Discover the Boosters of Myoblast Fusion

发现成肌细胞融合的促进剂

• 批准号: 10525471
• 负责人: Pengpeng Bi
• 金额: $ 16.61万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-16 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10525471
• 关键词: Ablation; Affinity Chromatography; Biochemical Genetics; Biological Assay; CRISPR library; CRISPR/Cas technology; Cell fusion; Cells; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Development; Dissection; Epitopes; Event; Evolution; Female; Fibroblasts; Foundations; Generations; Genes; Genetic; Genetic Complementation Test; Genetic Screening; Genetic Transcription; Genotype; Goals; Growth and Development function; Human; Human Development; Individual; Invertebrates; Knock-out; Knowledge; Ligation; Mass Spectrum Analysis; Membrane; Membrane Fusion; Membrane Proteins; Modeling; Molecular; Mus; Muscle; Muscle Development; Muscle Fibers; Muscle function; Musculoskeletal System; Myoblasts; Myopathy; Names; Output; Phenotype; Play; Process; Proteins; Proteomics; Reagent; Regulation; Research; Role; Series; Specificity; Statistical Data Interpretation; Surveys; System; Testing; Toxin; Transcriptional Activation; Validation; Work; base; cell type; comparative; design; experience; experimental study; fitness; gene discovery; gene function; genetic approach; genome-wide; human model; improved; innovation; insight; loss of function; male; muscular structure; mutant; myogenesis; novel; novel therapeutics; protein expression; protein protein interaction; reconstitution; transcriptome

Project Summary/Abstract Myoblast fusion unites the strength from otherwise mononucleate cellular compartments, thus representing a milestone of muscle evolution. Despite the clear significance, the genetic mechanism that drives the emergence of myoblast fusion has remained largely unknown. The central goal of this proposal is to understand the biochemical and genetic mechanisms of myoblast fusion. We recently uncovered the key roles and regulations of two muscle-specific membrane-coalescing agents Myomaker and Myomixer in human myoblasts. Our complementation tests suggested that although this duo is sufficient to induce cell fusion, it only occurs at low efficiency. As such, at least a third factor is required to constitute the highly efficient fusion system that is normally observed during human development. Our preliminary studies have delineated the expected roles of these mysterious factors that can be summarized as 1) like Myomixer, the unknown factor(s) should be able to induce cell fusion together with Myomaker; 2) When expressed together with Myomixer and Myomaker, the unknown factor(s) should induce more efficient fusion than by any two-factor combinations; 3) like Myomixer and Myomaker, the expression of the unknown factor(s) should also be controlled by MyoD the master regulator of myogenesis. These features provided a clear roadmap to streamline our experiment design that will ultimately identify such factors that we named Boosters. We propose to first unbiasedly identify the interactors of Myomixer and Myomaker given their close functional relationships with the Boosters. In parallel, we will also perform a genome-wide CRISPR knockout screen in unique models of human myoblasts to systematically identify the Booster genes. The fusogenic activity of the Booster candidates will be examined through complementary gain/loss-of-function tests in various cell types. Therefore, our study will integrate the strengths from a broad range of approaches including gene discovery, comparative proteomics, and gene function study. Our plan to systematically identify the unknown myoblast fusion factors exactly from human myoblasts also represents an innovation considering that previous studies have primarily focused on the mouse and invertebrate models. Ultimately, knowledge gained from this study will provide new insights into the mechanisms of intercellular fusion in general and may provide new strategies for improving human muscle development and growth.

项目摘要/摘要 成肌细胞融合使其他单核细胞隔室的强度结合在一起，因此 代表肌肉进化的里程碑。尽管显着意义，但遗传 驱动成肌细胞融合出现的机制在很大程度上尚不清楚。这 该建议的核心目标是了解 肌细胞融合。我们最近发现了两个肌肉特异性的关键作用和法规 人体肌细胞中的膜钙化剂myomaker和Myomixer。我们的 互补测试表明，尽管此二人足以诱导细胞融合，但仅 以低效率发生。因此，至少需要第三个因素才能构成高效的 通常在人类发展过程中观察到的融合系统。我们的初步研究 描述了这些神秘因素的预期作用，这些因素可以总结为1） Myomixer，未知因素应该能够与Myomaker一起诱导细胞融合。 2） 当与Myomixer和Myomaker一起表达时，未知因素应诱发 与任何两因素组合相比，融合效率更高； 3）像Myomixer和Myomaker一样 未知因素的表达也应由myod控制 肌发生。这些功能提供了清晰的路线图来简化我们的实验设计 最终将确定我们命名为助推器的这些因素。我们建议首先公正 鉴于他们与肌电肌和肌肌的相互作用者的交互者，鉴于他们与 助推器。同时，我们还将以独特的方式执行全基因组CRISPR敲除屏幕 人类肌细胞的模型系统地识别增强基因。融合活性 助推器的候选人将通过补充收益/功能丧失测试进行检查 各种细胞类型。因此，我们的研究将整合一系列广泛的优势 包括基因发现，比较蛋白质组学和基因功能研究在内的方法。我们的计划 还可以从人类成肌细胞中系统地识别未知的成肌细胞融合因子 考虑到以前的研究主要集中于小鼠，代表了一种创新 和无脊椎动物模型。最终，从这项研究中获得的知识将提供新的见解 总体上进入细胞间融合的机制，并可能提供新的策略 改善人类的肌肉发育和成长。