Molecular and biophysical principles of intermediate filament protein assembly

中间丝（IF）构成多细胞动物细胞中的细胞骨架丝系统。它们由一大组组织特异性纤维蛋白制成，其特征在于卷曲螺旋（CC）形成高度保守结构组织的中央α-螺旋结构域，尽管它们的一级氨基酸序列可能有很大差异。相应地，各个细丝的机械性能差异很大。IF蛋白已经被我们证明使用一个完全不同的组装途径相比，球状蛋白，如肌动蛋白和微管蛋白，微丝和微管的亚基，分别。这包括形成横向组装成反平行的，半交错的四聚体复合物的CC和这些四聚体的横向关联成全宽度的微丝，称为单位长度细丝（ULF），通过诱导的离子强度的变化。到目前为止，拉伸机制介导的纵向退火的超低频理解在微观层面上，可以通过数学建模来描述。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，我们计划联合收割机一套独特的互补生物化学和生物物理技术，以深入了解长丝形成的机制。原则上，我们可以将这个问题简化为对两个ULF本身的伸长的理解，因为在所有ULF都被消耗用于长丝形成之后，长丝的伸长也通过相同的端对端退火反应起作用。我们将采用高度纯化的重组蛋白在专门设计的微流控装置和观察蛋白质标记的荧光团在不同的网站结合荧光（交叉）相关光谱。由于波形蛋白中的一组突变在组装的各个不同阶段干扰组装，以及我们对CC架构的结构洞察，我们将设计应该在动力学上干扰组装过程的新突变。它们将首先通过分析超离心和电子显微镜进行表征，然后是微流体技术。在下一阶段，我们将研究波形蛋白与胶质细胞酸性蛋白（GFAP）的共组装，因为在胚胎发育过程中星形胶质细胞中发生了这种混合组装。在下一个层次的分析，我们将研究如何一个真正的IF相关蛋白，如桥粒斑蛋白影响组装时，绑定到一个图案化的表面。我们期望这些研究将揭示对IFs组装机制的全新见解，以及主要能够相互作用的蛋白质的协调平行组装。最后，我们将探讨IF相关蛋白质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.