Molecular Regulation of Myoblast Fusion

成肌细胞融合的分子调控

• 批准号: 6967397
• 负责人: HELEN S SINK
• 金额: $ 15.21万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-19 至 2006-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6967397
• 关键词: Drosophilidae; RNA interference; SDS polyacrylamide gel electrophoresis; cell fusion; cell membrane; cell morphology; confocal scanning microscopy; endocytosis; fluorescence microscopy; guanine nucleotide exchange factors; immunocytochemistry; immunoprecipitation; intermolecular interaction; membrane proteins; myoblasts; myogenesis; polymerase chain reaction; protein localization; protein structure function; tissue /cell culture; western blottings; yeast two hybrid system

DESCRIPTION (provided by applicant): Treatment strategies for Muscular Dystrophies will be improved by deepening understanding of how muscle formation is controlled and encouraged. The much needed identification and dissection of underlying molecular pathways is readily advanced through studies in model systems. This application focuses on investigating the mechanisms that facilitate the muscle forming process of myoblast fusion. Drosophila is used as the model system. Drosophila's Hibris (Hbs) and Sticks and stones (Sns) are transmembrane receptor-like proteins present on myoblasts. They are structural homologues. They are not functionally interchangeable. They are essential for successful myoblast fusion. No other model system or molecules provide such compelling, accessible entry points for dissecting the molecular processes supporting myoblast fusion. In this application, experiments are proposed in Specific Aim 1 to investigate how Hbs and Sns interact with a common downstream molecular target. The interactions will be assayed in vivo at the protein, genetic and cellular level through this aim. Specific Aim 2 capitalizes on observations that structural differences in the Hbs and Sns endodomains translate into differences in Hbs and Sns behaviors. The functions of the differing endodomain regions will be investigated using deletion and chimeric constructs in cellular and in vivo assays. Specific Aim 3 expands upon observations that Hbs and Sns are endocytosed, and that Hbs and Sns trans-endocytose a common extracellular ligand. Endocytosis takes Hbs and Sns from the membrane to the endosomes. Ligand trans-endocytosis by Hbs and Sns involves simultaneous transfer of membrane from the ligand-expressing cell, ligand internalization, and ligand re-presentation at the Hbs and Sns expressing cell's membrane. It is postulated that endoctyosis regulates Hbs and Sns levels at the membrane, while ligand trans-endocytosis mediates signal propagation and membrane destabilization. The putative functions for Hbs and Sns endocytosis and trans-endocytosis will be tested in vivo in this aim. In addition, molecular candidates will be tested in cell culture and in vivo for their ability to regulate Hbs and Sns endocytosis and trans-endocytosis. In summary, Hbs and Sns are key molecular players involved in signaling, endocytosis and trans-endocytosis in fusing myoblasts. Elucidating their actions and interactions will deepen our understanding of the roles these proteins and processes play in successful myoblast fusion.

描述（由申请人提供）：通过加深对如何控制和促进肌肉形成的理解，将改善肌营养不良症的治疗策略。通过模型系统的研究很容易推进对潜在分子途径的急需识别和剖析。该应用的重点是研究促进成肌细胞融合的肌肉形成过程的机制。果蝇被用作模型系统。果蝇的 Hibris (Hbs) 和 Sticks and Stones (Sns) 是成肌细胞上存在的跨膜受体样蛋白。它们是结构同系物。它们在功能上不可互换。它们对于成功的成肌细胞融合至关重要。没有其他模型系统或分子能够提供如此引人注目、易于理解的切入点来剖析支持成肌细胞融合的分子过程。在此应用中，具体目标 1 中提出了实验，以研究 Hbs 和 Sns 如何与常见的下游分子靶标相互作用。通过这一目标，将在体内蛋白质、遗传和细胞水平上对相互作用进行分析。具体目标 2 利用以下观察结果：Hbs 和 Sns 内域的结构差异转化为 Hbs 和 Sns 行为的差异。将在细胞和体内测定中使用缺失和嵌合构建体来研究不同内域区域的功能。具体目标 3 扩展了 Hbs 和 Sns 被内吞，以及 Hbs 和 Sns 反式内吞共同胞外配体的观察结果。内吞作用将 Hbs 和 Sns 从膜带到内涵体。 Hbs 和 Sns 的配体反内吞作用涉及膜从表达配体的细胞的同时转移、配体内化以及配体在表达 Hbs 和 Sns 的细胞膜上的重新呈现。据推测，内吞作用调节膜上的 Hbs 和 Sns 水平，而配体反式内吞作用介导信号传播和膜不稳定。为此目的，将在体内测试 Hbs 和 Sns 内吞作用和反式内吞作用的假定功能。此外，候选分子将在细胞培养和体内测试其调节 Hbs 和 Sns 内吞作用和反式内吞作用的能力。总之，Hbs 和 Sns 是参与成肌细胞融合过程中信号传导、内吞作用和反式内吞作用的关键分子参与者。阐明它们的作用和相互作用将加深我们对这些蛋白质和过程在成功的成肌细胞融合中所发挥的作用的理解。