PDF, English - main document Download (6MB) | Terms of use

Abstract

The organization of cells within tissues allows them to work together. Tight spatiotemporal control over cell-cell interactions is essential for individual cells to self-assemble and function as tissues. In addition, during many biological processes, such as embryogenesis and cancer development, cell-cell interactions undergo dynamic changes to alter their function. Analogously, in the context of bottom-up synthetic biology it is of interest to dynamically control the interactions between minimal synthetic cells and assemble them into precise multi-compartment prototissues with high spatiotemporal resolution. The aim of the first part of this thesis was to reversibly self-assemble different types of micrometer-sized colloids, which were used as synthetic cell-mimics, with high spatiotemporal resolution using visible light. Light provides a dynamic, non-invasive, and biocompatible control with high spatiotemporal precision. In order to control the self-assembly of cell-mimics, I functionalized them with photoswitchable proteins that specifically interact with each other under blue or red light. For this purpose I used several combinations of photoswitchable proteins that are dimerizing under blue light: heterodimerizing iLID and Nano proteins, nMagHigh and pMagHigh proteins, and homodimerizing VVDHigh protein. For the red light switchable proteins I used both the heterodimerization of phytochrome B (PhyB) and phytochrome interaction partner (PIF6) proteins and the homodimerization of Cph1 protein. All of these light dependent protein interactions enabled controlling the self-assembly of cell-mimics with light. Additionally, blue light dependent protein interactions are reversible in the absence of light with red light dependent interactions reversing under far-red light illumination. Consequently, the self-assembly of cell-mimics mediated by these protein interactions was also reversible. Additionally, the high specificity and the independent response of these protein interactions to blue or red light offers the potential to self-assemble a specific population of cell-mimics in the presence of others on demand. In multicellular organisms, cells do not just self-assemble but they also self-sort into precise arrangements in order to work together. As part of this thesis, I also mimicked the self-sorting behavior with synthetic compartments inspired by the observations in nature. Self-sorting is defined by the ability to distinguish between self and non-self, and happens in different modes depending on the interactions between the particles. One mode is social self-sorting, which leads to the separation of colloids into independent colloidal families and requires heterophilic and orthogonal interactions. In this part of the thesis, I used heterodimerization of two blue light switchable protein pairs, iLID/Nano and nMagHigh/pMagHigh, for the social self-sorting between four different populations of colloids within one mixture. Each protein pair specifically and orthogonally brings together two different subpopulations of colloids providing tight and reversible control over their self-sorting into two distinct families using blue light. On the other hand, asocial sorting is another mode of self-sorting, which requires homophilic interactions to bring together compartments of the same type into isolated aggregates. This could potentially be achieved by combining homodimerization of VVDHigh and Cph1 proteins under blue and red light respectively. Eventually, all the versatile and orthogonal light-switchable proteins and different dimerization modes have the potential to be incorporated together in different combinations to achieve the desired self-sorting outcome in complex prototissues. In the second part of the thesis, I addressed the spatiotemporally controlled formation of protein patterns on synthetic cell-mimics. Protein patterns and gradients on cell membranes are important during many biological processes to locally trigger events in multicellular structures with high spatiotemporal precision. To create and control protein patterns on synthetic membranes such as giant unilamellar vesicles (GUVs) with light, I used the blue light switchable heterodimerization of iLID and Nano proteins. For this purpose, the GUVs were functionalized with iLID. This allowed for the blue light mediated, reversible recruitment of a fluorescent protein (mOrange) fused to Nano using blue light with high spatiotemporal resolution. Further, this approach allowed scaling the size of protein patterns from the level of a single GUV to the level of a tissue-like GUV carpet. Hence, these photoswitchable proteins offer a versatile, reversible, dynamic, and non-invasive method to photopattern proteins with high spatiotemporal control that operates under mild conditions. Overall, photoswitchable proteins are important building blocks in the bottom-up synthetic biology toolbox. Incorporating them onto minimal synthetic cells can be used to self-assemble and self-sort different types of cell-mimics and to generate protein patterns, thus mimicking complex processes that occur in nature. Most importantly, these protein interactions provide high spatiotemporal precision and specificity to control these biomimetic processes. Ultimately, this concept can be transferred to assemble prototissues using various types of cell-mimics that host different functionalities, which would allow for controlling, how different synthetic cells work together in a prototissue.