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Abstract

Cells interact with neighboring cells and the extracellular matrix forming cell-cell and cell-matrix contacts, which are mainly mediated by cadherins and integrins, respectively. These processes are dynamic and spatially and temporally tightly regulated during many biological events including embryogenesis, wound healing and cancer development. Dynamic control of cell interactions is a key to understanding many underlying cellular processes, to achieving the bottom-up assembly of single cells into tissues and to developing medical implants. The challenge lies in controlling specific cell-cell and cell-material interactions both dynamically and reversibly with high spatiotemporal control in a non-invasive way over a long period of time. The aim of this thesis is to generate platforms where these cell interactions are controlled spatially, temporally, dynamically and reversibly using visible light responsive proteins from plants.

The recent developments in the field of optogenetics provide powerful tools to overcome above-mentioned challenges and they have been employed to control many intracellular signaling pathways. Among others cryptochromes and phytochromes were used in this study. Cryptochrome 2 (CRY2) is a blue light photoreceptor and it heterodimerizes with CIBN upon blue light irradiation. This interaction can be reversed to the ground state passively in the dark. Phytochrome B (PhyB) is a red and far-red light sensing protein and upon red light illuminations it interacts and forms heterodimers with PIF6. This heterodimerization can be reversed under far-red light or passively in the dark.

To control cell-cell interactions with light, the blue light dependent heterodimers, CRY2 or CIBN were expressed on the surfaces of the cells, which do not form any native cell-cell contacts. In equally mixed cultures of CRY2 and CIBN expressing cells, these cells form cell-cell interactions upon blue light illumination, which provides high high spatial and temporal control. These photoswitchable interactions are reversible in the dark, and can be repeatedly and dynamically switched on and off. These genetically encoded interactions can be sustained over a long time as they are genetically encoded and they respond to nontoxic low intensity blue light.

Towards controlling cell-material contacts of multiple cell types, one of the heterodimerization partners (CIBN or PIF6) was immobilized on non-adhesive glass surfaces. By expressing CRY2 or PhyB on the cell surfaces, their adhesion to CIBN and PIF6 functionalized substrates can be triggered under nontoxic low intensity blue and red light illumination, respectively. CRY2/CIBN and PhyB/PIF6 interactions are orthogonal to each other since they respond to only blue and red/far-red light, respectively. This orthogonality provides wavelength selective adhesion of one cell type to its complementary substrate in the presence of the other cell type. The ability of PhyB/PIF6 system to far-red light also makes orthogonal reversion of these adhesions possible while the other cell type (CRY2) remains adhered to its substrate. These photoswitchable cell-material interactions are reversible in the dark or under far-red light, and can be repeatedly and dynamically switched on and off.

Overall, this optogenetic approach to control cell interactions reflects the dynamic and reversible nature of cell-cell and cell-material interactions and provides the desired spatiotemporal control in a noninvasive manner. These blue and red/far-red light responsive proteins are genetically encodable; hence, they can be sustained over a long time. Finally, photoswitchable cell interactions will provide a new way of studying them and assembling cells into multicellular structures in the context of bottom-up tissue engineering.