Aug 25, 2023

Public workspaceGeneration of membrane tubules pulled from giant unilamellar vesicles (GUVs)

DOI
dx.doi.org/10.17504/protocols.io.j8nlkw7p5l5r/v1
  • Javier Espadas1,
  • Aurelien Roux1
  • 1Department of Biochemistry, University of Geneva, Geneva, 1211, Switzerland
xinbo.wang
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkw7p5l5r/v1
Protocol Citation: Javier Espadas, Aurelien Roux 2023. Generation of membrane tubules pulled from giant unilamellar vesicles (GUVs). protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw7p5l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 02, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 82801
Keywords: Generation of membrane tubules, giant unilamellar vesicles, ASAPCRN
Abstract
This protocol explains the methodology to generate lipid nanotubes pulled from giant unilamellar vesicles for fluorescence microscopy experiments.
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Materials
Materials:

  • Lipids:
Reagent1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1, 18:1 PC)Avanti Polar Lipids, Inc.Catalog #850375P
Reagent12-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)Avanti Polar Lipids, Inc.Catalog #840035
Reagent12-Dioleoyl-sn-glycero-3-phosphoethanolamine labeled with Atto 647NMerck MilliporeSigma (Sigma-Aldrich)Catalog #42247

  • Glass vials (2700 Supelco, Sigma-Aldrich).
  • ReagentSilica Beads Microspheres-NanospheresCatalog #140256-10
  • Parafilm.
  • Petri dish.
  • ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #650498
  • Closed glass micropipettes prepared using a P-1000 micropipette puller (Sutter Instruments, USA).
  • ReagentBovine serum albumin 2 mg/mlThermo Fisher ScientificCatalog #23209

Solutions:

  • Lipid films hydration buffer A: 25 mM HEPES 7.4.
  • Lipid films hydration buffer B: 1M Trehalose.
  • Working buffer:
A
20mM HEPES 7.4
150mM NaCl
2.5mM MgCl2
5% Glycerol
2mM DTT
Protocol
Protocol
3h 30m
3h 30m
Mix DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of Amount200 µL with chloroform and Amount0.5 g/L lipid final concentration in a glass vial.
Mix
Dry the lipid mixture in the glass vials for Duration02:00:00 in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.
2h
Add Amount200 µL of the lipid films hydration buffer A to the glass vial containing the dried lipid films.

Pipetting
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 Amount10 µL of MLVs with Amount2 µL of silica beads in an Eppendorf tube.

Mix
Deposit 6 drops of Amount2 µL 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 Duration01:00:00 in the vacuum chamber until the liquid is completely dried.

1h
Take one dried drop from the parafilm and insert it into a small plastic tip cutted at the thin end containing Amount6 µL of Concentration1 Molarity (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 Duration00:15:00 at Temperature60 °C attaching it to the cap of an Eppendorf with Amount500 µL destilled water inside by doing a small hole in the cap and inserting the cutted plastic tip.

15m
Incubation
Passivate the microscopy chamber by adding Amount200 µL solution of Amount2 g/L BSA for Duration00:15:00 .
15m
Pipetting
Remove the cutted plastic tip from the Eppendorf and put the thin part of the cutted tip in contact with the microscopy chamber containing Amount200 µL of working buffer until visually seeing how the beads are transferred from the tip to the observation chamber.
Note
Note: the microscopy chamber contains either Concentration20 nanomolar (nM) or Concentration0.5 micromolar (µM) of GFP-LRRK2 in the solution.

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.