This study investigates the interactions between septins and membranes using a supported lipid bilayer (SLB) model. The methodology involves creating a reaction chamber and observing septin assembly through total internal reflection fluorescence microscopy.
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.
To begin plasma cleaning of the slides, purge the plasma cleaner for 5 minutes with oxygen to remove air from the lines and chamber. Arrange dry cover glass slides into a ceramic cradle. While purging, stream an inert gas over the micro cover glass slides to remove dust and particulates.
Place the cradle at the back of the plasma chamber so that the coverslips are parallel with the long edge of the chamber. Run the plasma cleaner for 15 minutes with oxygen at maximum power.
For chamber preparation, cut off the cap just below the frosted part of a 0.2-milliliter PCR tube. Paint the rim of the PCR tube with UV-activated adhesive, avoiding the inside of the tube. Gently place the PCR tube glued down in the center of a plasma-cleaned coverslip, then, place the chamber under long-wavelength UV light for 5 to 7 minutes to cure the adhesive.
To form a bilayer, add the reagents to the well. Gently shake the chambers from side to side to disrupt the SUVs, and then, incubate at 37 degrees Celsius for 20 minutes.
After incubation, rinse the bilayer 6 times with 150 microliters of SLBB, by pipetting to wash away excess lipids. Then, wash the bilayer 6 times with 150 microliters of reaction buffer, before incubating with the septins.
On the last wash step, remove the reaction buffer, leaving 75 microliters in the well. Add 25 microliters of septins diluted in SSB, and image by TIRF microscopy.
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