Molecular and biophysical principles of intermediate filament protein assembly

点击翻译按钮获取中文摘要

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.