Single-molecule force spectroscopy studies of integrin-mediated cell signaling

Integrins constitute an important class of cell adhesion receptors,
as they bidirectionally transduce information between the cytoplasm
of biological cells and the surrounding extracellular matrix. By
means of atomic force microscopy, spectroscopic measurements of the
specific interactions of integrins with their corresponding ligands
were performed. Basically, these experiments allow deep insights
into cellular signal transduction, but despite sophisticated
vibration isolation systems the acquired data exhibit very low
signal-to-noise ratios that impair an accurate analysis. This
drawback was overcome by a novel post-processing algorithm, which
significantly reduces the noise and thus improves the
signal-to-noise ratio. Thereby, previously invisible signal
features can be revealed. Another important task when evaluating
this kind of experiments is the identification of steplike
transitions corresponding to unbinding events between the
receptor-ligand bonds. To this end, a technique has been developed
that can be adjusted to detect very low or narrow steps even if
they are smooth and hidden by noise. By applying the noise
reduction algorithm to force spectroscopy data obtained with living
T lymphocytes, the onset force required for the extraction of a
membrane tether could be observed for the first time. Using the
step detection method, strong evidence of sub-10-pN steps was
found. Moreover, it was shown that the chemokine SDF-1α leads to a
strengthening of individual bonds between VLA-4, one type of
integrins primarily involved in the early stages of
chemokine-induced lymphocyte adhesion, and its ligand VCAM-1. The
adhesion strengthening is accompanied by a stiffening of the
integrins’ environment. It is independent of an intracellular
binding site of VLA-4 to talin, the major intracellular factor
involved in integrin affinity modulation. Further, the functional
role of the integrin trans-membrane domains in receptor-ligand
interactions was explored by analyzing the effects of two mutations
of the integrin αvβ3 on cellular adhesion: a chimera encompassing
the strongly dimerizing trans-membrane domain of glycophorin A and
a point mutation known to induce trans-membrane domain
dissociation. The results show that both constructs provoke strong
cell adhesion. They correspond well to a three-state model of
integrin activation. A resting state is activated by intracellular
ligands to an intermediate state without trans-membrane domain
separation. The dimerizing chimera mimics the intermediate state,
which strengthens cellular adhesion.