Biomedical Engineering Reference
In-Depth Information
Although the first DFS studies on adhesion processes have been restricted to
investigate the interaction between individual biomolecules, more recently the DFS
capabilities have been extended to the study of the interactions between biomolecules
embedded within the cell surface. In such a way, more reliable information on the
behavior of biomolecules in physiological conditions can be extracted.
Baumgardner et al. have investigated the interaction between single recombi-
nant cadherin proteins that are ubiquitous, calcium-dependent homophilic molecules
involved in cell-cell adhesion (Baumgartner et al., 2000). Indeed, the cadherin-
cadherin interaction plays a crucial role in a multitude of physiological and patholog-
ical processes, including embryogenesis, motility, differentiation, and carcinogene-
sis. They found that unbinding forces for a single cadherin-cadherin association are
smaller than those previously obtained for other protein-protein complexes. Inter-
estingly, they put into evidence that the unbinding force increases when a longer
encounter time was applied; the encounter time being a delay time between the
approaching and the retraction phases. Such an effect has been put into relationship to
the occurrence of a cooperative interaction among cadherin molecules, likely respon-
sible for multiple-adhesion processes. The authors have deduced some criteria to dis-
criminate between specific and nonspecific unbinding events by taking into account
the nonlinear extension in the retraction curve due to the stretching of the PEG linker
used to bind cadherin to the AFM tip. More specifically, they have accepted only
those force curves displaying such a nonlinear trend, whose corresponding unbind-
ing length was consistent with that expected from the PEG stretching (see Figure 6.2
and Chapter 4). They found that the unbinding force as a function of the logarithm of
the loading rate follows a linear trend indicative of the presence of a single barrier in
the energy landscape, the corresponding parameters being reported in Table 6.5. The
rather high dissociation rate value is indicative of a transient complex, consistently
with the rapid remodeling of the cell shape to optimize the cellular adhesion at physi-
ological conditions. Interestingly, the authors have developed a procedure to roughly
estimate the association rate
k
on
from DFS data. In particular, they used the relation-
ship
k
on
5
,where
N
A
is the Avogadro number,
V
eff
is the effective volume
of a half-sphere, with radius
R
eff
around the tip, and
t
0
t
0
.
=
N
A
V
eff
/
5
is the time required for the
half-maximal binding probability that can be evaluated from
t
0
.
.
5
v
,where
v
is the approach speed of the cantilever. For the cadherin-cadherin association, they
obtained a
k
on
in the range 10
3
=
2
R
eff
/
10
4
M
−
1
s
−
1
. They have also derived an affinity
−
10
5
M. Such an approach allowed them to
analyze the affinity constant of the cadherin complexes as a function of the Ca
2
+
concentration. They found that the affinity drastically increased when the Ca
2
+
con-
centration exceeded a threshold at which the occurrence of some structural changes
were able to modulate the intercellular adhesion processes.
Panorchan et al. have applied DFS to investigate complexes between different
types of cadherin molecules (E- and N-cadherins), which were expressed on the
surface of living cells that, in turn, were immobilized on both the tip and the sub-
strate (see Figure 6.10; Panorchan et al., 2006). From the analysis of the unbinding
force as a function of the logarithm of loading rate, they found two distinct linear
k
off
in the range 10
3
constant
K
a
=
k
on
/
−
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