We develop and apply advanced atomic force microscopy (AFM) to unravel the physics behind biological systems, from single molecules to membranes and living cells.
Our research is based on imaging and force spectroscopy with conventional and high-speed atomic force microscopy (HS-AFM).
Our interdisciplinary group integrates members coming from physics, chemistry, engineering, and biology backgrounds.
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.
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 (Casuso et al., Nature Nanotechnology 2012) and the glass-like diffusion behavior of Pore Forming Toxins (Munguira & Casuso et al., ACS Nano 2016).
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). Now we are working on membrane tubulation due to Salmonella effector proteins.
Cellular to molecular 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).
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 are currently probing the binding strength of the streptavidin-biotin bond to compare them with simulations at the same pulling rates. 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 capacity of proteins to carry out different functions is related to their ability to undergo conformation changes, which depends on the flexibility of protein structures. We have applied PeakForce imaging mode to map quantitatively the flexibility of individual membrane proteins in their native, folded state at unprecedented submolecular resolution (Rico et al. Nano Letters 2011, Rico et al. Soft Matter 2013). We are now adapting HS-AFM to perform high-speed force mapping at unprecedented time resolution.
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. For example, we use PeakForce mechanical mapping and conventional force spectroscopy to determine the mechanical properties of lens cells and cells grown on micropatterns (Rigato et al. ACSNano 2015). We have recently adapted HS-AFM to probe the microrheology of living cells at high frequencies (Rigato et al. Nat Physics 2017, in press).
- 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
- Rigato, A., Miyagi, A., Scheuring, S. and Rico, F. (2017). High-frequency microrheology reveals cytoskeleton dynamics in living cells. Nature Physics (in press). And News and views by Klaus Kroy.
- 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
- Chiaruttini, N.*; Redondo-Morata, L.*; Colom, A.; Humbert, F.; Lenz, M.; Scheuring, S.; Roux, A.; Relaxation of loaded ESCRT-III spiral springs drives membrane deformation, Cell, 2015, 163 (4): 866-79 (*, equal contribution).
FM4B-Lab, U1006 INSERM & Aix-Marseille Unviersité
Parc Scientifique de Luminy, Bâtiment Inserm TPR2 bloc 5, case 909
163 avenue de Luminy, 13009 Marseille, France