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 成肌细胞融合将来自其他单核细胞隔间的力量结合在一起，因此 代表着肌肉进化的一个里程碑。尽管有明显的意义，但基因 驱动成肌细胞融合出现的机制在很大程度上仍不清楚。这个 这项建议的中心目标是了解糖尿病的生化和遗传机制。 成肌细胞融合。我们最近发现了两个肌肉特异性的关键作用和调节 人成肌细胞中的膜聚集剂Myomaker和Myomixer。我们的 互补试验表明，尽管这两个组合足以诱导细胞融合，但它只 发生的效率很低。因此，至少需要第三个因素才能构成高效的 通常在人类发育过程中观察到的融合系统。我们的初步研究已经 描绘了这些神秘因素的预期作用，可以概括为1)喜欢 Myomixer，未知因素(S)应该能与Myomaker一起诱导细胞融合；2) 当与Myomixer和Myomaker一起表达时，未知因素(S)应诱导 比任何双因素组合更有效的融合；3)像Myomixer和Myomaker一样， 未知因子(S)的表达也应受MyoD的调控 肌肉发生。这些功能为简化我们的实验设计提供了明确的路线图 最终将确定这样的因素，我们将其命名为助推器。我们建议首先不偏不倚地 确定Myomixer和Myomaker的相互作用因子，因为它们与 助推器。同时，我们还将在Unique中进行全基因组CRISPR基因敲除筛选 人类成肌细胞模型以系统地鉴定助推器基因。融合子活性 将通过补充性增益性/失效性测试来检查 各种细胞类型。因此，我们的研究将综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 为了系统准确地从人成肌细胞中鉴定未知的成肌细胞融合因子 考虑到之前的研究主要集中在老鼠身上，这是一项创新 以及无脊椎动物模型。最终，从这项研究中获得的知识将提供新的见解 研究细胞间融合的一般机制，并可能提供新的策略 改善人类肌肉的发育和生长。