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* These authors contributed equally
Here, a nanobar-supported lipid bilayer system is developed to provide a synthetic membrane with a defined curvature that enables the characterization of proteins with curvature sensing ability in vitro.
Membrane curvature plays important roles in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It is not only passively generated by cellular activities, but also actively regulates protein interactions and is involved in many intracellular signaling. Thus, it is of great value to examine the role of membrane curvature in regulating the distribution and dynamics of proteins and lipids. Recently, many techniques have been developed to study the relationship between the curved membrane and protein in vitro. Compared to traditional techniques, the newly developed nanobar-supported lipid bilayer (SLB) offers both high-throughput and better accuracy in membrane curvature generation by forming a continuous lipid bilayer on patterned arrays of nanobars with a pre-defined membrane curvature and local flat control. Both the lipid fluidity and protein sensitivity to curved membranes can be quantitatively characterized using fluorescence microscopy imaging. Here, a detailed procedure on how to form a SLB on fabricated glass surfaces containing nanobar arrays and the characterization of curvature-sensitive proteins on such SLB are introduced. In addition, protocols for nanochip reusing and image processing are covered. Beyond the nanobar-SLB, this protocol is readily applicable to all types of nanostructured glass chips for curvature sensing studies.
Membrane curvature is a critical physical parameter of a cell that occurs in a variety of cellular processes such as morphogenesis, cell division, and cell migration1. It is widely recognized now that membrane curvature is beyond a simple result of cellular events; instead, it has emerged as an effective regulator of protein interactions and intracellular signaling. For example, several proteins involved in clathrin-mediated endocytosis were found to preferentially bind to the curved membrane, resulting in the formation of a hotspot for endocytosis2. There are many different causes of membrane deformation such as membran....
1. Cleaning of nanochip
Nanobar design is recommended for probing positive curvature sensing proteins, which contains a half circle at each end with curvature defined by the nanobar width and one flat/zero curvature control locally at the center (Figure 2A,B). Successful formation of the SLB on nanobars results in evenly distributed lipid marker signals across the entire nanobar surface as shown in Figure 2C. Signals from multiple nanobars can be combined by averaging .......
The nanobar-SLB system described here offers a unique combination of the advantages in several existing in vitro assays. It efficiently reveals the preferential binding of proteins to highly curved membranes as the liposome floatation or sedimentation assay but requires much fewer samples and offers more accurately defined curvature on individual nanobars8,29. It also offers a wide range of precisely controlled curvature for simultaneous comparison as th.......
We thank Nanyang NanoFabrication Centre (N2FC) and the Centre for Disruptive Photonic Technologies (CDPT) at Nanyang Technological University (NTU) for supporting nanostructure fabrication and SEM imaging, the Protein Production Platform (PPP) at the School of Biological Sciences NTU for protein purification, and the School of Chemical and Biomedical Engineering NTU for the confocal microscope. This work is funded by the Singapore Ministry of Education (MOE) (W. Zhao, RG112/20, RG95/21, and MOE-T2EP30220-0009), the Institute for Digital Molecular Analytics and Science (IDMxS) supported by MOE funding under the Research Centres of Excellence scheme (W. Zhao), the Human....
Name | Company | Catalog Number | Comments |
Anhydrous Ethanol | Sigma-Aldrich | 100983 | |
Aluminum foil | Diamond | RN0879999FU | |
Amber Vial | Sigma-Aldrich | 27115-U | |
Brain PS: L-α-phosphatidylserine (Brain, Porcine) (sodium salt) | Avanti Polar Lipids, Inc. | 840032 | |
10 mL Beaker | Schott-Duran | SCOT211060804 | |
50 mL Beaker | Schott-Duran | SCOT211061706 | |
1000 mL Beaker | Schott-Duran | SCOT211065408 | The second container |
Chloroform | Sigma-Aldrich | V800117 | |
Cotton buds | Watsons | ||
18:1 DGS-NTA(Ni): 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) | Avanti Polar Lipids, Inc. | 790404 | |
Egg PC: L-α-phosphatidylcholine (Egg, Chicken) | Avanti Polar Lipids, Inc. | 840051 | |
F-BAR | Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore | Proteins and peptide | |
F-BAR+IDR | Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore | Proteins and peptide | |
GFP | Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore | Proteins and peptide | |
GFP-His | Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore | Proteins and peptide | |
GraphPad Prism | GraphPad | V9.0.0 | |
Hydrogen Peroxide, 30% (Certified ACS) | Thermo Scientific | H325-500 | |
IDR from human FBP17 | Sangon Biotech (Shanghai) Co., Ltd. | ||
ImageJ | National Institutes of Health | 1.50d | |
Laser Scanning Confocal Microscopy | Zeiss | LSM 800 with Airyscan | 100x (N.A.1.4) oil objective. |
Methanol | Fisher scientific | 10010240 | |
Mini-extuder | Avanti Polar Lipids, Inc. | 610000-1EA | |
1.5 mL Microtubes | Greiner | 616201 | |
MATLAB | Mathworks | R2018b | |
Nuclepore Hydrophilic Membrane,0.1 ÎĽm | Whatman | 800309 | |
Phosphate Bufferen Saline (PBS) | Life Technologies Holdings Pte Ltd. | 70013 | |
Polydimethylsiloxane (PDMS) Base | Dow Corning Corporation | SYLGARD 184 | |
Polydimethylsiloxane (PDMS) Crosslinker | Dow Corning Corporation | SYLGARD 184 | |
Plasma Cleaner | HARRICK PLASMA | PDC-002-HP | |
Quartz Nanochip | Donghai County Alfa Quartz Products CO., LTD | ||
Sodium Hydroxide | Sigma-Aldrich | 795429 | |
Sulfuric acid | Sigma-Aldrich | 258105 | |
Texas Red DHPE: Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium Salt | Life Technologies Holdings Pte Ltd. | T1395MP | |
Tweezer | Gooi | PDC-002-HP | |
Ultrasonic Cleaners | Elma | D-78224 | |
Voterx | Scientific Industries | G560E | |
Vacuum Desiccator | NUCERITE | 5312 | |
Water Bath | Julabo | TW8 |
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