Biology Reference
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of providing smaller contact areas at similar indentation forces than when
using cells immobilized on the cantilever pressed against a lat substrate.
For example, a pyramidal tip of semiopen angle of 35° indenting a cell of
1 kPa with a compression force of 50 pN will provide an area of contact
~0.3 μm 2 . It is thus easier to achieve single-molecule conditions than in
the previous coniguration. To further control contact conditions, modiied
cantilever tips with lat-ended cylindrical shape provide a constant and
known area of contact, independent of applied force and indentation. 32,45
In this coniguration, the formation of tethers may also be a problem.
However, tethers are normally less common in adherent cells than in blood
or suspended cells.
Using immobilized cells both on the substrate and on the cantilever is
probably the optimal coniguration for cell-cell adhesion studies, such as
in cadherin-cadherin adhesion measurements ( Fig. 11.4d ). Receptors and
ligands are in their native environment, fully functional and properly oriented.
However, the actual application of such a coniguration is complicated,
especially to obtain speciic single-molecule events. As one cell indents the
other, similar cell size and elasticity provides an even bigger area of contact,
in which multiple bonds are easily formed. More importantly, cells express
a wide variety of CAMs that can bind one or multiple ligands. Thus, it is
more dificult to discriminate between possible nondesired adhesion events
between molecules. A very elegant approach to prove speciicity is by using
the same cell line with knocked-out protein of interest, and then showing
that no adhesion is found on it. This set-up has been applied before to study
cell-cell adhesion at single 7,38 and multiple molecule levels. 15,46 It is also
possible to block undesired molecules using speciic antibodies.
Under this
coniguration, the probability of tether formation is even higher than in the
previous ones, as tethers can be extracted from both cells ( Fig. 11.4d ) .
In any of the four conigurations, control measurements are crucial to
prove the speciicity of the interaction. Different type of control approaches
can be used depending on the coniguration used and the biomolecules
involved. The most straightforward approach consists in blocking the speciic
receptor-ligand interaction with soluble ligand. Another approach is to use
AFM tips or substrates in which one of the molecules of interest has not been
immobilized. A more reliable approach is to block the receptor or the ligand
with speciic blocking antibodies. As mentioned before, some interactions
require the presence of multivalent ions, such as Ca 2+ or Mg 2+ . 5,6,8 Chelating
the required ions can also be used to prove speciicity. In the case of living
cells, an elegant approach is to knock out one of the proteins of interest. In
practice, a combination of these control experiments is recommended.
46
 
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