Biology Reference
In-Depth Information
biomembrane force probe, the optical and magnetic tweezers and atomic
force microscopy (AFM). These assays cover a wide range of forces and
length scales that are relevant to biology, going from small intermolecular
interactions to strong covalent bonds. As opposed to ensemble techniques,
single-molecule experiments can detect, localize and analyze individual
biomolecules in heterogeneous populations, thereby revealing rare events
that would otherwise be hidden. Notably, owing to its tiny scanning force
probe, AFM is the only force technique which can simultaneously localize,
manipulate and force probe single-molecules on microbial cells, thereby
enabling an important paradigm shift in microbiology. In this chapter, we
discuss recent progress made in using AFM to measure the adhesive and
mechanical properties of microbial cell envelope proteins.
15.2 BINDING STRENGTH OF CELL ADHESION PROTEINS
Cell adhesion proteins play essential roles in mediating cellular events
such as pathogen-host interactions and represent privileged targets for
anti-adhesion therapy. Advances in AFM-based single-molecule force
spectroscopy (SMFS)
have allowed researchers to measure the speciic
binding strength of various cell adhesion molecules (
Chapter 11
), including
selectins,
11,12
13,14
cadherins,
15
integrins,
16
proteoglycans
17
and bacterial adhesins.
18
Speciic molecular recognition forces are measured by recording force-
distance curves between the sample (cells or puriied receptors) and an
AFM tip modiied with appropriate ligands, and then assessing the binding
force between complementary molecules.
Notably, the spatial distribution
of the individual adhesion molecules can be mapped (
Chapter 12
).
To this end, force curves are recorded at multiple locations of the
19
plane to generate a spatially resolved force map in which the adhesion
force values are displayed as gray pixels. In single-cell force spectroscopy
(
Chapter 10
),
x
,
y
cells are attached to the cantilever to measure cell-cell or
cell-substrate adhesion forces.
20,21
15.2.1 Funconalized Tips
An important prerequisite for successful molecular recognition experiments
is to functionalize the AFM tip with ligands or receptors.
19
The forces which
immobilize the molecules have to be stronger than the intermolecular force
being studied and the attached biomolecules should have enough mobility
so that they can freely interact with complementary molecules. It is also
recommended to minimize the contribution of non-speciic adhesion to the
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