Intracellular Mechanical Fingerprinting: Identifying the proteins controlling the intracellular active mechanics

• Applicant: Professor Dr. Timo Betz
• Subject Area: Biophysics
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 516046415

Deciphering the intracellular mechanics and active force generation in living cells is key for the quantitative understanding of many fundamental cellular processes. Despite more than two decades of research, we still have only very little knowledge about the intracellular mechanics, not even to mention how the mechanical properties are controlled by the cell. Using a recently established “fingerprint” of intercellular active mechanics, we will combine active and passive optical tweezer measurements with a synergistic bottom up and top down approach to determine which proteins do control the intracellular viscoelastic properties and the active force generation in the cytoplasm. While the bottom up approach will directly determine the effect of pharmacological driven protein and functional alterations, we will use a top down approach to determine which proteins correlate with changes in the mechanical properties. In this sense we will push the recently introduced idea of a "mechanome" to a new level. The bottom up approach will rely on HeLa cells that are combined with known inhibitors, but also with modern CRISPR/dCas9 based protein expression engineering. The CRISPR approach will allow to also up and downregulate any target protein additionally to the proteins which can be targeted by known drugs. In the top down approach, we will follow the mechanical fingerprint during the differentiation of iPSCs to three different cell lines. Our preliminary experiments show that indeed there are important differences in the intracellular mechanics when comparing neurons, muscle cells and lung epithelial cells. During the differentiation into these three different cell lines, we will obtain the proteome on a regular base, and then compare the protein expression level of the full proteome to the mechanical fingerprint. Using modern correlation approaches we will determine protein candidates that are responsible for the different parameters of the mechanical fingerprint. These candidates are then checked in both the iPSC and the HeLa context to determine which protein has the capacity to alter which mechanical property. Overall this approach has the potential add deep insights to the new field of mechanomics by providing clear quantitative data that relates protein expression patterns to intracellular mechanics.

• DFG Programme: Research Grants