Spontaneous and induced front-rear polarization and a subsequent asymmetric actin cytoskeleton is a crucial event leading to cell migration, a key process involved in a variety of physiological and pathological conditions such as tissue development, wound healing, and cancer. Migration of adherent cells relies on the balance between adhesion to the underlying matrix and cytoskeleton-driven front protrusion and rear retraction. A current challenge is to uncouple the effect of adhesion and shape from the contribution of the cytoskeleton in regulating the onset of front-rear polarization. Here, we present a minimal model system that introduces an asymmetric actin cytoskeleton in synthetic cells, which are resembled by giant unilamellar lipid vesicles (GUVs) adhering onto symmetric and asymmetric micropatterned surfaces. Surface micropatterning of streptavidin-coated regions with varying adhesion shape and area was achieved by maskless UV photopatterning. To further study the effects of GUV shape on the cytoskeletal organization, actin filaments were polymerized together with bundling proteins inside the GUVs. The micropatterns induce synthetic cell deformation upon adhesion to the surface, with the cell shape adapting to the pattern shape and size. As expected, asymmetric patterns induce an asymmetric deformation in adherent synthetic cells. Actin filaments orient along the long axis of the deformed GUV, when having a length similar to the size of the major axis, whereas short filaments exhibit random orientation. With this bottom-up approach we have laid the first steps to identify the relationship between cell front-rear polarization and cytoskeleton organization in the future. Such a minimal system will allow us to further study the major components needed to create a polarized cytoskeleton at the onset of migration.

Bottom‐up synthetic biology has reconstituted processes like adhesion, cortex formation, or division of giant lipid vesicles (GUVs), which all rely on changes in the vesicle morphology. However, oftentimes, GUV morphologies and shape transitions are described qualitatively, which makes it difficult to quantitatively compare results from different studies and to advance precision engineering. Herein, open‐source software package GeoV for the 3D reconstruction and analysis of GUV shapes from confocal microscopy z‐stacks is presented. The accuracy of the reconstruction by comparing the output for the Hausdorff distance of the surface, the curvature, and the bending energy to ground truth data for simulated shapes of different complexities is quantified. Next, GeoV on a variety of confocal microscopy datasets, including z‐stacks from spherical GUVs and GUVs deformed with DNA origami, adherent GUVs, DNA droplets, and cells, is tested. Additionally, the effect of membrane‐binding DNA origami on the vesicle shape, volume, and bending energy is quantified. It is found that osmotic deflation and attachment of DNA origami can increase the bending energy of GUVs by a factor of 10. All in all, GeoV as an open‐source software package for the quantitative analysis of confocal microscopy data for bottom‐up synthetic biology is provided.

Cell-extracellular matrix (ECM) adhesion mediated by integrins is a highly regulated process involved in many vital cellular functions such as motility, proliferation and survival. However, the influence of lateral integrin clustering in the coordination of cell front and rear dynamics during cell migration remains unresolved. For this purpose, we describe a novel protocol to fabricate 1D micro-nanopatterned stripes by integrating the block copolymer micelle nanolithography (BCMNL) technique and maskless near UV lithography-based photopatterning. The photopatterned 10 μm-wide stripes consist of a quasi-perfect hexagonal arrangement of gold nanoparticles, decorated with the RGD (arginine-glycine-aspartate) motif for single integrin heterodimer binding, and placed at a distance of 50, 80, and 100 nm to regulate integrin clustering and focal adhesion dynamics. By employing time-lapse microscopy and immunostaining, we show that the displacement and speed of fibroblasts changes according to the nanoscale spacing of adhesion sites. We found that as the lateral spacing of adhesive peptides increased, fibroblast morphology was more elongated. This was accompanied by a decreased formation of mature focal adhesions and stress fibers, which increased cell displacement and speed. These results provide new insights into the migratory behavior of fibroblasts in 1D environments and our protocol offers a new platform to design and manufacture confined environments in 1D for integrin-mediated cell adhesion.