Molecular pathways maintaining homeostasis in the vertebrate muscle sarcomere

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中的质量控制机制，重叠的肌球蛋白和肌动蛋白丝由其他蛋白质连接，这些蛋白质在三维中为复合物提供强度和弹性，同时允许纤维的急剧收缩。即使是这些蛋白质复合物的组装或稳定性中的微小缺陷也会损害它们的功能。存在高度调节的途径来控制新生结构组分的丰度，以及组分添加到肌节的严格顺序。肌肉中最丰富的蛋白质组分是众所周知的，但对调节组装、维持功能性收缩状态以及在收缩组分变性或受损时修复或替换收缩组分的因子知之甚少。这些组装因子是我们实验室最近的重点，旨在实现两个目标。(i)利用遗传学来揭示这一途径的新成分，以及（ii）确定这些成分如何调节骨骼肌和心肌中的肌节图案化和组装，并在体内测试这些模型的预测。后生动物肌肉发生的过程是非常保守的，这使我们能够扩展我们在C。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，基因组编辑技术（CRISPR/Cas9）的最新发展使这个系统变得更加强大。第一个目的是确定非结构质量控制组分（如unc45b和smallp1b）在肌节的模式化中发挥什么作用，并确定它们是否通过泛素介导的蛋白质降解起作用。这将监测在早期肌节组装过程中组分的空间和时间表达，并检查动物中肌节结构突变的主要组分。在第二个目标中，我们将使用现有的肌肉突变体（其中同源基因未被识别）和来自影响不同肌节组装步骤的肌肉突变体的转录数据集的分析，寻找在这个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来产生突变体不可用的疑似肌节组装组分的敲除（例如肌节蛋白和一些E3连接酶）。将测试这些突变体由于组装缺陷而导致的异常肌节结构。

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.