In vitro Reconstitution of Cytoskeletal Networks inside Phase Separated Giant Unilamellar Vesicles (GUVs).

Biomimetic lipid membranes in the form of giant unilamellar vesicles (GUVs) are commonly used to mimic cellular membrane behavior because of the ease of protein reconstitution inside GUVs, visualization, as well as understanding cellular membrane-protein dynamics. However, cell membranes comprise lipid rafts (or domains) arising from the presence of different lipids in the cellular membrane. Such increased complexity in model systems can be incorporated to result into phase separated GUVs, where lipid composition can be finely tuned. While encapsulation methods for the generation of homogeneous GUVs are widely known, methods to encapsulate proteins within phase separated GUVs are limited. Here, this protocol presents a simplified one-pot production of phase separated GUVs, comprised of liquid-disordered (Ld) and liquid ordered (Lo) domains, efficiently encapsulating different cytoskeletal proteins, i.e., FtsZ and actin, making the method a versatile tool for minimal cell production. Specifically, this approach uses an emulsion transfer protocol to produce GUVs with a high encapsulation efficiency. In this method, a lipid-monolayer is first generated by emulsifying a protein solution in a lipid/oil mixture, where lipids of varying phase transition temperatures are chosen to yield phase separation in the resultant GUVs. This emulsion is transferred gently on top of a lipid-in-oil solution in another tube, resulting in the formation of a water-oil interface. The solution is then centrifuged at elevated temperatures (ideally at 37 °C to retain protein activity), after which GUVs are collected for imaging. This method simplifies the in vitro reconstitution of cytoskeletal proteins within phase separated GUVs without using a cumbersome laboratory setup, and thus serves as a convenient method for studying the mechanics of cytoskeletal-membrane interactions in confinement.