Discover the Boosters of Myoblast Fusion

发现成肌细胞融合的促进剂

• 批准号: 10705662
• 负责人: Pengpeng Bi
• 金额: $ 19.93万
• 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/10705662
• 关键词: Ablation; Affinity Chromatography; Biochemical; Biological Assay; CRISPR library; CRISPR/Cas technology; Cell fusion; Cells; Child; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Development; Dissection; Epitopes; Event; Evolution; Experimental Designs; 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; Specific qualifier value; Specificity; Statistical Data Interpretation; Surveys; System; Testing; Toxin; Transcriptional Activation; Validation; Work; cell type; comparative; design; evidence base; 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.

项目概要/摘要 成肌细胞融合结合了单核细胞区室的力量，因此 代表肌肉进化的里程碑。尽管具有明确的意义，但遗传 驱动成肌细胞融合出现的机制仍然很大程度上未知。这 该提案的中心目标是了解生物化学和遗传机制 成肌细胞融合。我们最近发现了两种特定肌肉的关键作用和调节 人类成肌细胞中的膜聚结剂 Myomaker 和 Myomixer。我们的 互补测试表明，尽管这对组合足以诱导细胞融合，但它仅 发生在低效率的情况下。因此，至少需要第三个因素才能构成高效的 在人类发育过程中通常观察到的融合系统。我们的初步研究有 描述了这些神秘因素的预期作用，可概括为 1） Myomixer，未知因子应该能够与Myomaker一起诱导细胞融合； 2） 当与 Myomixer 和 Myomaker 一起表达时，未知因子应诱导 比任何二因素组合更有效的融合； 3）像Myomixer和Myomaker一样， 未知因子的表达也应受 MyoD 的主调节器控制 肌生成。这些功能为简化我们的实验设计提供了清晰的路线图 最终将确定这些因素，我们将其命名为助推器。我们建议首先公正地 鉴于 Myomixer 和 Myomaker 的密切功能关系，识别它们的交互者 助推器。与此同时，我们还将以独特的方式进行全基因组 CRISPR 敲除筛选 人类成肌细胞模型系统地识别助推器基因。融合活性 的助推器候选者将通过补充性获得/功能丧失测试进行检查 各种细胞类型。因此，我们的研究将整合来自广泛领域的优势 方法包括基因发现、比较蛋白质组学和基因功能研究。我们的计划 系统地鉴定来自人类成肌细胞的未知成肌细胞融合因子 考虑到之前的研究主要集中在小鼠身上，这代表了一项创新 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 深入了解细胞间融合的一般机制，并可能提供新的策略 改善人体肌肉的发育和生长。