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Light Controlled Cell-Cell Adhesions and Self-Sorting in Multicellular Structures Dissertation z ur Erlangung des Grades Doktor der Naturwissenschaften Am Fachbereich Biologie Der Johannes Gutenberg -Universität Mainz vorgelegt von Samaneh Rasoulinejad Geboren in Tehran, Iran Mainz, 2020 Page ii Date of oral examination: 02.12.2020 Page iii Declaration I hereby declare that I wrote the dissertation submitted without any unauthorized external assistance and used only sources acknowledged in this work. All textual passages which are appropriate verbatim or paraphrased from published and unpublished texts, as well as all information obtained from oral sources, are duly indicated and listed in accordance with bibliographical rules. In carrying out this research, I complied with the rules of standard scientific practice as formulated in the statutes of Johannes Gutenberg-University Mainz to ensure standard scientific practice. _____03.12.2020________ _________________________ Samaneh Rasoulinejad Page iv Page v Abstract The self-organizing of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. In this thesis, I demonstrated how two cells types can be independently controlled with blue and red light in order to control their self-assembly and self-sorting into distinct multicellular assemblies. For this purpose, different cell-cell interactions between the same or different cell types were developed such that they were individually triggerable with different colors of light, reversible in the dark and provided non-invasive and temporal control over the cell-cell adhesions. In multicellular mixtures, upon orthogonal photoactivation each cell type self-assembled independently and cells sorted out into separate assemblies based on specific self-recognition. Moreover, this work highlights the importance of the cell-cell interactions dynamics and tuning them by pulsed light activation in the formation of kinetically and thermodynamically controlled multicellular architectures. Only if cell-cell adhesions were strong and dynamic enough compact spheroids and sorting behavior of different cell types as predicted by the differential adhesion hypothesis were observed. The here developed photoswitchable cell-cell interactions and the formed self-sorted multicellular architectures with programmable organization provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them. Page vi Zusammenfassung Die Selbstorganisation verschiedener Zelltypen zu multizellulären Strukturen und ihre Organisation in räumlich-zeitlich kontrollierten Mustern sind sowohl eine Herausforderung als auch extrem leistungsfähig, um zu verstehen, wie Zellen innerhalb von Geweben und für das Bottom-up-Tissue Engineering funktionieren. In dieser Arbeit habe ich gezeigt, dass ich nicht nur die Selbstorganisation von zwei Zelltypen zu multizellulären Architekturen mit blauem und rotem Licht unabhängig voneinander kontrollieren kann, sondern auch ihre Selbstsortierung in verschiedene Zusammensetzungen erreichen kann. Die Interaktionen waren individuell mit verschiedenen Lichtfarben auslösbar, im Dunkeln reversibel und bieten eine nicht- invasive und zeitliche Kontrolle über die Zell-Zell-Verwachsungen. Bei multizellulären Mischungen hat sich bei orthogonaler Photoaktivierung jeder Zelltyp unabhängig voneinander selbst assembliert und die Zellen auf der Grundlage einer spezifischen Selbsterkenntnis in separate Baugruppen sortiert. Darüber hinaus führt die Bedeutung dynamischer Interaktionen und die Abstimmung der Dynamik der Zell-Zell-Interaktionen durch Pulslichtaktivierung zu der kompakten Struktur und darüber hinaus zu der mehrzelligen Architektur der Strukturen der differenziellen Adhäsionshypothese. Diese selbstsortierten mehrzelligen Architekturen bieten uns ein leistungsstarkes Werkzeug zur Herstellung gewebeähnlicher Strukturen aus mehreren Zelltypen und zur Untersuchung der Prinzipien, die sie steuern. Page vii Publications 1) Orthogonal Blue and Red Light Controlled Cell-Cell Adhesions enable Sorting- out in Multicellular Structures Rasoulinejad S., Mueller M., Nzigou, M. B., Wegner, S. V. (2020) ACS Synth. Biol., 9, 2076–2086 (2020), doi:10.1021/acssynbio.0c00150 2) Blue Light Switchable Cell–Cell Interactions Provide Reversible and Spatiotemporal Control Towards Bottom‐Up Tissue Engineering Yüz S. G.*, Rasoulinejad S.*, Mueller M., Wegner A. E., and Wegner, S. V Adv. Biosyst., 3, 1800310 (2019), doi: 10.1002/adbi.201800310 3) The importance of cell-cell interaction dynamics in bottom-up tissue engineering: Concepts of colloidal self-assembly in the fabrication of multicellular architectures. Mueller M., Rasoulinejad S., Garg S., Wegner, S. V. Nano Lett., 20, 2257–2263 (2020), doi: 10.1021/acs.nanolett.9b04160 4) Green light lithography: a general strategy to create active protein and cell micropatterns. Xu D., Bartelt, S. M., Rasoulinejad S., Chen F., Wegner, S. Mater. Horizons, 6, 1222–1229 (2019), doi: 10.1039/c9mh00170k 5) The dynamic differential adhesion hypothesis: the role of the cell-cell adhesion dynamics in cell sorting Rasoulinejad S., Wegner, S. V. (2020), Submitted 6) Spatiotemporal control over multicellular migration using green light reversible cell-cell interactions Nzigou, M. B, Bijonowski, B.t M., Rasoulinejad S., Mueller M., Wegner, S. V. (2020) Accepted to Adv. Biosyst. Page viii Table of contents Declaration ................................................................................................................. iv Abstract ...................................................................................................................... vi Publications .............................................................................................................. viii Table of contents ....................................................................................................... ix List of figures ............................................................................................................. xii List of tables ............................................................................................................. xiv List of abbreviations ................................................................................................. xv Chapter 1: Introduction .............................................................................................. 1 1.1 Bottom-up tissue engineering ......................................................................... 2 1.2 Cell-sorting and self-assembly of cells ............................................................ 3 1.2.1 Sorting through specific cell-cell adhesions and tissue affinity ................. 4 1.2.2 Sorting by differences in cell adhesion strength (Steinberg’s Differential Adhesion Hypothesis) ........................................................................................... 6 1.2.3 Differential Interfacial Tension Hypothesis (DITH) ................................... 9 1.2.4 Differential Surface Contraction Hypothesis (DSCH) ............................. 10 1.2.5 Sorting based on differences in velocity ................................................. 11 1.2.6 Leading questions in cell sorting ............................................................ 12 1.3 Cadherins as cell-cell adhesion molecules and their role in cell-sorting ........ 13 1.4 Candidates for controlling cell-cell contact .................................................... 15 1.4.1 Chemical modifications .......................................................................... 15 1.4.2 Light controlled interactions ................................................................... 17 1.4.3 Biological modifications ......................................................................... 17 1.5 Photoswitchable proteins .............................................................................. 18 1.5.1 Blue light responsive receptors .............................................................. 19 1.5.2 Red light responsive receptors .............................................................. 21 Chapter 2: Materials and Methods ........................................................................... 24 Page ix 2.1 Materials ....................................................................................................... 24 2.1.1 General laboratory equipment ............................................................... 24 2.1.2 Microscopes .......................................................................................... 25 2.1.3 Software ................................................................................................ 25 2.1.4 Bacteria and cell lines ............................................................................ 25 2.1.5 Antibodies .............................................................................................. 26 2.1.6 Chemicals .............................................................................................. 26 2.1.7 Biochemicals ......................................................................................... 27 2.1.8 Primer sequences .................................................................................. 28 2.1.9 Plasmid.................................................................................................

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