Norland optical adhesive

Lipid bilayers supported on silica microspheres were prepared as previously described (Bridges et al., 2016; Cannon et al., 2019) . SUVs were adsorbed onto silica microspheres (Bangs Laboratories) by incubating 50 nM of lipid with different bead sizes (with a summed surface area of 440 mM 2 ) for hour at room temperature with gentle rotation. Microspheres were pelleted, then resuspended in in Pre-Reaction buffer (33 mM KCl, 50 mM HEPES pH 7.4) to wash away excess SUVs. This wash step was repeated four additional times.
Planar supported lipid bilayers were prepared in a similar process as reported previously (Bridges et al., 2014) . No. 1.5 coverslips were first cleaned with oxygen plasma (PE25-JW, Plasma Etch). Chambers for lipid bilayers cut from PCR tubes were glued (Norland optical adhesive, Thor Labs) onto cleaned coverslips. SUVs were incubated at 37°C in chambers on coverslips suspended in Supported Lipid Bilayer buffer with additional 1 mM CaCl2 for 20 minutes (1 mM final lipid concentration in 50 μl solution). After vesicle fusion, lipid bilayers were washed six times with 150 μl of Supported Lipid Bilayer buffer to rinse away excess SUVs. Immediately prior to adding septins, bilayers were further washed with a 150 μl of buffer containing 50 mM Hepes pH7.4, 1 mM BME, and 0.01% BSA six times.

No 1.5H cover glass (Matsunami Cover Glass Ind., Inc) were cleaned with oxygen plasma for 15 min at maximum power (PE25-JW, Plasma Etch). Chambers modified from PCR tubes (Bridges et al., 2014) were adhered onto cleaned cover glass under UV light for 5 min (Norland optical adhesive, Thor Labs). SUVs were incubated at 37°C on cover glass within chambers, diluted in Supported Lipid Bilayer buffer (with additional 1 mM CaCl 2 ) to a final lipid concentration of 1 mM μl for 20 minutes. Ultra-pure, distilled water (Millipore Sigma) was necessary for any solution used in preparation or experiments with planar supported lipid bilayers to obtain reproducible results. After vesicle fusion, supported lipid bilayers were washed six times with 150 μl of Supported Lipid Bilayer buffer (without CaCl 2 ) to rinse away excess SUVs.
Immediately prior to adding septins or extracts, lipid bilayers were rinsed six times with a 150 μl of Reaction Buffer (50 mM Hepes pH7.4, 1 mM BME, and 0.13% low fatty-acid BSA [product no. A4612, lot no. SLBM2472V, Millipore Sigma]). The product number and lot number of low fatty-acid BSA were important for reconstitution assay reproducibility.