TROPOMODULIN FUNCTION IN HEART DEVELOPMENT

心肌中完全排列的肌原纤维的形成代表了对真核细胞中超分子组装的戏剧性检查；发生这种组装的机制仍然不完全清楚。这项研究的长期目标是确定在肌原纤维组装过程中调节收缩蛋白相互作用的分子成分和机制。这项建议的目的是利用一种新的心肌原纤维组装模型来确定原调节蛋白在调节细丝组装和组织中的功能(S)及其特定的分子相互作用，该模型忠实地概括了体内肌原纤维组装。我的职业研究发展计划包括建立一个跨学科的心肌研究计划，将包括通过正规课程工作、与同事的互动和实践努力获得分子发育生物学和先进显微技术(包括实时荧光图像分析)方面的专业知识。亚利桑那大学将提供所需的所有资源。这将使在活细胞和生物体范围内研究细胞骨架蛋白成为可能。其目的是：(1)研究肌原纤维组装在分化心肌细胞中的作用。来自禽类胚胎心脏前区域的外植体将被用来研究肌原纤维蛋白的出现和组装的时间模式。2)研究原调节蛋白-原肌球蛋白的相互作用，并通过微量注射功能阻断抗体或过度表达截短的原调节蛋白来鉴定肌动蛋白细丝封顶所需的其他分子相互作用。细丝的长度和组织将使用共聚焦(3D)、偏振、去卷积和双光子显微镜进行检查。将使用视频显微镜研究收缩功能；3)利用新的渗透性细胞模型和绿色荧光标记蛋白的表达，确定组装原调节蛋白所需的原调节蛋白和原肌球蛋白异构体的结构域。4)确定原调节蛋白与一个可能的心脏星云蛋白样分子相互作用的功能意义。了解细丝组装的机制并确定关键调节蛋白如原调节蛋白在调节肌动蛋白细丝动态中的作用，对于未来考虑各种类型的心脏病，包括家族性肥厚性心肌病中的肌病的分子基础至关重要。

The formation of perfectly aligned myofibrils in cardiac muscle represents a dramatic examine of supramolecular assembly in eukaryotic cells; the mechanisms by which this occurs are still incompletely understood. The long term objective of this research is to identify the molecular components and mechanisms that regulate contractile protein interactions during myofibril assembly. The goal of this proposal is to define the function(s) of tropomodulin, and its specific molecular interactions, in regulation of thin filament assembly and organization using a novel model of de novo cardiac myofibril assembly that faithfully recapitulates myofibril assembly in vivo. My career research development plan, which encompasses the establishment of an interdisciplinary cardiac muscle research program, will include gaining expertise in molecular developmental biology and advanced microscopy techniques (including real-time fluorescence image analysis) by formal course work, interactions with colleagues and hands-on efforts. All the recourses needed will be available at the University of Arizona. This will allow for the investigations of cytoskeletal proteins in live cells and within the context of organisms. The Aims are: (1) To investigate myofibril assembly in differentiating cardiac myocytes. Explants from precardiac regions of avian embryos will be used to examine the temporal pattern of appearance and assembly of myofibril proteins. 2) To examine tropomodulin-tropomyosin interactions and to identify other molecular interactions required for actin filament capping by microinjecting function-blocking antibodies or overexpression of truncated tropomodulins that can act as dominant negative inhibitors. Thin filament length and organization will be examined using confocal (3D), polarization, deconvolution and two-photon microscopy. Contractile function will be studied using video microscopy; 3) To identify the domains of tropomodulin and of tropomyosin isoforms that are required for the assembly of tropomodulin using a novel permeabilized cell model and expression of green fluorescent- tagged proteins. 4) To identify the functional significance of the interaction of tropomodulin with a putative cardiac nebulin- like molecule. Understanding the mechanisms of thin filament assembly and determining the roles of key regulatory proteins such as tropomodulin in modulating actin filament dynamics are pivotal for future considerations of the molecular bases for myopathies seen in various types of heart disease, including familial hypertrophic cardiomyopathies.