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Projekt Druckansicht

Molekulare und biophysikalische Prinzipien der Assemblierung von Intermediärfilament-Proteinen

Antragstellerinnen / Antragsteller Professor Dr. Harald Herrmann; Professorin Dr. Sarah Köster
Fachliche Zuordnung Strukturbiologie
Förderung Förderung von 2012 bis 2016
Projektkennung Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 227073266
 
Erstellungsjahr 2018

Zusammenfassung der Projektergebnisse

Intermediate filaments (IFs) form, besides actin filaments and microtubules, the third protein filaments system in the cytoskeleton of eukaryotes. The composite network of all three filaments types enable the cell to adapt to a large variety of mechanical challenges. Therefore, a precise knowledge of the mechanical behavior of each component is key to understanding cell mechanics. By contrast to actin filaments and microtubules, IFs assembly in a strictly hierarchical manner, rather than polymerizing from globular monomers. Another important particularity is the cell-type specific manner in which IFs are expressed, e.g. vimentin in mesenchymal cell and keratin in epithelial cells. Within this project we were able to elucidate several important aspects of this peculiar IF assembly pathway on all hierarchical levels, i.e. lateral association, longitudinal annealing and network formation, following the hypothesis that the specific architecture of IFs gives rise to their superb mechanical properties. We employed a variety of complementary methods to assess the relevant length scales for each studied process. (i) Using static and dynamic light scattering, we revealed time scales, on which the lateral assembly dominates (< 1 min), and time scales, on which the elongation reaction dominates (> 10 min), which some overlap in between (given the salt and protein concentrations used in our experiment); Monte-Carlo (MC) simulations showed protein specific elongation speeds (vimentin vs. keratin 8/18). (ii) Using fluorescence microscopy, we were able to directly show subunit (tetramers, octamers…) exchanges from fully assembled, mature filaments. The occurrence of such exchanges depends on how smoothly assembled the filaments are, which, in turn, is related to the assembly conditions. As the assembly environment is likely to be well-controlled in cells, we expect, that cells use this aspect to control the local architecture of IF networks. (iii) Small-angle X-ray scattering (SAXS) on different protein systems (vimentin, keratin 8/18) and after addition of different ions (K+, Mg2+, Co(NH3)63+) showed that the specific charge and hydrophobicity pattern along IFs plays an important role in their ability to form networks and in the architecture of these networks. We also employed more complex interaction partners such as synemin and nestin. Notably, both proteins do not form hetero-dimers with vimentin and desmin, indicating a different type of integration into IFs. The giant plakin plectin binds in its dimeric form strongly to IFs, and thereby serves as a potent cross-bridging and spacing factor for IFs. (iv) From a methods point of view, we have developed a setup that combines fluorescence fluctuation spectroscopy (FCS, PCH) with microfluidics and thus allows us now to study the temporal evolution of the emergence of protein assemblies and aggregates.

Projektbezogene Publikationen (Auswahl)

 
 

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