To reduce the surface interaction to ESCRT tubes, we have used a lipid bilayer on top of freshly cleaved mica, as modified from literature (21, 54, 55). The lipid bilayer was formed by absorption of large unilamellar vesicles (LUVs) onto freshly cleaved mica. To prepare LUVs, the lipids, DOPC, and DOPS from Avanti, polar lipids were used. Sixty percent DOPC and 40% DOPS (mol:mol) containing 1 mg/ml of total lipids were mixed in 200 μl of chloroform in a small glass vial. Next, chloroform was evaporated using argon gas while slowly rotating the vial to produce a lipid film on the glass wall. Last, the film was completely dried in a vacuum desiccator for 30 to 45 min. Rehydration of the lipid film was performed with 200 μl of buffer B [10 mM Hepes (pH 7.4), 100 mM NaCl, and 50 mM sucrose]. The mixture was immediately vortexed for 30 s, followed by three cycles of freeze-thaw using liquid nitrogen. Small aliquots of LUVs were stored at −20°C and used within 1 month after preparation. LUVs were thawed at room temperature and diluted to a concentration of 0.2 mg/ml in the recording buffer C [10 mM Hepes, 10 mM tris-HCl (pH 7.5), and 150 mM NaCl] before use. Two microliters of diluted LUVs was incubated on a freshly cleaved, 1-mm-diameter mica disc attached to a small glass rod (HS-AFM sample stage) (56). After incubation for 5 to 10 min, the surface was cleaned three to five times with cleaning buffer D [10 mM tris-HCl (pH 7.5), 100 mM NaCl, and 10 mM MgCl2].

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