Molekulare und biophysikalische Prinzipien der Assemblierung von Intermediärfilament-Proteinen
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)
- Competitive counterion binding regulates the aggregation onset of vimentin intermediate filaments, Israel Journal of Chemistry (2014) 56, 614–621
C. Dammann, H. Herrmann, S. Köster
(Siehe online unter https://doi.org/10.1002/ijch.201400153) - Direct Observation of Subunit Exchange Along Mature Vimentin Intermediate Filaments, Biophysical Journal (2014) 107, 2923–2931
B. Nöding, H. Herrmann and S. Köster
(Siehe online unter https://doi.org/10.1016/j.bpj.2014.09.050) - Dynamics of counterion-induced attraction between vimentin filaments followed in microfluidic drops, Lab on a Chip 14 (2014), 2681 – 2687
C. Dammann and S. Köster
(Siehe online unter https://doi.org/10.1039/c3lc51418h) - Impact of ion valency on the assembly of vimentin studied by quantitative small angle x-ray scattering, Soft Matter 10 (2014) 2059 – 2068
M. E. Brennich, S. Bauch, U. Vainio, T. Wedig, H. Herrmann, and S. Köster
(Siehe online unter https://doi.org/10.1039/c3sm52532e) - Analysis of distinct molecular assembly complexes of keratin K8 and K18 by hydrogen-deuterium exchange, Journal of Structural Biology (2015) 192, 426-440
A. Premchandar, A. Kupniewska, K. Tarnowski, N. Mücke, M. Mauermann, M. Kaus- Drobek, A. Edelman, H. Herrmann, and M. Dadlez
(Siehe online unter https://doi.org/10.1016/j.jsb.2015.10.001) - Intermediate filament mechanics in vitro and in the cell: From coiled coils to filaments, fibers and networks, Current Opinion in Cell Biology (2015) 32, 82-91
S. Köster, D. Weitz, R. Goldman, U. Aebi and H. Herrmann
(Siehe online unter https://doi.org/10.1016/j.ceb.2015.01.001) - Physical Properties of Cytoplasmic Intermediate Filaments, BBA Molecular Cell Research (2015) 1853, 3053-3064
J. Block, V. Schroeder, P. Pawelzyk, N. Willenbacher and S. Köster
(Siehe online unter https://doi.org/10.1016/j.bbamcr.2015.05.009) - The assembly of simple epithelial keratin filaments: Deciphering the ion-dependence in filament organization, Biomacromolecules (2015) 16, 3313-3321
C. Hemonnot, M. Mauermann, H. Herrmann and S. Köster
(Siehe online unter https://doi.org/10.1021/acs.biomac.5b00965) - Both monovalent cations and plectin are potent modulators of mechanical properties of keratin K8/K18 networks, Soft Matter (2016) 12, 6964-6974
I. Martin, M. Moch, T. Neckernuss, S. Paschke, H. Herrmann, and O. Marti
(Siehe online unter https://doi.org/10.1039/c6sm00977h) - In vitro Assembly Kinetics of Cytoplasmic Intermediate Filaments: A Correlative Monte Carlo Simulation Study, PLoS One (2016) 11, e0157451
N. Mücke, S. Winhein, H. Merlitz, J. Buchholz, J. Langowski, and H. Herrmann
(Siehe online unter https://doi.org/10.1371/journal.pone.0157451) - Intermediate Filaments: Structure and Assembly, Cold Spring Harbour Perspectives in Biology (2016), a018242
H. Herrmann and U. Aebi
(Siehe online unter https://doi.org/10.1101/cshperspect.a018242) - Lateral association and elongation of vimentin intermediate filament proteins: a time resolved light scattering study, Proceedings of the National Academy of Sciences of the USA (2016), 113, 11152-11157
C. G. Lopez, O. Saldanha, K. Huber and S. Köster
(Siehe online unter https://doi.org/10.1073/pnas.1606372113) - Structural Dynamics of the Vimentin Coiled-Coil Contact Regions involved in Filament Assembly as revealed by Hydrogen-Deuterium Exchange, Journal of Biological Chemistry (2016) 291, 24931–24950
A. Premchandar, N. Mücke, J. Poznański, T. Wedig, M. Kaus-Drobek, H. Herrmann, and M. Dadlez
(Siehe online unter https://doi.org/10.1074/jbc.M116.748145) - αB-crystallin is a sensor for assembly intermediates and for the subunit topology of desmin intermediate filaments, Cell Stress Chaperones (2017) 22, 613-626
S. Sharma, G.M. Conover, J.L. Elliott, M. Der Perng, H. Herrmann H, and R.A. Quinlan
(Siehe online unter https://doi.org/10.1007/s12192-017-0788-7) - An image-based small molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells, FASEB Journal (2018)
K-P. Trogden, R.A. Battaglia, P. Kabiraj, V.J. Madden VJ, H. Herrmann, and N.T. Snider
(Siehe online unter https://doi.org/10.1096/fj.201700663R)
