TROPOMODULIN FUNCTION IN HEART DEVELOPMENT

原调节蛋白在心脏发育中的功能

• 批准号: 6536541
• 负责人: Carol C Gregorio
• 金额: $ 8.95万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6536541
• 关键词: actins; cardiac myocytes; cell differentiation; chick embryo; complementary DNA; cytoskeletal proteins; electron microscopy; green fluorescent proteins; immunoprecipitation; intermolecular interaction; microfilaments; microinjections; molecular assembly /self assembly; monoclonal antibody; myofibrils; myosins; protein isoforms; protein protein interaction; protein structure function; radiotracer; striated muscles; tropomyosin; video microscopy; western blottings

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.

心肌中完全比对的肌原纤维的形成代表了对真核细胞中超分子组件的戏剧性检查。发生这种情况的机制仍未完全理解。 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。 该提案的目的是使用从头心脏肌动纤维组装的新型模型来定义对薄丝组装和组织的特定分子相互作用的功能，该功能忠实地概括了体内的肌原纤维组件。 我的职业研究开发计划包括建立跨学科的心肌研究计划，包括通过正式课程工作，与同事和手提工作的互动以及与同事的互动以及与同事和手法相互作用获得分子发育生物学和高级显微镜技术方面的专业知识（包括实时荧光图像分析）关于努力。 亚利桑那大学将提供所有所需的回报。 这将允许对活细胞和生物体中的细胞骨骼蛋白进行研究。 目的是：（1）研究以区分心肌细胞的肌原纤维组件。 来自鸟类胚胎的part体区域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）检查肌动蛋白 - 肌球蛋白相互作用并确定通过微注射功能阻断抗体或过度表达截短的肌蛋白的过表达所需的其他分子相互作用，这些抑制蛋白可以充当主要的负抑制剂。 将使用共聚焦（3D），极化，反卷积和两光子显微镜检查细丝长度和组织。 收缩功能将使用视频显微镜研究； 3）使用新型的透化细胞模型和绿色荧光标记的蛋白的表达来鉴定托型瘤蛋白组装所需的tropomodulin和tropomyosin同工型的结构域。 4）确定托托瘤蛋白与推定心脏雾蛋白的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在调节肌动蛋白丝动力学中的关键调节蛋白的作用，这是至关重要的，对于将来考虑了在包括家族性肥大性心肌病在内的各种类型心脏病中的分子碱基。