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（IF）蛋白结蛋白是肌节外细胞骨架的主要成分之一。在胚胎发生和发育的后期阶段，该系统通过额外的IF-蛋白（即波形蛋白、突触蛋白和突触蛋白）被加强和区域功能化，因为这些蛋白质都与整个丝或在战略重要位点（如costamers）共定位。尽管波形蛋白似乎与结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，它既不单独形成IF蛋白的主要分子复合物，也不与波形蛋白或结蛋白复合形成卷曲螺旋，而是以高亲和力结合形成细丝。根据syncoilin的结构域组织，这大大偏离了标准的IF-蛋白质，我们预计这种蛋白质也有类似的分子情景。因此，我们希望研究结蛋白与synemin和syncoilin在分子水平上采用分析超离心和化学交联技术的相互作用。为此，我们将从这两种蛋白质产生特定的重组亚结构域，并确定它们与结蛋白的结合特性。从这项研究开始，我们将确定仍然与结蛋白结合的最小肽，并确定它们在结蛋白分子上的结合位置。此外，我们将调查是否双重绑定的synemin和syncoilin以及plectin和这些分子中的任何一个细丝正在发生。然后，我们将采用这些蛋白质的结合模块来研究它们对组装动力学以及结蛋白丝的稳定性的影响，无论是在体外还是在成肌细胞到肌管转换的细胞系统中。

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.