<em>In Vitro</em> Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

Abstract

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such systems allows for detailed studies of their underlying mechanisms which would not be feasible in vivo. Here, we provide a protocol for the in vitro reconstitution of the MinCDE system of Escherichia coli, which positions the cell division septum in the cell middle. The assay is designed to supply only the components necessary for self-organization, namely a membrane, the two proteins MinD and MinE and energy in the form of ATP. We therefore fabricate an open reaction chamber on a coverslip, on which a supported lipid bilayer is formed. The open design of the chamber allows for optimal preparation of the lipid bilayer and controlled manipulation of the bulk content. The two proteins, MinD and MinE, as well as ATP, are then added into the bulk volume above the membrane. Imaging is possible by many optical microscopies, as the design supports confocal, wide-field and TIRF microscopy alike. In a variation of the protocol, the lipid bilayer is formed on a patterned support, on cell-shaped PDMS microstructures, instead of glass. Lowering the bulk solution to the rim of these compartments encloses the reaction in a smaller compartment and provides boundaries that allow mimicking of in vivo oscillatory behavior. Taken together, we describe protocols to reconstitute the MinCDE system both with and without spatial confinement, allowing researchers to precisely control all aspects influencing pattern formation, such as concentration ranges and addition of other factors or proteins, and to systematically increase system complexity in a relatively simple experimental setup.

Understanding T cell signaling using membrane reconstitution

“…SUVs and LUVs can be readily prepared using either sonication or extrusion using commercially available devices. Various methods for preparing GUVs and SLBs have been described . Despite their demonstrated utilities and exciting potential, membrane reconstitution assays have limitations, which are listed below.…”

Reconstitution and Coupling of DNA Replication and Segregation in a Biomimetic System

“…After oxygen plasma treatment, microfluidic devices were bound on a glass slide. Lipid‐bilayer PDMS microcompartments were prepared as previously described …”

Design of Sealable Custom-Shaped Cell Mimicries Based on Self-Assembled Monolayers on CYTOP Polymer

“…A flat CYTOP was prepared by spin-coating a film of CYTOP (809M); diluted 1:10 in volume in solvent CT-SOLV180, at 3000 rpm for 40 s; and a reaction chamber assembled, as described previously. 22…”

“…Small unilamellar vesicles (SUVs) composed of DOPC or DOPC/DOPG (1,2-dioleoyl- sn -glycero-3-phospho-(1′- rac -glycerol)) (7:3 molar ratio), containing additional 0.005 mol % Atto655-DOPE (Atto-TEC GmbH, Siegen, Germany), were prepared at a concentration of 4 mg mL –1 in buffer M, as described elsewhere. 22 Shortly, lipids dissolved in chloroform were dried under a nitrogen stream, and vials were placed in a desiccator to remove residual chloroform for at least 30 min. Afterward, lipids were slowly rehydrated in buffer M and SUVs were generated by sonication in a water bath (model 1510; Branson) until the solution appeared clear.…”