Force microscopy @DyNaMo

Membrane remodeling 

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. 

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; Rico et al. 2019). 

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.

Membrane mechanics

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.

Cell Mechanics

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).