PROBING CELLULAR ADHESION AT THE SINGLE-MOLECULE LEVEL - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 236

probable rupture force depends on the loading rate. Thus, DFS experiments
require the acquisition of many rupture events at various loading rates
( Fig. 11.5 ) . In practice this involves the acquisition of sets of force-distance
curves at various retraction speeds, usually ranging three or more orders
of magnitude ( Fig. 11.5a ) . The rupture force distributions will provide a
measure of the most probable rupture force at each loading rate ( Fig. 11.5b ) .
A plot of the most probable rupture force versus loading rate is named the
dynamic force spectrum of the interaction ( Fig. 11.5c ) . In the next section we

will describe how to obtain the intrinsic parameters of the interaction from
such dynamic force spectra and the force-dependent lifetime ( Fig. 11.5d ) .

(a) (b)

(c) (d)

Figure 11.5. Dynamic force spectroscopy of integrin α 4 β 1 binding to vascular cell
adhesion molecule-1 measured on living monocytic cells. (a) Representative examples
of force curves showing single-molecule rupture events at three different retraction
speeds. The determination of the rupture force ( f r ) and the effective stiffness ( k s ) is
shown. The loading rate can be extracted by multiplying k s by the retraction speed.
(b) Rupture force distributions at three loading rates. (c) Dynamic force spectrum of
the interaction, i.e. most probable rupture (±SEM) forces as a function of loading rate
(open circles). The solid line represents the best it to the Bell-Evans model Eq. (11.6) ,
that leads to γ = 5.5 ± 0.5 Å and τ 0 = 3.3 ± 1.3 seconds. (d) Force-dependent lifetime
(solid line) calculated using the itted parameters in Eq. (11.2) . The open symbols
show the results obtained from directly computing the lifetime-force response from
the rupture force distribution at moderate loading rate (middle histogram in a) using
the new approach introduced by Dudko and coworkers, Eq. (11.7) . 58

It is important to mention that pulling from a receptor-ligand complex
at constant retraction speed not always leads to constant loading rate. For
example, when using long linkers to tether the biomolecules to the tip,
constant retraction speeds give rise to nonlinear responses that can be
described by different models, such as the freely jointed chain model.

In the case of living cells, the situation is even more complex. As mentioned
before, some receptors are linked to the cytoskeleton via different molecules.
If this link is stronger than the receptor-ligand bond, the force response at a

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