Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres
Transcript
Septins are key cytoskeletal proteins that bind to curved plasma membrane surfaces.
To observe in vitro septin assembly on microspheres, first, take a suspension of uniformly sized, microscopic silica beads, acting as solid support for bilayer formation. Vortex this suspension to break any bead clusters.
Supplement it with small unilamellar vesicles, SUVs – spherical vesicles surrounded by a single lipid bilayer. Incubate with constant agitation, preventing microspheres from settling.
During incubation, the outer polar head groups facilitate the adsorption of SUVs onto the hydrophilic microsphere surface. This interaction causes SUVs to rupture, followed by the fusion of SUV lipids onto the curved microsphere surface as a continuous layer, resulting in a spherical, supported lipid bilayer, SLB, formation.
Wash to remove excess unbound lipids and resuspend in reaction buffer. Transfer the spherical SLB suspension onto a small region of a polyethylene glycol-coated surface, to prevent non-specific adsorptions. Add a fluorescently-labeled septin suspension to the chamber and incubate.
Septins form hetero-oligomers that recognize the positive curvature of spherical SLBs, polymerizing to form filaments. Eventually, septin filaments interact with each other, leading to the formation of higher-order structures.
Observe septin-coated SLBs under a high-resolution fluorescence microscope. The presence of fluorescence indicates septin’s curvature-sensitive deposition.
