Planar Supported Lipid Bilayer Assay: A Microscopy-Based In Vitro Technique to Investigate Septin Assembly on Biological Membrane Mimics
Transcript
Septins are cytoskeletal proteins comprising a GTP-binding domain flanked by terminal amino and carboxyl domains, with an inherent ability to self-assemble into filaments and higher-order structures at the inner plasma membrane for diverse cellular functions.
To study septin-membrane interactions in vitro using a biological membrane mimic, obtain a cut microcentrifuge tube. Apply ultraviolet adhesive over the uncut, flat rim and position it onto a plasma-treated, hydrophilized glass coverslip. Upon ultraviolet light exposure, the adhesive cures, creating an open reaction chamber.
Transfer monodispersed zwitterionic unilamellar lipid vesicles of desired lipid composition, supplemented with mono- and divalent cation-containing buffer, into the chamber. Cations facilitate lipid vesicles to adsorb and accumulate onto the coverslip. Gently shake the chamber, initiating vesicle rupture, and incubate.
The ruptured vesicles unfold and fuse to the coverslip via their hydrophilic head groups, forming planar lipid bilayer patches. The patches' edges trigger remaining vesicles to rupture, generating a continuous planar-supported lipid bilayer, SLB. Remove excess unadsorbed vesicles with suitable buffer.
Next, add fluorescently-labeled septin suspension with crowding agent-containing buffer and incubate. Crowding agents enable septins to deposit onto the SLB-coated coverslip. Under optimal conditions, septins may associate with the lipids in the SLB, assembling into organized structures.
Selectively illuminate the coverslip using total internal reflection fluorescence microscopy to excite adhered fluorescently-labeled septins on the SLB to observe septin assembly.
