Impact of MFM disease mutations on the assembly mechanism and network formation of muscle-specific intermediate filament proteins

在肌肉中，中间丝蛋白（IF）是肌外细胞骨架的主要成分之一。在胚胎发育和后期发育阶段，该系统被额外的if蛋白（即vimentin， synemin和syncoilin）强制执行和区域功能化，因为这些蛋白都与整个丝或具有战略意义的重要位点（如共聚体）共定位。尽管波形蛋白似乎与聚乳酸蛋白形成真正的共聚物，但与synemin和syncoilin的情况尚不清楚。对于synemin，我们有证据表明它既不能单独形成if蛋白的主要分子复合物，也不能与vimentin或desmin复合物形成卷曲的线圈。相反，它以高亲和力结合形成细丝。根据syncoilin的结构域组织与标准if -蛋白的结构域组织有很大的差异，我们预计该蛋白也会有类似的分子结构。因此，我们希望利用分析超离心和化学交联技术在分子水平上研究地精蛋白与synsynmin和syncoilin的相互作用。为此，我们将从这两个蛋白中生成特异性重组子结构域，并确定它们与desmin的结合特性。从这项研究开始，我们将确定仍然结合到desmin的最小肽，并确定它们在desmin分子上结合的位置。此外，我们将研究是否双结合的synemin和syncoilin以及plectin和这些分子中的任何一个到细丝正在发生。然后，我们将使用这些蛋白质的结合模块来研究它们在体外和成肌细胞到肌管转化的细胞系统中对组装动力学的影响以及聚丝的稳定性。

In muscle, the intermediate filament (IF) protein desmin is one of the major components of the extra-sarcomeric cytoskeleton. During embryogenesis and later stages of development, this system is enforced and regionally functionalized by additional IF-proteins, namely vimentin, synemin and syncoilin, as these proteins all colocalize either with the entire filament or at strategically important sites such as costamers. Whereas vimentin appears to form authentic copolymers with desmin, the situation with synemin and syncoilin is not clear. For synemin we have evidence that it does not form the principal molecular complex of IFproteins, i.e. the coiled coil, neither on its own nor in complex with vimentin or desmin.Instead, it binds with high affinity to forming filaments. According to the domain organization of syncoilin, which deviates considerably from that of standard IF-proteins, we expect a similar molecular scenario for this protein too. Therefore, we want to investigate the interaction of desmin with both synemin and syncoilin at the molecular level employing analytical ultracentrifugation and chemical cross-linking techniques. For this purpose, we will generate from these two proteins specific recombinant subdomains and determine their binding properties to desmin. Starting from this investigation, we will determine the minimal peptides that still bind to desmin and determine where on the desmin molecule they bind. In addition, we will investigate if dual binding of both synemin and syncoilin as well as plectin and either of these molecules to filaments is taking place. We will then employ the binding modules of these proteins to investigate their impact on the assembly kinetics as well as the stability of desmin filaments, both in vitro and in a cellular system of myoblast-to-myotube conversion.