Aug 25, 2023
Generation of membrane tubules pulled from giant unilamellar vesicles (GUVs)
- Javier Espadas1,
- Aurelien Roux1
- 1Department of Biochemistry, University of Geneva, Geneva, 1211, Switzerland
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, giant unilamellar vesicles for fluorescence, giant unilamellar vesicle, lipid nanotube, generation of membrane tubule, membrane tubule, microscopy experiment, lipid, fluorescence
Abstract
This protocol explains the methodology to generate lipid nanotubes pulled from giant unilamellar vesicles for fluorescence microscopy experiments.
Attachments
733-1844.docx
16KB
Materials
Materials:
- Lipids:
1,2-dioleoyl-sn-glycero-3-phosphocholine (18:1, 18:1 PC)Avanti Polar Lipids, Inc.Catalog #850375P
12-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)Avanti Polar Lipids, Inc.Catalog #840035
12-Dioleoyl-sn-glycero-3-phosphoethanolamine labeled with Atto 647NMerck MilliporeSigma (Sigma-Aldrich)Catalog #42247
- Glass vials (2700 Supelco, Sigma-Aldrich).
- Silica Beads Microspheres-NanospheresCatalog #140256-10
- Parafilm.
- Petri dish.
- ChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #650498
- Closed glass micropipettes prepared using a P-1000 micropipette puller (Sutter Instruments, USA).
- Bovine 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
3h 30m
Mix DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of 200 µL with chloroform and 0.5 g/L lipid final concentration in a glass vial.
Dry the lipid mixture in the glass vials for 02:00:00 in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.
2h
Add 200 µL 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 10 µL of MLVs with 2 µL of silica beads in an Eppendorf tube.
Deposit 6 drops of 2 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 01:00:00 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 6 µL of 1 Mass Percent 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 00:15:00 at 60 °C attaching it to the cap of an Eppendorf with 500 µL destilled water inside by doing a small hole in the cap and inserting the cutted plastic tip.
15m
Passivate the microscopy chamber by adding 200 µL solution of 2 g/L BSA for 00:15:00 .
15m
Remove the cutted plastic tip from the Eppendorf and put the thin part of the cutted tip in contact with the microscopy chamber containing 200 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 20 nanomolar (nM) or 0.5 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.