Cichos
The understanding of amyloid fibril formation as a fundamental process of protein aggregation was hampered by the heterogeneity of the ensemble that was commonly studied. It was particularly difficult to extract information on the molecular details of fibril formation, such as primary and secondary nucleation, as well as varying growth rates from an ensemble that averaged over different species. The aim of this project was to contribute with microscopic studies of fibril formation and growth to the overall picture of amyloid formation. Building on the developed technique of thermophoretic trapping and fluorescence imaging, we aimed to constrain single seed fibrils spatially to directly image their growth, branching, or fragmentation in real time, and investigate their dependence on the protein structure and other factors that modify fibril growth. This was intended to provide a microscopic foundation for the growth dynamics of kinetic models described in the literature. In particular, the use of the sensitive dependence of the rotational diffusion coefficient on the length of the fibril allowed for growth studies over time periods of many 10 minutes with a resolution below the diffraction limit of optical microscopy.
The experiments were planned in four steps. Firstly, we established a model system that demonstrated growth and fragmentation of fibrils, which allowed us to reconstruct ensemble averages from single fibril measurements. In the second step, we applied the measurement technique to insulin, which, according to literature, was supposed to exhibit fibril growth and fragmentation. We aimed to identify the suggested surface-catalyzed secondary nucleation for Aβ40 fibrils. Finally, in collaboration with our partners in the CRC (A03, A06, A12), we explored the influence of structural modifications in the proteins on fibril growth.
Highlighted Publications:
- Martin Fränzl, Frank Cichos, Hydrodynamic manipulation of nano-objects by optically induced thermo-osmotic flows Nature Communications 13, 656 (2022)
- Martin Fränzl, Tobias Thalheim, Juliane Adler, Daniel Huster, Juliane Posseckardt, Michael Mertig, and Frank Cichos, Thermophoretic trap for single amyloid fibril and protein aggregation studies Nat. Methods 16, 611 (2019)
- M. Braun, A. P. Bregulla, K. Günther, M. Mertig, and F. Cichos, Single Molecules Trapped by Dynamic Inhomogeneous Temperature Fields Nano Lett 15, 5499 (2015)