Shedding Light on Cell-Material Interactions with Coiled Coil-based Molecular Force Sensors

• Applicant: Professorin Dr. Kerstin Blank
• Subject Area: Biophysics ; Biochemistry ; Statistical Physics, Nonlinear Dynamics, Complex Systems, Soft and Fluid Matter, Biological Physics
• Term: from 2021 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 449551016

Mammalian cells utilize a large variety of different mechanosensors and actuators to detect and respond to mechanical signals. At the interface between cells and their extracellular matrix (ECM), mechanosensing involves receptor-mediated force transduction at focal adhesion complexes. It is widely accepted that many focal adhesion proteins (e.g. integrins) undergo force-induced conformational changes that trigger downstream signaling. Little is currently known, however, about the magnitude of forces required to cause these conformational changes. This proposal aims at the development of a new generation of peptide-based molecular force sensors (MFSs). These MFSs convert the experienced force into a fluorescence readout and thus allow for the detection of forces acting across cell surface receptors.Inspired by natural, mechanoresponsive ECM proteins, the proposed project focuses on the development of peptide-based MFSs that contain coiled coils (CCs) as their key molecular building block. These CCs will be mechanically calibrated at the single-molecule level and, subsequently, be equipped with the fluorescence reporter system. More specifically, the following steps will be taken:1. Starting with a thermodynamically highly stable CC, a library of CCs with different mechanical stabilities will be established while keeping their thermodynamic stability unaffected. These CCs will be mechanically characterized with dynamic single-molecule force spectroscopy to determine their mechanical stabilities (i.e. rupture forces) as a function of the applied force loading rate.2. These mechanically calibrated CCs will then be converted into MFSs that report on their mechanical state with a specific fluorescence signal. For this purpose, Förster resonance energy transfer (FRET) will be utilized. The donor-acceptor pair will be introduced such that the intact MFSs show a high FRET efficiency while FRET is absent once the MFSs have been broken apart.3. These new MFSs will then be tested in cell culture applications with the goal of detecting the molecular forces acting between integrins and their extracellular ligands. To answer a critically important biological question and to obtain new insights into integrin-mediated mechanotransduction at the cell-ECM interface, the interplay between different integrin subclasses will be investigated in time-dependent cell adhesion experiments.In summary, this technology oriented proposal aims at the development of a new ECM-inspired MFS design that allows for the sensitive detection of molecular forces. The ultimate goal is to establish real-time force maps with the highest possible spatial resolution. Once established, this powerful MFSs platform will be introduced into mechanically-controlled ECM-mimicking hydrogels to obtain smart 3D cell culture systems for biomedical applications.

• DFG Programme: Research Grants
• International Connection: Austria