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is similar to the forces measured between HBHA and heparin molecules.
Adhesion maps recorded across A549 cells showed fairly homogeneous
contrast, indicating that the receptors were widely and homogeneously
exposed. The speciicity of the measured interaction was conirmed by
showing a dramatic reduction of both the adhesion frequency and adhesion
force values when the cell surface was treated with heparinase. Strikingly,
at large pulling velocities, constant force plateaus were seen in most curves
(
Fig. 15.4c
)
. This indicated that stressed HSPG receptors detached from the
cytoskeleton, therefore leading to the extraction of membrane tethers or
nanotubes (
Fig. 15.4d
)
. These membrane structures have been observed in
liposomes and different cell types, including red blood cells, neutrophils,
neurons, ibroblasts as well as mesendoderm, epithelial and endothelial
cells.
34,35
Tether formation may play a role in pathogen-host interactions
since the invasion mechanisms of pathogens such as
Salmonella
and
Shigella
are known to involve the production of large membrane projections and the
formation of membrane-bound vacuoles.
15.2.4.3 Homophilic HBHA
-
HBHA interacons
The N-terminal domains of HBHA contain a predicted coiled-coil region
involved in homophilic interactions that may potentially contribute to
bacterial aggregation and to the formation of polymeric HBHA structures.
32
Until now, detailed information on the forces of such homophilic interactions
was lacking. In this context, SMFS could reveal the molecular forces driving
HBHA-HBHA interactions, both on model surfaces and on live mycobacteria.
36
Histidine-tagged proteins were attached, via their C-terminal or N-terminal
end, to gold-coated AFM tips and supports
(
Fig. 15.5a
). Force-distance curves
recorded between HBHA exposing their N-terminal regions showed a bimodal
distribution cantered at 68 ± 2 pN and 130 ± 14 pN (
Fig. 15.5b
)
. These maxima
were attributed to the formation of one or two HBHA dimers, resulting from
speciic coiled-coils interactions. Most adhesion peaks displayed non-linear
elongation forces that were best described by the worm-like chain (WLC)
model, classically used to model the unfolding of polypeptide chains. Hence,
elongation forces were attributed to the unfolding of
A
-helices of the coiled-
coil domain.
When the forces between the lysine-rich C-terminal domains were
measured, binding events showed a much broader distribution. The lack
of a well-deined maximum, together with the larger average binding force
values, suggests these forces do not primarily originate from speciic coiled-
coil interactions. Rather, the main contribution is probably due to multiple
intermolecular electrostatic bridges between the cationic groups of the
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