This article discusses the assembly of septins, key cytoskeletal proteins, on supported lipid bilayers (SLBs) formed on microspheres. The methodology involves the use of silica beads and small unilamellar vesicles (SUVs) to create a stable environment for observing septin polymerization and filament formation.
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.
First, vortex the bottle containing silica microspheres for 15 seconds. Then, bath-sonicate for one minute, and vortex again for 15 seconds to break any clusters. Mix the beads with SLBB and 10 microliters of 5-millimolar SUVs in a low-adhesion microcentrifuge tube.
Incubate the bead-lipid mixture for one hour at room temperature, on a shaker to prevent sedimentation. In the meantime, thaw a PEGylated coverslip, and glue a cut 0.2-milliliter PCR tube to it. After incubation, centrifuge the beads for 30 seconds at the designated RCF.
After the first spin, remove 50 microliters of supernatant. Then, add 200 microliters of PRB and mix by vigorous pipetting. For the next rounds, remove 200 microliters of PRB, and add another 200 microliters after the second and third spin, and add 220 microliters of PRB after the fourth spin.
If doing a competition assay with multiple bead sizes, prepare the reaction by mixing equal volumes of each bead size, and diluting 29 microliters of this bead mixture with 721 microliters of reaction buffer. Then, add 75 microliters of diluted beads and 25 microliters of the protein diluted in SSB to the wells and mix.
If measuring septins at a steady state, incubate the mixture at room temperature for one hour, and then, image by either near-TIRF or confocal microscopy.
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