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Abstract

Polarity and spatial molecular organization are key features found in life at scales from the single cell to the whole organism. It determines cell fate in asymmetric cell division as well as the direction in migrating cells. Polarity in cells can generally be defined as a result of an asymmetric distribution of molecules and evolves in directly observable changes such as rearrangement of cell shape and actin distribution. Cell deformation, induced by adhesion to micropatterned surfaces, is used to standarize and normalize cells and can control polarity depending on the underlying shape of the micropatterns. The controlled deformation induced by micropatterning, which can lead to defined polarity in natural cells, is applied in this work to bottom-up synthetic cell models based on giant unilamellar vesicles (GUVs) to create a simple induced-polarity system. GUVs were ad- hered to micropatterned surfaces using biotin and streptavidin as linking systems, as well as complementary DNA oligomers, leading to a controlled deformation of the vesicles on differently shaped micropatterns. While GUVs have been deformed before using tools like microfluidic chips, micropatterns allow for the controlled deformation of large amounts of vesicles in a similar manner to natural cells. The deformation of adherent GUVs is quantified on stripes of 15 μm in length and 5 μm in width. Here, it is observed that GUVs deform most when the GUV size is comparable to the size of the underlying micropat- terns. The effects of the GUV deformation on encapsulated actin bundled with fascin is investigated, as the organization of parallel stress fibers is a hallmark in mesenchymal cell migration. In adherent GUVs, I could show for line shaped micropatterns that actin bundles orientate along the major axis due to confinement. Additionally, I demonstrate using a simple theoretical model, that actin bundles, modeled as rigid rods, are most affected inside of GUVs adhered to line-shaped micropatterns, compared to other patterns commonly used to deform cells, such as cross or crossbows. In the next step, I polymerized actin in the presence of dextran as a crowding agent inside of the GUVs, which leads to the generation of cortex-like structures. These synthetic actin cortices organize due to the high membrane curvature at the adhesion site, where the GUV membrane transitions from the adherent to the free part, leading to spiraling actin patterns in the GUVs. Synthetic actin cortices further inhibit GUV deformation, when adhered to micropatterned surfaces. I show, that the deformability is only reduced when both components, actin and crowders are present in the GUVs. This indicates that the cortex-structure leads to the decreased deformability of the GUVs. These results are confirmed using real-time deformability cytometry (RTDC), which provides data for large populations of GUVs. Last, I try to use phase separated GUVs as a model system for polarity. Lipid phase separation creates domains with distinct physical and chemical properties of the membrane, which allows for the selective functionalization of a single phase. Using adhesive micropatterns, the biotin-rich phase accumulates at the micropatterned surface, as was reported before. Ad- ditionally, the geometric change in the adherent GUV leads to a cap-formation, where a domain of the minority phase is stably localized on top of the deformed GUV. I use Monte Carlo simulations to demonstrate that the cap-formation can be explained by line-tension. To conclude, I show that micropatterns provide a tool in bottom-up synthetic biology to controllably adhere and deform GUVs. Further, I show that the geometrical cues lead to changes in actin organization inside of the GUV and that synthetic actin cortices can hinder the GUV deformation. Ultimately, I establish that the arrangement of domains within phase-separated GUVs can be attained through adhesion and deformation of GUVs on micropatterned surfaces. For prospective applications, this organization could serve as a scaffold, leveraging the controlled localization of domains to organize proteins inside synthetic cells leading to polarity formation. Additionally, it may facilitate interfacing synthetic and natural cells, as synthetic actin cortices can provide mechanical cues, which, when combined with the spatially controlled presentation of ligands at the surface, open new ways to modulate and create signaling in such combined systems.