Our research is based on imaging and force spectroscopy with high-speed atomic force microscopy (HS-AFM). We divide our work in two main areas.
Dynamics in biological membranes by HS-AFM imaging
Goal: The understanding of relevant biophysical processes of the cell membrane.
How: Simultaneous characterization of structure, dynamics and mechanics of biomolecules with submolecular resolution and sub-second time resolution.
Tool: Our main work tool is the high-speed atomic force microscope (HS-AFM). This is a young and still semi-prototype technique. We perform important development activities with the goal of increasing the information flow on the biomolecular activity obtained by HS-AFM.
How it works: The HS-AFM is a miniaturization of conventional AFM where the dynamic components (AFM probe and sample scanner) have been reduced to the minimal possible size achieving x1000 faster imaging speeds than conventional AFM.
Lipid-mediated protein interactions
In 1972 the fluid-mosaic membrane model was proposed by Nicolson to depicture a highly organized crowded and clustered mosaic of lipid membranes. The model keeps updating as new data is available, stressing the fluctuating membrane domains, protein complexes, cooperative events and anomalous diffusion. Our approach is unique to tackle this complexity, as it provides direct and unlabeled observations. We have used high acquisition rates to analyze the influence of membrane crowding on the motion of individual OmpF proteins in the membrane. Glass-like diffusion was identified.
Characterization of the motion of membrane proteins using high-speed atomic force microscopy
We are interested in how the membrane properties constrain the actions of proteins involved in membrane deformation and restructuration. By using simple lipid membrane models, we characterize dynamic biological processes occurring in the membrane, as is the Endosomal Sorting Complex Required for Transport (ESCRT). We previously deciphered how the polymerization of ESCRT drives membrane deformation (Chiaruttini & Redondo-Morata et al., Cell 2015).
Glasslike Membrane Protein Diffusion in a Crowded Membrane
‘Lysenin toxin membrane insertion is pH-dependent and non-cooperative´ Munguira, I. Casuso (co-first autor), H. Takahashi, S. Scheuring 2017 Biophysical J 113(9), 2029-2036´
´Glasslike Membrane Protein Diffusion in a Crowded Membrane´ I. Munguira, I. Casuso (co-first autor), H. Takahashi, F. Rico, A. Miyagi, M. Chami, S. Scheuring 2016 ACS nano 10 (2), 2584-2590
´A hybrid high-speed atomic force–optical microscope for visualizing single membrane proteins on eukaryotic cells´ A. Colom, I. Casuso, F. Rico, S. Scheuring 2013 Nature communications 4, 2155
Molecular to cellular mechanics
The second part of our research involves the development and application of atomic force microscopy (AFM) to probe the mechanical properties of single molecules, membranes and living cells. It is divided in three main axes that interlace and interact with each other.
High-speed force spectroscopy (HS-FS) of single protein unfolding and receptor-ligand complexes
We use HS-AFM to perform force spectroscopy measurements on single biomolecules at high velocities with microsecond time resolution. We have recently adapted the HS-AFM system to allow force spectroscopy measurements at the speed of molecular dynamics (MD) simulations. We unfolded titin immunoglobulin domains at speeds up to ~4 mm/s. Experimental unfolding forces compared well with in silico experiments on the same titin domain (Rico et al. Science 2013; Takahashi et al. ACS Nano, 2018).
We go now a step forward in the adaptation and application of HS-AFM and its microsecond time force response. We combine molecular dynamics and high-speed force spectroscopy to unravel the binding strength of receptor-ligand bonds. We have recently probed the binding strength of the streptavidin-biotin bond and compare it with simulations at the same pulling rates revealing heterogenous and rate-dependent unbinding pathways (Rico et al. PNAS 2019).
The final goal is to apply HS-FS to probe the interaction of adhesion molecules. The combination of HS-FS and MD simulations provides an atomic description of the unbinding process based on experimental results.
The mechanical properties of living cells are crucial for biological function. We apply different methods based on AFM combined with other techniques to probe the mechanics of cells under various conditions. We have recently adapted HS-AFM to probe the microrheology of living cells at high frequencies (Rigato et al. Nat Physics, 2017). Microrheology over a wide dynamic range provides mechanistic understanding of cell mechanics and a univocal fingerprint, applicable to diagnosis or prognosis of disease.
- Rico, F*¶, A Russek¶, L González, H Grubmüller*, and S Scheuring*. “Heterogeneous and Rate-Dependent Streptavidin–Biotin Unbinding Revealed by High-Speed Force Spectroscopy and Atomistic Simulations.” PNAS 116, 14, 6594–6601 2019 https://doi.org/10.1073/pnas.1816909116
- F Sumbul, A Marchesi, F Rico*. History, Rare And Multiple Events Of Mechanical Unfolding Of Repeat Proteins. J Chem Phys, 148(12), 123335 2018
- Takahashi¶, F Rico¶, C Chipot , and S Scheuring. α-Helix Unwinding as Force Buffer in Spectrins. ACS Nano, (in press) 2018. DOI: 10.1021/acsnano.7b08973
- Rico F, L González, I Casuso, M Puig, and S Scheuring. High-speed force spectroscopy unfolds titin at the velocity of molecular dynamics simulations. Science 342 (6159), 741-743 2013