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

Opfer, Jan (2012): Single-molecule force spectroscopy studies of integrin-mediated cell signaling. Dissertation, LMU München: Fakultät für Physik

Abstract

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.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 530 Physik
• Fakultäten: Fakultät für Physik
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 12. September 2012
• 1. Bericht­erstatter:in: Gottschalk, Kay-Eberhard