PROBING CELLULAR ADHESION AT THE SINGLE-MOLECULE LEVEL - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 232

(a) (b)

(c) (d)

Figure 11.4. Tip and substrate conigurations for single-molecule measurements of
cell adhesion. Schematic examples of retraction force-time curves are shown for each
case. (a) Protein on tip and substrate. (b) Cell on cantilever and protein on substrate.
(c) Cell on substrate and protein on tip. (d) Cells on substrate and cantilever. The
force-time curves in a-c represent rupture events typical of force measurements at
constant retraction speed in which the receptor and ligand are irmly attached to
the substrate (tip, dish, or cell cytoskeleton). The rupture forces ( f r ) are measured
as the force jump relative to noncontact (grey dashed line) plus a viscous drag
correction, 42,43 while the loading rate ( r f ) is estimated from the slope prior to
rupture. The dynamic force spectra are obtained by measuring the most probable
rupture force at different loading rates ( Fig. 11.5 ) . The force-time curve in (d) shows
an example of an adhesion event in which membrane tethers are formed through
detachment of the receptor and ligand from the underlying cells' cytoskeletons. In
that case, the force jump ( f tether ) represents the force required to extract the tethers
and it is applied to the receptor-ligand bond. The measured lifetime ( τ bond ) is thus the
lifetime of the bond at the applied tether force. Tether forces mainly depend on the
friction between the membrane and the cytoskeleton and on the retraction speed. At
different retraction speeds, the tether forces vary allowing us to estimate the bond
lifetime at various force levels. 44

mentioned before, it is important to use coatings that will not activate cells,
such as certain antibodies, as this would modify the adhesive properties
of the cells and, perhaps, the afinity of the adhesion molecules. The main

advantage of using living cells is that membrane proteins are in their native
environment, being thus fully functional. The main practical drawback is that
cells are complex systems and a wide variety of other proteins are normally
expressed in the cell surface. This can lead to undesired or multiple binding
to the ligand of interest, which is dificult to discriminate and isolate. In
addition, measurements in cells present normally more unspeciic binding.
Thus, blocking strategies and control measurements are particularly

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