TROPOMODULIN FUNCTION IN HEART DEVELOPMENT

心肌中完美排列的肌原纤维的形成代表了真核细胞中超分子组装的戏剧性检查；这种现象发生的机制仍未完全了解。本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。本研究的目标是定义原调节蛋白的功能，及其特定的分子相互作用，在细丝组装和组织的调节中，使用一种新的新心肌肌原纤维组装模型，忠实地概括了肌原纤维在体内的组装。我的职业研究发展计划包括建立一个跨学科的心肌研究项目，将包括通过正式的课程学习、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镜技术（包括实时荧光图像分析）方面的专业知识。所有需要的资源都可以在亚利桑那大学获得。这将允许在活细胞和生物体的背景下的细胞骨架蛋白的调查。目的是：(1)研究心肌细胞分化过程中肌原纤维的组装。从禽类胚胎的心脏前区提取的外植体将用于检查肌原纤维蛋白的外观和组装的时间模式。2)检测原调节性蛋白与原肌球蛋白的相互作用，并通过微注射功能阻断抗体或过表达截断的原调节性蛋白来确定肌动蛋白丝盖顶所需的其他分子相互作用，这些原调节性蛋白可以作为显性负抑制剂。细丝的长度和组织将使用共聚焦（3D），偏振，反褶积和双光子显微镜检查。使用视频显微镜研究收缩功能；3)利用一种新的渗透细胞模型和绿色荧光标记蛋白的表达，鉴定原调节性蛋白和原肌球蛋白同型异构体的结构域，这些结构域是原调节性蛋白组装所必需的。4)确定原调节蛋白与假定的心脏星云样分子相互作用的功能意义。了解细丝组装的机制和确定关键调节蛋白（如原调蛋白）在调节肌动蛋白丝动力学中的作用，对于未来考虑各种类型心脏病（包括家族性肥厚性心肌病）中所见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.