PROBING CELLULAR ADHESION AT THE SINGLE-MOLECULE LEVEL - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 228

10-20%. 19 Force curve measurements have been extensively used to probe
the viscoelastic properties of cells, the mechanical unfolding of proteins and
the disruption forces of receptor-ligand bonds. 20,21 There are various types of
force curves that will be described in the following sections.
11.3 SINGLEMOLECULE MEASUREMENTS OF CELL
ADHESION
As mentioned before, cellular adhesion is ultimately mediated by individual
receptor-ligand interactions that are subjected to mechanical stress. Thus,
it is relevant to understand the forced dissociation behaviour of individual
receptor-ligand complexes on living cells. The AFM is particularly suitable
for probing adhesion interactions one molecule at a time, given its high
spatial resolution and fast time response. To that end, the AFM is used in
force spectroscopy mode to measure the interaction forces between two
adhering surfaces. The biomolecules of interest have to be immobilized
on opposing surfaces (AFM tip and substrate or cell, Fig. 11.3 ) that will be
brought into contact and allowed to interact. Then, the bond will be forcibly
dissociated by pulling from receptor and ligand. Figure 11.4 shows four
possible conigurations to probe cell adhesion interactions with the AFM at
the single-molecule level together with a schematic example of a resulting
force curve. The various approaches have advantages and disadvantages
and the inal application will determine the best coniguration to be used.
In this section, we will describe the experimental set-up for single-molecule
force measurements of cell adhesion using the AFM, including the necessary
coating of cantilevers and substrates. The second part of this section will
describe different force spectroscopy approaches, such as dynamic force
spectroscopy (DFS) and force clamping techniques, which can be used
to characterize biological bonds. In the last section we will introduce the
theoretical framework to interpret force spectroscopy measurements.
11.3.1 Experimental Set-Up
Force spectroscopy measurements can be carried out on puriied proteins
immobilized on the tip and substrate, on proteins expressed on the surface
of living cells or a combination of both. The use of a living cell has the main
advantage of having the protein in its native environment, which means the
protein is fully functional. Some CAMs on the cell membrane are dynamically
linked to the cytoskeleton and the pulling response of a protein with or
without link to the cytoskeleton varies importantly. In some cases, e.g. when