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Photo-Switchable Proteins for Cell-Matrix Adhesion

Ricken, Julia

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Abstract

The adhesion of cells to each other and to a scaffolding matrix is a major feature of multi-cellular organisms. Since misregulation can have fatal consequences, thereto related biological processes, such as wound healing, differentiation and embryogenesis, are tightly regulated in space and time. To get an understanding in these systems it is of big interest to generate dynamically regulated platforms. In order to dynamically and spatiotemporally control integrin-mediated cell adhesion to the extracellular matrix (ECM) several systems have been establish, which can be controlled by an outer stimulus. Nevertheless, these methods are limited by a variety of drawbacks like low resolution, the use of photo-toxic UV-light or tedious synthesis of chemical building blocks. In this thesis, a novel optogenetic tool to control cell-matrix adhesion, combining in a new manner the approaches from material science with optogenetics is presented. By incorporation of naturally occurring ligands for cell-matrix receptors, namely intergrins, like the RGD-sequence or the PHSRN-sequence into optogenetic proteins, they can be applied to mediated cell-surface adhesion. Surfaces coated with the engineered proteins can be switched either once or reversibly, by visible light in their ability to mediate cell adhesion. First, the green light switchable protein CarH from T. thermophilus in order to switch surfaces from an adhesive to a non-adhesive state was employed. The protein monomers form tetramers when the cofactor Cobalamin is incorporated and disassemble into dimers upon green light illumination. By incorporation of the RGD motif into the protein a cell adhesive building block was generated that can be immobilized on surfaces. Upon disassembly of the tetramer, the monomers presenting the RGD motif to the cell are removed and the surface is switched to the non-adhesive state.

In this work, the immobilization of proteins on the surfaces as well as their ability to bind integrin and disassemble under green light in vitro was shown. Further, these results were confirmed in cell adhesion experiments. For the first time, I designed and implemented a green light-switchable system for cell-surface adhesion based on altered ligand presentation. In the second part of my thesis, the blue light-switchable protein domain LOV2 from A. sativa was employed to build a reversible system. This protein domain consists of a small protein core with a long C-terminal Ja-helix, which reversibly unwinds, when illuminated with blue-light. A library of 40 LOV2RGD mutants by insertion of the RGD motif at different positions in the Ja-helix and by combination with further complementing mutations, like the PHSRN motif, was designed. The RGD peptide is caged by the helical conformation in dark and is presented to cells upon unwinding of the Ja-helix under blue light illumination. The library was narrowed to 8 promising mutants through characterization of the ability of the LOV2RGD mutants to bind integrin in dark and under blue light illumination in fluorescence anisotropy measurements. The selected mutants were evaluated for their ability to mediate cell adhesion depending on their switching state, showing significant difference in the spreading area of the cells. Additionally, the reversibility of cell adhesion on such substrates was determined. Finally, the most promising two candidates were evaluated in live cell experiments, showing a significant decrease in the spreading area in dark but not under blue light illumination.

In summary, two protein-based approaches for the dynamic control of cell adhesion using light of two different wavelengths were designed and implemented. The green light-dependent CarH based system can be used to capture and release cells on command, whereas the LOV2 domain-based approach resembles natural timescales for adhesion and can therefore be used in investigations of signaling cascades or other processes involved in cell-matrix adhesion. In conclusion, the herein presented systems can overcome most of the existing drawbacks due to their sensitivity to visible light, their facile way to get produced in high amounts and their high spatial and temporal control.

Item Type: Dissertation
Supervisor: Spatz, Prof. Dr. Joachim P.
Place of Publication: Heidelberg
Date of thesis defense: 25 January 2019
Date Deposited: 29 Apr 2019 16:56
Date: 2019
Faculties / Institutes: Fakultät für Chemie und Geowissenschaften > Institute of Organic Chemistry
Fakultät für Chemie und Geowissenschaften > Institute of Inorganic Chemistry
Fakultät für Chemie und Geowissenschaften > Institute of Physical Chemistry
Subjects: 540 Chemistry and allied sciences
570 Life sciences
660 Chemical engineering
Controlled Keywords: Proteindesign, Zelladhäsion, Fokale Adhäsion, RGD-Sequenz, Extrazelluläre Matrix, Integrine
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