Reconstitution and anchoring of cytoskeleton inside giant unilamellar vesicles

Among the requirements for all life forms is the ability to self-replicate. In eukaryotic cellular systems, this division is achieved through cytokinesis, and is facilitated by the (re)arrangement and interaction of cytoskeletal proteins with lipids and other proteins localized to the plasma membrane. A fascinating challenge of modern synthetic biology is the bottom-up reconstitution of such processes for the generation of an artificial cell. One crucial step towards this goal is the functional reconstitution of the protein-anchoring machinery to facilitate cytokinesis into lipid vesicles. True to the ideal of a minimal cell-like system, we here describe the formation of an actin-based cytoskeleton within giant unilamellar vesicles (GUVs) made from porcine brain lipid extracts. We demonstrate that the actin filaments are localised and anchored to the interior walls of the GUVs through the spectrin/ankyrin proteins, and produce tightly packed actin bundles. These studies allow for the examination of cytoskeletal rearrangements within a cell-like model membrane system and represent important first steps in reconstituting the minimal machinery required for the division of an artificial cell. In addition, the study of such minimal systems can shed light on protein functions that are commonly unobservable or hidden within the overwhelming complexity of cells.