QUANTIFYING CELL ADHESION USING SINGLE-CELL FORCE SPECTROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 212

contact zone, and the probability that these bonds can resist large rupture
forces decreases ( Fig. 10.5 ) . 48 Since the total number of receptors and their
binding strengths contribute to F D , it is most commonly used to quantify the

overall cell adhesion. This overall cell adhesion is deined as the sum of all
adhesive interactions established between cell and substrate.

10.3.3.2 Analyzing discrete force steps

F-D curves usually display small discrete force steps ( Fig. 10.4b ) that can be
distinguished into jumps and tethers ( Fig. 10.4 ). A non-linear force loading
typically precedes jump-events, whereas a force plateau is detected prior to
tether-events ( Fig. 10.6 ) . Force gradient prior rupture (>0 for jumps, ≈0 for
tethers) along with the distance at which force jumps occur can be used to
distinguish jump- and tether-events. Separating jump- and tether-events is
necessary, since they contribute to different detachment scenarios ( Fig. 10.6 ) .
Jump-events can be interpreted as the unbinding of single or few receptors

from the substrate. The non-linear force loading prior to rupture suggests
that the probed receptors are connected to the actin cytoskeleton 49 ( Fig.
10.6a ) . To give an example, integrins often localize to specialized complexes
involving assemblies of cytoskeletal linker and signalling proteins. Stretching
these membrane-cytoskeleton linkers leads to a non-linear force increase
prior bond rupture. 49 The magnitude of the force step relects the stochastic
survival of this ligand-receptor bond under an increasing force load. 48,49 The
ensemble of jump-events can provide information on the afinity and avidity
of receptors.
Tether-events can be found at pulling distances up to several tens of

micrometers after the major rupture peak. The force plateau preceding the
force step originated from the extraction of membrane tethers (membrane
nanotubes) from the cell membrane ( Fig. 10.6b ). Membrane tethers are
formed when receptors that are not or weakly connected to the cytoskeleton
are pulled from the cell membrane ( Fig. 10.6b ) . 49 Alternatively, membrane
tethers can form by “unspeciic” interactions established between membrane
and AFM tip. Speciic blocking experiments may be suitable to show
unambiguously by which of the two binding events membranes tethers are
formed. 50
The force plateau measured upon extraction of a membrane tether shows
that the force required to extract the tether is constant over long extraction
lengths. The membrane tether restoring force depends on the extraction
velocity and does not relect the strength of the receptor-ligand bond
anchoring the membrane tether at its tip. 49,51 Thus, native cell membranes
establish force-clamped membrane tethers that can be employed to measure