Periodic Reporting for period 1 - minimal-phagocyte (Reconstitution of the basic molecular mechanism of phagocytosis – a bottom-up synthetic biology approach)
Periodo di rendicontazione: 2016-09-01 al 2018-08-31
Phagocytosis is the process by which a cell engulfs another cell or particle greater than 0.5 mm by invagination of the cell membrane around the object, and subsequent abscission of the membrane-coated object to form an intracellular, membrane-bound organelle. Phagocytosis is of fundamental importance to both single-cell organisms (for feeding), and multicellular organisms (for defending against pathogens and tissue re-modelling). The key aim of this action was to define the “minimal phagocyte” – i.e. the minimal number of components that enable a cell to carry out this process of phagocytosis –– to allow us (1) to fully capture its essential features, (2) to recreate it in vitro, and (3) to modulate it. Beyond increasing our knowledge about one of the most basic features of living cells, this project was aimed at generating knowledge with the potential to aid the design of novel biotechnological applications, such as improved drug delivery by preventing or promoting phagocytic uptake.
In summary, this project proposed to construct this “minimal phagocyte” by building a bio-inspired in vitro system capable of phagocytosis from the bottom up by reconstituting a minimal, dynamic actin cytoskeleton in receptor-carrying, cell-sized lipid vesicles to create an artificial phagocyte (i.e. a vesicle capable of taking up a particle). While even after the conclusion of the action, this goal – a complete minimal model system for phagocytosis – remains unachieved, key technologies were developed that not only will allow constructing of this minimal phagocyte in the years to come, but that will also benefit other, related research endeavours.
In summary, this project proposed to construct this “minimal phagocyte” by building a bio-inspired in vitro system capable of phagocytosis from the bottom up by reconstituting a minimal, dynamic actin cytoskeleton in receptor-carrying, cell-sized lipid vesicles to create an artificial phagocyte (i.e. a vesicle capable of taking up a particle). While even after the conclusion of the action, this goal – a complete minimal model system for phagocytosis – remains unachieved, key technologies were developed that not only will allow constructing of this minimal phagocyte in the years to come, but that will also benefit other, related research endeavours.
We were able to establish a workflow for encapsulating all components of the minimal actin cytoskeleton in cell-sized lipid vesicles using a method referred to as electroformation. Equally, we succeeded in binding particles to a membrane with artificial receptors (transmembrane peptides). However, before these two methodologies could be combined for the minimal phagocyte, it became clear that two main obstacles remained: (1) the symmetric functionalisation of the vesicle membrane and (2) generating stable but yet porous vesicles. The initially symmetric functionalisation of the vesicle membrane caused the problem that the actin cytoskeleton was bound to both the inside and the outside of the vesicle (whereas only binding to the inside was desired). Therefore, we developed a microfluidic device for trapping vesicles with high efficiencies, and using this trap to remove the actin specifically from the outer surface of the vesicles (publication accepted; presented at >3 international conferences). The experiment further required the insertion of membrane pores to trigger the cytoskeleton dynamics inside the vesicle upon particle binding. However, despite investigating numerous pore formatting agents, pore formation was either highly inefficient, or greatly destabilising the vesicles. We therefore had to find conditions to maintain vesicle stability in the presence of membrane pores, which required both tuning membrane composition and the concentration of the pore forming agent. These results will be published in a scientific journal. Overcoming these two experimental challenges left no time left to investigate the constituted system in detail. Over the course of this action, we also developed a method to generate liposomes with defined contents (e.g. actin cytoskeleton proteins) by emulsion transfer in microtitre plates (https://doi.org/10.1088/1367-2630/aabb96(si apre in una nuova finestra)) and while we did not study the effects of a minimal acto-myosin network on actin-driven membrane deformations in lipid vesicles, we did explore myosin mediated actin turnover on supported lipid bilayers (https://www.biorxiv.org/content/early/2018/05/04/312512(si apre in una nuova finestra)).
During this research project, it became clear that its progress was greatly hampered by the lack in reproducibilty and reliability of pre-existing technologies for the reconstitution of functional actin networks inside of lipid vesicles. Thus, it was concluded that significant improvements to these methods were required to allow them to be combined for more complex projects, such as the minimal phagocyte. This work done in the course of this Marie Curie Action has thus invested time and effort in the reliable fabrication of lipid vesicles of a desired composition and with stable membrane pores as well as establishing a microfluidic platform for handling these vesicles. Meanwhile, the cytoskeletal processes that underpin phagocytosis were studied in a simpler model membrane system as originally intended. While the objectives of this project have not been fully reached within the project’s time line, the research results and developed methods have placed the goal of a minimal model of phagocytosis within much closer reach, and with its potential for informing novel therapies. All results of the project were shared with the scientific community (via conferences and research articles) and the general public (via open access articles, open access pre-print servers and the host institution’s open day).