Generation of membrane tubules pulled from giant unilamellar vesicles (GUVs)

Mix DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of 200µL with chloroform and 0.5g/L lipid final concentration in a glass vial.

Dry the lipid mixture in the glass vials for 2h 0m 0s in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.

Add 200µL of the lipid films hydration buffer A to the glass vial containing the dried lipid films.

Vortex the glass vials until visually seeing complete resuspension of the dried lipid films in the solution (seen by an increase in the turbidity of the lipid solution) forming the multilamellar vesicles (MLVs).

Mix 10µL of MLVs with 2µL of silica beads in an Eppendorf tube.

Deposit 6 drops of 2µL each containing the mixture of MLVs and silica beads on a parafilm slide placed in the bottom of a petri dish.

Dry the drops for 1h 0m 0s in the vacuum chamber until the liquid is completely dried.

Take one dried drop from the parafilm and insert it into a small plastic tip cutted at the thin end containing 6µL of 1Molarity (M) trehalose solution until visually seeing how the dried beads get to the thin bottom.

Incubate the cutted plastic tip containing the drop and the trehalose for 0h 15m 0s at 60°C attaching it to the cap of an Eppendorf with 500µL destilled water inside by doing a small hole in the cap and inserting the cutted plastic tip.

Passivate the microscopy chamber by adding 200µL solution of 2g/L BSA for 0h 15m 0s.

Remove the cutted plastic tip from the Eppendorf and put the thin part of the cutted tip in contact with the microscopy chamber containing 200µL of working buffer until visually seeing how the beads are transferred from the tip to the observation chamber.

Gently stir the microscopy chamber to promote the detachment of the hydrated lipid films from the silica beads, leading to the formation of the GUVs.

Place a closed micropipette in the micro-positioning system (MP-285, Sutter Instrument, Novato, CA, USA), and use it to approach the pipette to the GUV membrane.

Touch the GUV membrane with the pipette and then move back the pipette until a lipid nanotube is pulled from the GUV.

Wait until protein coverage reaches the steady state in both the GUV and the pulled membrane nanotube.