Molecular and biophysical principles of intermediate filament protein assembly

在后生动物细胞中，中间丝构成了一个细胞骨架丝系统。它们是由一大群组织特异性纤维蛋白组成的，这些蛋白的特征是形成一个高度保守的结构组织的中心α-螺旋结构域，尽管它们的主要氨基酸序列可能有很大的不同。相应地，各个细丝的机械性能也有很大差异。如果我们已经证明蛋白质使用与球状蛋白质完全不同的组装途径，如肌动蛋白和微管蛋白，它们分别是微丝和微管的亚单位。这包括CCS的形成，这些CCS横向组装成反平行的、半交错的四聚体复合体，以及通过诱导离子强度的变化，这些四聚体横向缔合成全宽微丝，称为单位长度细丝(ULF)。到目前为止，超低碳纤维纵向退火的延伸率机制是在微观水平上被理解的，并且可以用数学模型来描述。然而，定义这种相互作用的分子和生物物理参数才刚刚开始出现。因此，我们计划结合一套独特的互补生化和生物物理技术，以深入了解细丝形成的机制。原则上，我们可以将这个问题归结为对两个ULF的伸长的理解，以及在所有ULF被消耗用于灯丝形成后，通过相同的端到端退火反应来发挥作用的灯丝的伸长。我们将在专门设计的微流控设备中使用高纯度的重组蛋白质，并结合荧光(交叉)相关光谱观察不同位置标记有荧光团的蛋白质。由于波形蛋白的一组突变会在组装的不同阶段干扰组装，以及我们对CC架构的结构洞察，我们将设计新的突变，这些突变应该会在动力学上干扰组装过程。它们将首先通过分析超速离心法和电子显微镜进行表征，然后是微流控技术。在下一阶段，我们将研究波形蛋白和胶质纤维酸性蛋白(GFAP)的共组装，因为这种混合组装发生在胚胎发育期间的星形胶质细胞中。在下一层次的分析中，我们将研究真正的IF相关蛋白，如桥粒蛋白，在结合到有图案的表面时如何影响组装。我们预计，这些研究将揭示对IF本身的组装机制以及主要能够相互作用的蛋白质的协调平行组装的全新见解。最终，我们将探索IF相关蛋白如何作为复杂网络生成的拓扑种子。

Intermediate filaments (IFs) constitute a cytoskeletal filament system in metazoan cells. They are made from a large group of tissue-specific fibrous proteins that are characterized by a coiled-coil (CC) forming central alpha-helical domain of highly conserved structural organization, although their primary amino acid sequence may differ considerably. Correspondingly, the mechanical properties of the individual filaments differ largely. IF proteins have been shown by us to use a completely different assembly pathway compared to globular proteins such as actin and tubulin, the subunits of microfilaments and microtubules, respectively. This includes the formation of CCs that laterally assemble into anti-parallel, half-staggered tetramer complexes and a lateral association of these tetramers into full width mini-filaments, called unit-length filaments (ULFs), through an induced change in the ionic strength. Up to now, the elongation mechanism as mediated by longitudinal annealing of ULFs is understood at the microscopic level and can be described by mathematical modelling. However, the molecular and biophysical parameters that define this interaction are only beginning to emerge. Therefore we plan to combine a distinct set of complementary biochemical and biophysical techniques in order to get insight into the mechanics of filament formation. In principle, we can reduce this problem to the understanding of the elongation of two ULFs proper, as also the elongation of filaments, after all ULFs have been consumed for filament formation, functions by the same end-to-end annealing reaction. We will employ highly purified recombinant proteins in specifically designed microfluidic devices and observe proteins labeled with fluorophores at distinct sites in combination with fluorescence (cross) correlation spectroscopy. Due to a panel of mutations in vimentin that interfere with assembly at various different stages of assembly as well as our structural insight into the CC architecture, we will design new mutations that should kinetically interfere with the assembly process. They will first be characterized by analytical ultracentrifugation and electron microscopy followed by microfluidic techniques. In a next stage, we will investigate the coassembly of vimentin with glial fibrillary acidic protein (GFAP), as such a mixed assembly occurs in astrocytes during embryogenesis. At a next level of analysis, we will investigate how an authentic IF-associated protein such as desmoplakin influences assembly when bound to a patterned surface. We expect that these investigations will reveal completely new insights into the assembly mechanism of IFs as such, as well as into the coordinated parallel assembly of proteins that are principally able to interact with each other. Eventually, we will explore how IF-associated proteins may serve as topological seeds for the generation of complex networks.