray in transfected cardiomyocytes. The third specific aim is to analyze the role of a new Z-band protein, NspI-1, in the transition of Z-bodies to Z-bands of mature myofibrils and in the maintenance of Z-band structure. Nspl-1 may be a potentially important molecule for linking Z-bands, costameres, intermediate filaments, and membranes. The experimental approaches include the use of plasmids encoding members of the group of Green Fluorescent Proteins ligated to sarcomeric proteins and mutated domains for transfections of cardiomyocytes. Images of live cells will be analyzed by optical techniques, such as Fluorescence Recovery after Photobleaching (FRAP), confocal and deconvolution microscopy. These advanced optical methods coupled with molecular and biochemical techniques will be used to test hypotheses about the formation of myofibrils and the effects of mutant Z- band titin fragments on the dynamics of proteins interacting with them. These approaches should yield new insights into the role of the Z-band in basic and pathologic processes in myofibril assembly in cardiomyocytes.