Spherical Supported Lipid Bilayer Assay: A Technique to Observe the Septin Assembly on Microspheres

Protocolo

1. Spherical supported lipid bilayers

NOTE: This protocol uses silica microspheres suspended in ultrapure water at 10% density. For any work on the kinetic parameters of protein assembly, it is important to strictly control the total membrane surface area between experiments and curvatures. Table 1 shows the corrected volumes of beads and buffer to maintain 5 mm² of total membrane surface area.

1. Bilayer formation
   NOTE: As for planar bilayers, it is important to have high-quality SUVs for robust bilayer formation. Table 1 lists the volume of beads from a 10% density solution and their respective SLBB volumes that are used to obtain a final surface area of 5 mm² for a range of bead diameters.
   1. Silica microspheres (also referred to as beads) tend to settle and clump together; to mix the beads and break up any clusters, vortex the bottle for 15 s, then bath-sonicate for 1 min, and vortex again for 15 s.
   2. Mix the appropriate volume of beads (Table 1) with the corresponding volume of SLBB and 10 µL of 5 mM SUVs in a 0.5 mL low-adhesion microcentrifuge tube.
   3. Rock the bead-lipid mixture end-over-end for 1 h at room temperature. Ensure that this incubation is done on a rotator to prevent sedimentation.
2. Prepare chambers on PEGylated coverslips
   NOTE: As for planar bilayers, homemade chambers from plastic PCR tubes are used to support the reaction volumes, but other low-adhesion materials, such as silicone wells, may work just as well.
   1. While the beads are rocking, thaw a PEGylated coverslip at room temperature. Cut a 0.2 mL PCR tube and glue it to the coverslip as described. For 24 mm x 50 mm slides, up to 10 chambers can feasibly fit on one slide.
      NOTE: This protocol uses 24 mm x 50 mm PEGylated glass coverslips that are prepared in advance. Briefly, glass coverslips are thoroughly cleaned through sonication in acetone, methanol, and 3 M KOH. Coverslips are then functionalized with n-2-aminoethyl-3-aminopropyltrimethoxysilane and covalently linked to mPEG succinimidyl valerate. Finished PEGylated coverslips are stored vacuum-sealed at −80 °C. While PEGylated glass coverslips are suggested here, other means of passivation, such as BSA or poly-L-lysine methods, may be effective.
3. Washing away excess lipids
   NOTE: This step functions to remove excess SUVs and perform a buffer exchange. Beads are washed 4x in total with pre-reaction buffer (PRB: 33.3 mM KCl, 50 mM HEPES, pH 7.4). Table 2 lists the spin speeds in RCF for a range of bead diameters.
   1. Spin beads at the designated RCF for 30 s (Table 2). If using multiple bead sizes, keep beads rocking until it is their time to be washed.
      NOTE: It is not worth sedimenting larger beads in the same spin as smaller beads with the smaller bead sedimentation velocity to save time. This can cause beads to stick to each other and compromise the integrity of the bilayer.
   2. After the first spin, remove 50 µL of supernatant and add 200 µL of PRB. After the second and third spins, remove 200 µL and add 200 µL of PRB. After the fourth spin, remove 200 µL and add 220 µL of PRB. Pipette vigorously for each wash.
      NOTE: The final resuspension volume is different than the washes. Do not vortex the beads after incubation with the lipids as this can leave gaps in the bilayers. To avoid sedimentation while working with the other bead sizes or setting up other parts of the assay, place the bead mixtures back on the end-over-end rotator.
4. Preparing the reaction
   1. If doing a competition assay with multiple bead sizes, make a 1:1 mixture by mixing equal volumes of each bead size together. Then, mix 29 µL of the desired bead mixture (or of a single bead) with 721 µL of RXN buffer.
      NOTE: It is important to thoroughly mix the beads at each step with a pipette to avoid dense clusters of beads; this will result in a more even bead distribution and accurate total surface area.
   2. Mix 75 µL of diluted beads and 25 µL of protein diluted in SSB to the wells.
      NOTE: The authors typically image yeast septin complexes at 1-50 nM.
   3. If measuring septins at a steady state, incubate at room temperature for 1 h, and then image by either near-TIRF or confocal microscopy.
      NOTE: This reaction is designed to preserve the total membrane surface area of the reaction at 5 mm² and to produce a final reaction condition of 100 mM KCl, 50 mM HEPES, 1 mg/mL BSA, 0.1% methylcellulose, and 1 mM BME.

Table 1: Normalized volumes of microspheres. In order to maintain an equal surface area of each bead size and to keep the total membrane surface area consistent between experiments, volumes for each bead size and buffer that normalized the total surface area were calculated.

Bead Diameter (µm)	Volume of well-mixed beads (µL)	Volume of SLB buffer (µL)	Volume of SUVs (µL)
6.46	8.94	61.1	10
5.06	7	63	10
3.17	4.39	65.6	10
0.96	1.33	68.7	10
0.54	0.75	69.3	10
0.31	0.43	69.6	10

Table 2: Sedimentation velocities for microspheres of varying diameters. For each bead diameter, the shown minimum sedimentation velocities were used to pellet the beads for washing away unbound liposomes

Bead diameter (µm)	Sedimentation velocity (RCF)
0.31	4.5
0.54	4.5
0.96	2.3
3.17	0.8
5.06	0.3

Materiales

Name	Company	Catalog Number	Comments
0.2 mL PCR Tubes with flat cap, Natural	Watson	137-211C(EX)
0.5 mL low adhesion tubes	USA Scientific	1405-2600
Beta mercaptoethanol (BME)	Sigma-Aldrich	M6250-100ML
Bovine Serum Albumin (BSA)	Sigma-Aldrich	A4612-25G
Coverglass for making PEGylated coverslips	Thermo Scientific	152450	Richard-Allan Scientific SLIP-RITE Cover Glass 24x50 #1.5
DOPC	Avanti Polar Lipids	850375
Egg Liss Rhodamine PE	Avanti Polar Lipids	810146
HEPES	Sigma Aldrich	H3375-1KG
Methyl cellulose 4000cp	Sigma-Aldrich	M052-100G
Mini centrifuge
Non-Functionalized Silica Microspheres	Bangs Laboratories, Inc.	Depends on size: SS0200*-SS0500*	Silica in aqueous suspension
Optical Adhesive	Norland Thorlabs	NOA 68	Flexible adhesive for glass or plastics
Potassium chloride	VWR	0395-1kg
Sonicator bath	Branson	1510R-MT	Bransonic Ultrasonic cleaner. 50-60 Hz. Output: 70W
Soy PI	Avanti Polar Lipids	840044
Tabletop centrifuge	Eppendorf	22331
UV Lamp	Spectroline	ENF-260C	115 Volts, 60 Hz, 0.20 AMPS