A Force Spectroscopy Setup to Mimic Cellular Interaction Processes
by Bärbel Lorenz
Date of Examination:2012-06-26
Date of issue:2013-06-21
Advisor:Prof. Dr. Andreas Janshoff
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Jörg Großhans
Persistent Address:
http://dx.doi.org/10.53846/goediss-3895
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Abstract
English
The main objective of this thesis was to establish a biophysical model system that permits to quantify forces involved in cell-cell contacts and cell-substrate-adhesion in a native-like fashion. For this purpose, colloidal probe microscopy was modified to mimic two opposing membranes, a situation very much like that encountered in cell-cell recognition. Membrane coated probes allow to capture the impact of lateral mobility, multiple bonds, and merging of bilayers as encountered in fusion events. In the first part, the strong non-covalent interaction between His-tagged peptides embedded in a lipid bilayer and Ni-NTA bearing lipids in the opposing membrane were probed. Hemifusion as well as full fusion events were frequently observed depending on the compression forces. Upon retraction from the surface, membrane nanotubes were formed that allowed to carry out force clamp measurements. The constant force of a tether permits to measure the lifetime of individual bonds within the contact zone. The second part was devoted to weak non-covalent interactions playing a pivotal role in the formation of focal contacts or cell-cell recognition. Many of these bonds in parallel provide the necessary strength to attach cells to surfaces or other cells forming tissue. Early metazoae such as sponges use carbohydrate moieties to form reversible bonds between each other a prerequisite for self-recognition, a crucial advantage in evolution. Membrane-based colloidal probe microscopy was used to quantify the forces between sulfated carbohydrates as they are found in the marine sponge Microciona prolifera organized in small clusters. The interaction was found to be highly calcium dependent and reversible. In the third part of the project, the heterodimeric coiled-coil interaction between peptides, which serve as minimum model for the SNARE-mediated membrane fusion, was studied. We found that the membrane interaction forces are significantly smaller than expected. By performing topographical imaging and photobleaching experiments of the membrane, we could attribute the low adhesion forces to a lipopeptide cluster formation prior to the formation of the coiled-coils. We conclude that efficient membrane docking, which is the prerequisite for fusion events, does not only depend on the concentration of peptides on the surface but is decisively controlled by the lateral organization of peptides in the membranes. The fourth part of the thesis dealt with interactions found between highly evolved eukaryotic cells. The homomeric recognition of E-cadherins from L-cells was probed on the level of single molecule to shine light on the assembly scheme used by cells to ensure a firm connection between them. Force spectroscopy revealed two distinguishable modes of interactions that could be presumably attributed to cis-interaction between adjacent cadherins. While the homomeric EC12 recognition was barely detectable, EC15 displayed forces beyond the background suggesting that the length of the molecule plays an important role. In conclusion, a versatile lab-on-a-probe setup has been introduced that allows studying a large variety of interactions between membranes and that is essentially mimicking cell-cell interactions with reduced complexity.
Keywords: Force Spectroscopy; Lipid Membranes; Cellular Interactions; Functionalized Membranes; Mimicry
Schlagwörter: Force Spectroscopy; Lipid Membranes; Cellular Interactions; Functionalized Membranes; Mimicry