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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MECHANICS (Mechanics of cells: the role of intermediate filaments)

Teaser

The mechanical properties of each of the over 200 cell types in the human body are perfectly well adapted to their function. The large variety of viscoelastic profiles, ranging from soft brain cells to stiff cartilage, and the temporal variability in the mechanical stress...

Summary

The mechanical properties of each of the over 200 cell types in the human body are perfectly well adapted to their function. The large variety of viscoelastic profiles, ranging from soft brain cells to stiff cartilage, and the temporal variability in the mechanical stress response when stationary cells begin to migrate, e.g. in embryogenesis, wound healing or cancer metastasis, is reflected in a surprisingly small number of molecular building blocks. Three distinct filament systems, actin filaments, microtubules and intermediate filaments (IFs), self-organize into a wealth of structural units, collectively termed the cytoskeleton. The main molecular players of this remarkable composite material are largely known. However, from a physics point of view, in particular IFs are poorly understood, despite their importance in health and disease and astonishing mechanical properties, like extreme extensibility and high flexibility. It is not known, how these properties are encoded in the molecular interactions of the protein filament and how they feed into the mechanical behavior of a whole cell. The aim of the proposed research is thus to establish a structure-mechanics-function relationship for this important component of the cytoskeleton. The genetic complexity of the IF protein family with 70 members that are expressed in a tissue specific manner requires a strategic approach involving well-defined model systems and the combination of in vitro and cell work. Direct mechanical testing by applying stress and in situ high-resolution imaging will link mechanical properties to molecular interactions in the hierarchical IF architecture. The results of these in vitro studies will be related to cell experiments to decipher the link between IF type and cell mechanics. The work program will lead to models that predict, how modifications, e.g., in the type of IF protein or specific charge interactions, are associated with changes in cell mechanics and eventually in cell function.

Work performed

First reporting period (05/2017 - 10/2018):

During the first reporting period, we purchased and set up the necessary instrumentation, a quadruple optical trap with integrated confocal laser microscope and microfluidic chip. Three PhD students and a technician were hired in the beginning of the funding period. An administrator was also hired. The technician was later moved to a permanent position within the institute when the PI was promoted to a full professor position. In September 2018, a postdoc was hired.
A number of technical issues had to be solved concerning the optical trap setup. Now the instrument is working mostly smoothly and data are being acquired on a daily basis. There will be a free software and hardware update next spring, which will increase the quality of the data and the ease of use even more.
Expression and purification of recombinant protein was established in our lab and we are now able to produce and handle different intermediate filament proteins, including directed mutagenesis. Cell culture was also established, in particular using IF-free fibroblasts and we have performed first tests using mutant cells lines including wildtype and mutated protein.
Experiments were carried out on the following topics:
- comparison of the mechanical properties of vimentin and keratin IFs
- influence of ionic environments on different types of IFs
- influence of the pH on different types of IFs
- interactions between two single IFs, influecne of ionic and pH environement
- influence of physophorylations on IF assembly
- test expeirments with cells in optical traps
- theoretical modeling of the above situations

Final results

The PRIMARY GOAL of this research program is to establish a convincing structure-mechanics-function relationship for IFs and thus provide the missing link between IF protein mechanics and cell mechanics. The large variety of IF proteins that are expressed in different cells of the human body on the one hand and the versatile viscoelastic properties of the cells on the other hand lead to the hypothesis that the type (or combination) of IF protein(s) in a cell is, at least in parts, responsible for the mechanical properties of the cell. In turn, I assume that the mechanical properties are directly encoded in the hierarchical structure of the filaments. Both aspects are accessible using biophysical methods. Reaching this goal is important for at least three reasons:
1. A thorough understanding of the physical principles underlying the processes in a healthy cell is the necessary prerequisite for investigating situations in disease. Long alpha-helices are abundant in mechanically relevant proteins and our findings may be generalized in this respect.
2. Apart from the importance in biomedicine, I also expect this work to open up new opportunities for materials research. Apparently, the special hierarchical architecture of IFs leads to astonishing viscoelastic properties. If we were able to understand how these structural elements work individually and in concert (e.g. the high stretchability), mimicking the mechanical properties and development of equally remarkable materials might be possible.
3. As much as physics can help biology (see aspect 1), the other way around, highly complex biological systems, like IFs, provide a wealth of intriguing soft condensed matter physics problems, which can be studied on accessible time, length and force scales.