Single-molecule recognition force spectroscopy of transmembrane transporters on living cells

Abstract

Atomic force microscopy (AFM) has proven to be a powerful tool in biological sciences. Its particular advantage over other high-resolution methods commonly used is that biomolecules can be investigated not only under physiological conditions but also while they perform their biological functions. Single-molecule force spectroscopy with AFM tip-modification techniques can provide insight into intermolecular forces between individual ligand-receptor pairs of biological systems. Here we present protocols for force spectroscopy of living cells, including cell sample preparation, tip chemistry, step-by-step AFM imaging, force spectroscopy and data analysis. We also delineate critical steps and describe limitations that we have experienced. The entire protocol can be completed in 12 h. The model studies discussed here demonstrate the power of AFM for studying transmembrane transporters at the single-molecule level.

Investigating cell mechanics with atomic force microscopy

“…Using this method, researchers have shown that the specificities of E-and N-cadherins are distinct ( preferring homophilic bonds), alongside producing different adhesive and compliant bonding forces. Similarly, ligand binding to atomic force microscope tips allows for examination of specific cellular molecules and their interactions [162]. Recently, biotin/streptavidin functionalization has shown that tension regulates actin depolymerization [163].…”

Atomic Force Microscopy (AFM) in biomedical research

“…The application of adhesion force mapping is an expanding fi eld where the power of the technique is limited by the stability and effectiveness of the functionalized probe. Procedures to functionalize probes require expertise across multiple disciplines; however, these techniques are now starting to become more standardized in practice (Heinisch J 2010, Puntheeranurak T 2011, allowing non-specialist AFM experimentalists to apply these adhesion mapping procedures to their research.…”

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor

“…In the AFM measurement, the CD3ε CD -conjugated cantilever was firstly approached to PMSs and remained still at a constant force of 100 pN for 1 s to allow the protein-membrane interaction. Similar low contact forces have been widely used by the AFM community to study physiological systems with membranes to avoid disrupting membrane structures [59][60][61]. We analyzed the mechanical properties of PMSs by fitting the contact region in the approaching trace using the Hertz model (Supplementary information, Figure S1B).…”