Final Report Summary - SYNAD (Synthetic Biology Approach to Adhesion-Mediated Environmental Sensing) Cellular adhesion to the extracellular matrix is involved in nearly every cellular response in vivo. These responses, in turn, affect nearly all facets of cell’s life, including, but not limited to, migration, cell proliferation and differentiation. Although attaining a fundamental characterization of these cellular functions is a compelling goal, the extensive complexity of these processes has hindered a full understanding. Therefore, the central goal of SynAd interdisciplinary research was the development of synthetic cell model systems, which could serve as platforms for bottom-up assembly of specific sets of minimal adhesion-associated protein complexes and testing their functional role in initiating a signalling response.Cell-matrix adhesion sites are composed of various protein complexes, therefore, during starting period of the project we performed a detail cellular adhesion study to elucidate the most crucial protein complexes that will be used within the project as building blocks to assemble minimal synthetic adhesions. This knowledge led us to successful establishment of purification protocols for most prominent adhesion associated proteins, including cytoplasmic tails of different integrin proteins, αIIbβ3 integrin, vinculin, focal adhesion kinase, kindlin, G actin, myosin and tubulin. The next step was to adapt these purification procedures to the requirements of lipid biochemistry so that the different proteins can be incorporated into lipid-based protocells. A major challenge during this transfer process was to maintain the biological activity of the different proteins, so that they can fulfil their native functions, e.g. cell adhesion or formation of a cytoskeleton, when being reconstituted into a lipid-based synthetic cell. To achieve that we developed a modular engineering approach based on droplet-based microfluidics for sequential bottom-up assembly of bio inspired synthetic cells which can perform cellular function such as adhesion and migration. We have succeeded to combine the lipids and polymer-based microfluidic droplets to generate mechanically and chemically stable and, therefore, manipulable cell-like compartments, called droplet-stabilized giant unilamellar vesicles (dsGUVs). Importantly, the enhanced stability of dsGUVs and implementation of the picoinjection microfluidic technology enabled the sequential and precise loading of such compartments with purified proteins without compromising their functionality as synthetic cells. It is worth noting that these compartments do not assemble correctly by simply mixing the components. We have managed to combine, simultaneously and under identical conditions, molecular systems that do not function together by purely mixing them. This is the first time that a real bottom-up, step-by-step construction of a complex synthetic cell with desired features has been accomplished. The outcomes of this research provided crucial information for the successful reconstitution of cytoskeletal, transmembrane and adhesion-associated proteins within the microfluidic-generated synthetic cells. To allow determination of biochemical reactions dynamics within the synthetic cells we reconceptualised fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), converting them into a powerful detection methods for droplet-based microfluidics. This was achieved by several simple changes in the way FCS data is been acquired and interpreted. This is the first time that FCS is applied as a real-time detection system for flowing droplets, instantly detecting heterogeneities in their content. Moreover, to pave the way towards investigations in which synthetic cell interactions with the extracellular matrix will be crucial, we developed microfluidic and bulk technologies to release the functional synthetic cells from the stabilizing polymer/oil environment into a physiologically relevant aqueous phase. This methodology allowed us to validate the functionality of the reconstituted proteins in physiological conditions. For example, spreading/adhesion behaviour of the released integrin-functionalized synthetic cells was investigated on constructed natural and synthetic matrices. Moreover, we showed that the developed synthetic cells are capable of self-assemble different cytoskeletal and transmembrane proteins, and, as a consequence, generate cellular functions such as adhesion, migration and self-propelling.
