Biochemistry
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Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions
Chapters
Summary July 28th, 2022
Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.
Transcript
This protocol will guide those working on membrane binding proteins to determine how membrane shape plays a role in the organization of protein assemblies. The primary advantage of this technique is its versatility for protein type, membrane composition, and membrane geometry. It also allows interrogation of how a lipid bilayer can alter properties of associated proteins.
To identify a good membrane for the experiment, we suggest establishing a checklist that might include lipid diffusion rates, mobility of the protein of interest, and whether there are unburst liposomes or fused liposomes stuck to the surface. To begin plasma cleaning of the slides, purge the plasma cleaner for five 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 glue down in the center of a plasma cleaned coverslip, then place the chamber under long wavelength UV light for five to seven 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 six times with 150 microliters of SLBB by pipetting to wash away excess lipids, then wash the bilayer six 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. 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 five 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. TIRF micrographs of planar SLBs incubated with septins shown in green showed homogeneous protein distribution.
SLBs made with poorly cleaned glass coverslips showed holes or gaps in the bilayer in regions of low septin. Unburst and tubulated liposomes may accumulate on the surface if old liposomes are used or if washes are not stringent. In micrographs of lipid-coated microspheres visualized using rhodamine PE showed smooth bilayer deposition.
The spherical supports were evenly coated by the membrane and there were few bead clusters. Insufficient washing of excess lipids caused uneven membrane coating as observed by lipid clumps and improper handling caused gaps in the bilayer. Insufficient mixing of beads caused clumping which is not ideal for measuring the total protein absorption.
Using the supported lipid bilayers presented here, other methods such as fluorescence lifetime imaging microscopy, mass cytometry of membranes, and high-speed atomic force microscopy can be performed to examine different protein-protein or protein-membrane dynamics.
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