Jun 02, 2026

Characterization of VPS13Cs binding to GUVs

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbwko1gpk/v1
  • 1Departments of Neuroscience and of Cell Biology, Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815;
  • 3Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA
Dazhi Li
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DOI: https://dx.doi.org/10.17504/protocols.io.81wgbwko1gpk/v1
Protocol Citation: Xinbo Wang, Dazhi Li, Pietro De Camilli, Karin Reinisch 2026. Characterization of VPS13Cs binding to GUVs. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbwko1gpk/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: October 19, 2025
Last Modified: June 02, 2026
Protocol  Integer ID: 230233
Keywords: Lipid transport protein, VPS13C, GUV, important for lysosomal membrane damage repair, lysosomal membrane damage repair, lysosome contact site protein, giant unilamellar vesicle, characterization of vps13c, membrane, organellar membrane, membrane packing defect, vps13c, recognizing membrane packing defect, guvs vps13c, lipid, atg2-c region
Funders Acknowledgements:
Michael J. Fox Foundation
Grant ID: ASAP-000580
NIH-R35
Grant ID: GM131715
NIH
Grant ID: DA018343
Abstract
VPS13C is a ER-lysosome contact site protein that transports lipids between the two organellar membranes and important for lysosomal membrane damage repair. We employed structure-function analysis of purified VPS13C and uncovered novel regulatory mechanisms. Here, we describe the protocol to characterize VPS13C's binding to giant unilamellar vesicles (GUVs), where we discovered VPS13C's binding to membrane depends on its ATG2_C region recognizing membrane packing defects.
Materials
POPC (Cat. #850457) Lissamine Rhodamine PE (Rhod-PE; Cat. #810150)
DOPS (Cat. #840035, Avanti Polar Lipids)
Cy5-DOPE (Cat. #810335, Avanti Polar Lipids)
DOG (Cat. #800811, Avanti Polar Lipids)
APTES ((3-aminopropyl) trimethoxysilane; Cat. #A3648, Sigma Aldrich)
glutaraldehyde (GA; Cat. #G6257, Sigma Aldrich)
ammonium persulfate (APS; Cat. #248614, Sigma Aldrich) N,N,N′,N′-tetramethylethylenediamine (TEMED; Cat. #1610801, Bio-Rad)
acrylamide (AA; 40% w/v, Sigma Aldrich, A4058) N,N′-methylenebisacrylamide (BAA; 2% w/v, Cat. #M1533, Sigma Aldrich)
Protocol materials
Glutaraldehyde solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #G6257
N,N’-methylenbisacrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1533
TEMEDBio-Rad LaboratoriesCatalog #1610801
16:0-18:1 PC (POPC)Avanti Polar Lipids, Inc.Catalog #850457
12-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)Avanti Polar Lipids, Inc.Catalog #840035
Rhod-PEAvanti Polar Lipids, Inc.Catalog #810150
18:1 Cy5 PEAvanti Polar LipidsCatalog #810335
18:1 DG (format: Chloroform)Avanti Polar LipidsCatalog #800811C
Preparation of giant unilamellar vesicles (GUVs)
3h 25m
GUVs were generated as previously described.
Citation
Parigoris E, Dunkelmann DL, Silvan U (2020). Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels. Bio-protocol.
https://doi.org/10.21769/BioProtoc.3807
LINK


Sonicate coverslips in distilled water and ethanol, then incubate in 10% APTES in ethanol for 00:30:00 at room temperature.

30m
Wash coverslips with ethanol and distilled water, then incubate in 2.5% Glutaraldehyde solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #G6257 in PBS for 00:30:00 .

30m
Wash coverslips with distilled water, dry, and store at 4 °C .

Prepare the PAA gel solution by mixing 250 μL acrylamide, 10 μL bis-acrylamideN,N’-methylenbisacrylamideMerck MilliporeSigma (Sigma-Aldrich)Catalog #M1533 , and 729 μL H2O. Degas the solution under vacuum for 00:30:00 .

30m
Initiate polymerization by adding 10 μL APS and 1 μL TEMEDTEMEDBio-Rad LaboratoriesCatalog #1610801 .

Place 30 μL gel solution on a clean glass plate and lay the treated coverslip on top. Allow the gel to polymerize for 00:15:00 .

15m
Remove coverslips, wash with water, and dry at 50 °C for 00:30:00 .

30m
Prepare lipid mixtures in chloroform at 1 mg/mL. For POPC-GUVs, use 89.5% POPC16:0-18:1 PC (POPC)Avanti Polar Lipids, Inc.Catalog #850457 , 10% DOPS12-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt)Avanti Polar Lipids, Inc.Catalog #840035 , and 0.5% Rhod-PE Rhod-PEAvanti Polar Lipids, Inc.Catalog #810150 or Cy5-DOPE18:1 Cy5 PEAvanti Polar LipidsCatalog #810335 .

For DOG-GUVs, use 59.5% POPC, 10% DOPS, 30% DOG18:1 DG (format: Chloroform)Avanti Polar LipidsCatalog #800811C , and 0.5% Rhod-PE or Cy5-DOPE.

30m
Place the gel-coated coverslips in 6-well plates and apply 20 μL lipid solution to each gel. Spread evenly and evaporate the solvent under vacuum for 00:40:00 .

40m
Add 1 mL buffer containing 500 mM sucrose and 25 mM HEPES, pH 7.4, then incubate Overnight to form GUVs.

Collect GUVs by gentle pipetting and transfer to clean 1.5 mL tubes.
Confocal fluorescence microscopy of protein–GUV association
2h 20m
Add 1 μL GUVs to a BSA-passivated 35 mm glass-bottom MatTek dish.
Incubate with 4 μL purified protein at 300 nM working concentration, or with cell lysate, for 00:10:00 at Room temperature .

10m
Acquire images using the spinning-disk super-resolution microscope.
2h
Analyze images in ImageJ.
10m
Citations
Step  1
Parigoris E, Dunkelmann DL, Silvan U. Generation of Giant Unilamellar Vesicles (GUVs) Using Polyacrylamide Gels.
https://doi.org/10.21769/BioProtoc.3807