Molecular pathways maintaining homeostasis in the vertebrate muscle sarcomere

维持脊椎动物肌肉肌节稳态的分子途径

• 批准号: RGPIN-2018-06684
• 负责人: Pilgrim, David
• 金额: $ 4.66万
• 依托单位: University of Alberta
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744809
• 关键词: Molecular; pathways; maintaining; homeostasis; vertebrate

Our lab studies the molecular pathways that control and effect cell determination and differentiation. Of particular interest are the mechanisms of quality control in the metazoan muscle sarcomere, overlapping myosin and actin filaments that are joined by other proteins that give strength and elasticity to the complex in three dimensions while allowing the dramatic contraction of the fibers. Even minor defects in the assembly or stability of these protein complexes can compromise their function. There are highly regulated pathways that control the abundance of nascent structural components, and a strict order in which components are added to the sarcomere. The most abundant protein components of muscle are well known, but little is understood about the factors that regulate assembly, maintain the functional contractile state, and repair or replace the contractile components as they denature or are damaged. These assembly' factors have been the recent focus of our lab towards two goals. (i) use genetics to uncover novel components of this pathway, and (ii) determine how these components regulate sarcomere patterning and assembly in skeletal and cardiac muscle, and test predictions of these models in vivo. The process of metazoan myogenesis is remarkably conserved, allowing us to extend our initial work in C. elegans to vertebrate muscle. The zebrafish is our experimental system of choice for the experiments described here, and recent developments in genome editing technology (CRISPR/Cas9) has made this system even more powerful. The first aim is to determine what role the non-structural quality control components (such as unc45b and smyd1b) play in patterning of sarcomeres, and to determine whether they work via ubiquitin-mediated protein degradation. This will monitor spatial and temporal expression of the components during early sarcomere assembly, and examine sarcomere structure in animals mutant for the major components. In the second aim, we will search for novel factors that work in this process, using existing muscle mutants (where the cognate gene is not identified) and analysis of transcript datasets derived from muscle mutants affecting different sarcomere assembly steps. In the last aim, we will use CRISPR/Cas9 to generate knockouts of suspected sarcomere assembly components for which mutants are not available (e.g. myomesin, and some E3 ligases). These mutants will be tested for abnormal sarcomere structure due to defects in assembly.

我们的实验室研究控制和影响细胞决定和分化的分子途径。特别令人感兴趣的是后生动物肌肉肌节的质量控制机制，重叠的肌球蛋白和肌动蛋白细丝被其他蛋白质连接在一起，这些蛋白质在三维空间赋予复合体强度和弹性，同时允许纤维的戏剧性收缩。即使这些蛋白质复合体在组装或稳定性上的微小缺陷也会损害它们的功能。有高度调控的途径来控制新生结构成分的丰度，并有严格的顺序将成分添加到肌节上。肌肉中最丰富的蛋白质成分是众所周知的，但人们对调节组装、维持功能性收缩状态以及在收缩成分变性或受损时修复或替换收缩成分的因素知之甚少。这些组装的因素一直是我们实验室最近朝着两个目标的重点。(I)使用遗传学来发现这一途径的新组件，以及(Ii)确定这些组件如何调节骨骼肌和心肌中的肌节图案和组装，并在体内测试这些模型的预测。后生动物的肌肉发生过程非常保守，这使得我们可以将最初在线虫身上的工作扩展到脊椎动物的肌肉。斑马鱼是我们在这里描述的实验中选择的实验系统，最近基因组编辑技术(CRISPR/Cas9)的发展使这个系统变得更加强大。第一个目标是确定非结构性质量控制成分(如unc45b和smyd1b)在肌瘤图案化中所起的作用，并确定它们是否通过泛素介导的蛋白质降解发挥作用。这将监测这些成分在早期肌节组装过程中的空间和时间表达，并检查主要成分突变动物的肌节结构。在第二个目标中，我们将使用现有的肌肉突变(同源基因尚未确定)和分析来自影响不同肌节组装步骤的肌肉突变的转录本数据集，寻找在这一过程中起作用的新因素。在最后一个目标中，我们将使用CRISPR/Cas9来产生疑似肌节组装组件的敲除，这些组件是没有突变的(例如，myomesin和一些E3连接酶)。这些突变体将接受由于组装缺陷而导致的异常肌节结构的测试。