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

• Final Report Year: 2024

Final Report Abstract

Mechanical signals play a fundamental role in regulating cell function in health and disease. Mechanosensing and transduction are key regulatory processes where these signals are transmitted across focal adhesion (FA) sites from the intracellular to the extracellular environment and vice versa. Unravelling the underlying molecular mechanisms is crucial for understanding cell function and, on the long-term, for disease diagnosis and treatment. The goal of this technology oriented project was to develop a new generation of peptide-based molecular force sensors (MFSs) for visualizing and quantifying molecular forces between cell surface receptors, located in FA sites, and their extracellular ligands. These MFSs are intended to convert cell forces into a fluorescent readout, detectable with optical microscopy typically used for observing cellular structures. Inspired by extracellular matrix proteins, these MFSs consist of mechanically calibrated coiled coil (CC) structures and need to be equipped with a ligand and a fluorescent reporter system based on Förster resonance energy transfer (FRET). A key design feature of the CC-based MFS is that they possess similar thermodynamic and kinetic stabilities while their rupture forces are different over a range of pulling speeds. This is achieved using one and the same CC structure and applying force in different pulling geometries. To establish CCs as MFSs, they were first mechanically calibrated with singlemolecule force spectroscopy. Next, two series of experiments were performed to (1) introduce a donor-acceptor pair to distinguish the folded CC structure (intact MFS; high FRET efficiency) and the dissociated CC structure (broken MFS; no FRET) and (2) to equip the CC with a celladhesive ligand for proof-of-concept cell culture experiments. Testing both organic fluorophores and fluorescent proteins as donor-acceptor pairs, it was shown that the FRET efficiency reports on the structural state of the CC; however, optimization of the labelling strategy is still needed and no cell culture experiments with FRET-labelled CCs have been performed yet. Cell culture experiments were performed with unlabelled CCs, using MFSs targeting integrin receptors (RGDS ligand). Fibroblast cells were seeded onto RGDS-MFS functionalized surfaces, grown for 30 to 120 min, fixed and immunostained to visualize the nucleus, actin filaments and FA sites (p-paxillin). Clear differences were observed between cells grown on RGDS-MFS functionalized and control surfaces with covalently coupled RGDS. Results so far suggest that the MFSs tested lack the necessary mechanical stability to resist cell-generated forces impeding long-term cell attachment. Future work will focus on the development of stronger MFSs, the introduction of the FRET reporter system and live cell imaging to observe MFS rupture in real-time. We anticipate that this MFS platform can aid the analysis of cells with different mechanical profiles as they may occur in disease.

Publications

• Shedding Light on Cell-Material Interactions with Coiled Coil-based Molecular Force Sensors (Dec 2021) Lorentz Workshop on Cancer in a Physical Context: from Understanding to Therapeutics, online (invited lecture, Kerstin Blank)
  Kerstin Blank
  
• 2D Mechanoresponsive Surfaces for Measuring Cellular Traction Forces (Sept 2022) German Biophysical Society Meeting 2022, Konstanz/Germany (poster, Russell Wilson)
  Russell Wilson
  
• Coiled Coil Molecular Force Sensors for Measuring Cellular Force & Attachment (Sept 2022) Alpbach Workshop Coiled-Coil, Fibrous & Repeat Proteins, Alpbach/Austria (contributed talk, Russell Wilson)
  Russell Wilson
  
• Shedding Light on Cell-Material Interactions with Coiled Coil-based Molecular Force Sensors (March 2022) Nanoengineering for Mechanobiology (N4M), Camogli/Italy (invited lecture, Kerstin Blank)
  Kerstin Blank
  
• Coiled Coils as Molecular Force Sensors - from molecular mechanisms towards applications in cell biology (Aug 2023) 11th International Conference on Biological Physics, Seoul/Korea (invited lecture, Kerstin Blank)
  Kerstin Blank
  
• Coiled coils as molecular force sensors: from molecular mechanisms and design to biological applications (Dec 2023) 12th Austrian Peptide Symposium, Vienna/Austria (contributed talk, Russell Wilson)
  Russell Wilson
  
• Kleine Kräfte, große Wirkung: mechanische Prozesse in unserem Körper (May 2023) MINT TANK Summer School, Linz/Austria (Kerstin Blank)
  Kerstin Blank
  
• Coiled coils as mechanoresponsive sensors for measuring cell forces and attachment (July 2024) Gordon Research Conference on Multiscale Mechanochemistry and Mechanobiology, Lewiston (ME)/USA (poster, Russell Wilson)
  Russell Wilson
  
• Coiled coils as mechanoresponsive sensors for measuring cell forces and attachment (July 2024) Gordon Research Seminar on Multiscale Mechanochemistry and Mechanobiology, Lewiston (ME)/USA (contributed talk, Russell Wilson)
  Russell Wilson
  
• Coiled Coils as Molecular Force Sensors (Feb 2024) XXIII. Annual Linz Winter Workshop, Linz/Austria (contributed talk, Kerstin Blank)
  Kerstin Blank
  
• Coiled Coils as Molecular Force Sensors: from Molecular Mechanisms to Applications in Cell Biology (July 2024) Biophysics Austria Conference 2024: Advancing Science, Salzburg/Austria (contributed talk, Kerstin Blank)
  Kerstin Blank
  
• Kleine Kräfte, große Wirkung: mechanische Prozesse in unserem Körper (Feb 2024) Rotary Club Linz, Linz/Austria (Kerstin Blank)
  Kerstin Blank
  
• Kleine Kräfte, große Wirkung: mechanische Prozesse in unserem Körper (Feb 2024) Traumberuf Technik, Linz/Austria (Kerstin Blank)
  Kerstin Blank
  
• Structure-Mechanics Relationships of Heterodimeric Coiled Coils (March 2024) Spring Meeting of the German Physical Society, Berlin/Germany (contributed talk, Kerstin Blank)
  Kerstin Blank
  
• Structure-Mechanics Relationships of Heterodimeric Coiled Coils (Sept 2024) 73rd Annual Meeting of the Austrian Physical Society, Linz/Austria (contributed talk, Kerstin Blank)
  Kerstin Blank
  
• What do Bubble Tea and Tissue Regeneration have in Common? (May 2024) Long Night of Research 2024, Linz/Austria (Russell Wilson)
  Russell Wilson