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;发生这种情况的机制仍然不完全清楚。 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组分和机制。 该提案的目标是使用一种新的从头心脏肌原纤维组装模型来定义原调节蛋白的功能及其特定的分子相互作用，以调节细丝组装和组织，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，其中包括建立一个跨学科的心肌研究计划，将包括获得专业知识，在分子发育生物学和先进的显微镜技术（包括实时荧光图像分析）通过正式的课程工作，与同事的互动和动手的努力。 亚利桑那大学将提供所需的所有资源。 这将允许活细胞和生物体背景下的细胞骨架蛋白的研究。 目的：（1）研究心肌细胞分化过程中肌原纤维的组装。 将使用来自禽类胚胎心前区的外植体来检查肌原纤维蛋白的外观和组装的时间模式。 2)研究原调节蛋白-原肌球蛋白相互作用，并通过显微注射功能阻断抗体或过表达可作为显性负抑制剂的截短原调节蛋白来鉴定肌动蛋白丝帽所需的其他分子相互作用。 细丝长度和组织将使用共焦（3D），偏振，去卷积和双光子显微镜检查。 将使用视频显微镜研究收缩功能; 3）使用新的透化细胞模型和绿色荧光标记蛋白的表达来鉴定原调节蛋白和原肌球蛋白同种型的结构域，所述结构域是原调节蛋白组装所需的。 4)确定原调节蛋白与假定的心脏星云蛋白样分子相互作用的功能意义。 理解细丝组装的机制和确定关键调节蛋白如原调节蛋白在调节肌动蛋白丝动力学中的作用对于将来考虑在各种类型的心脏病（包括家族性肥厚性心肌病）中观察到的肌病的分子基础是关键的。

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.