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 Frontier Science Program Foundation (W. Zhao, RGY0088/2021), the NTU Start-up Grant (W. Zhao), School of Chemical and Biomedical Engineering NTU for the research scholarship (X. Miao), and China Scholarship Council for the research scholarship (J. Wu).

1. Cleaning of nanochip
<ol>
	<li>Place the nanochip in a 10 mL beaker with the patterned side facing up. 
	NOTE: This quartz nanochip has been fabricated via electron beam lithography as described before21. The geometry and arrangement of the nanostructure on the chip can be custom designed. The sizes of the gradient nanobars used here are 2000 nm in length, 600 nm in height, and 100 to 1000 nm in width (100 nm step-set).</li>
	<li>Carefully add 1 mL of 98% sulfur...

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</ol>

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...

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 membrane pulling by the cytoskeletal forces, the presence of lipid asymmetry with different sized head groups, the existence of transmembrane proteins with conical shape, the accumulation of membrane-shaping proteins like BAR-domain proteins (named after Bin, amphiphysin, and Rvs proteins), and the insertion of amphipathic helices domain into the membrane1. Interestingly, these proteins and lipids not only deform the membrane but can also sense the membrane curvature and exhibit preferential accumulation1. Therefore, it is crucial to study whether and how membranes with different curvatures alter the distribution and dynamics of proteins and lipids attached to them and the potential impacts on the related intracellular processes.
Many techniques have been developed to analyze the interaction between curved membrane and proteins in both live cell and in vitro systems. The live cell system provides a real cell environment with rich lipid diversity and dynamic protein signaling regulation2,3,4,5,6,7. However, such a sophisticated system is difficult to study due to the uncertainties and fluctuations in the intracellular environment. Hence, the in vitro assays using an artificial membrane composed of known lipid species and purified proteins have become powerful reconstitution systems to characterize the relationship between proteins and curved membranes. Traditionally, liposomes of different diameters are generated by extrusion to detect curvature-sensitive proteins via either a co-sedimentation assay using centrifugal force or a co-flotation assay with a density gradient to avoid protein aggregation8,9. However, the curvature of the extruded liposomes is limited by the available pore size of the membrane filter used in the extruder10. Single liposome curvature (SLiC) assay has been proven to overcome this limitation, in which liposomes with different diameters are fluorescence-labeled and immobilized onto the surface so that the curvature can be marked by the fluorescent intensity11. However, strong variability in lipid composition has been observed in small vesicles, which affects the accuracy of the curvature measurement12. Tether-pulling experiments provide a more accurate measurement of the curvature on the transient tether pulled from giant unilamellar vesicles (GUVs) using an optical tweezer, where the curvature can be well controlled by the membrane tension generated13,14. This method is suitable to study either positive- or negative-curvature sensing proteins, but is constrained by the throughput of tube generation10. Supported membrane tubes (SMrT) assay affords simultaneous generation of multiple membrane tubes that are extruded from the same lipid reservoir by microfluidic flows. Nevertheless, the membrane curvature varies intrinsically along the nanotube, which compromises the accuracy of fluorescence-intensity-based curvature measurement15,16. In comparison, using small unilamellar vesicles (SUVs, diameter &#60;100 nm17) to form a supported lipid bilayer (SLB) on surfaces containing designed topographies generated a single bilayer membrane with curvatures predetermined by nanofabrication or nanomaterials in high accuracy18,19,20.
Here, we present a protocol for the formation of the SLB on fabricated nanochip surfaces with nanobar arrays and how it can be used to probe the curvature sensitivity of proteins in vitro. As shown in Figure 1, there are six essential components of the assay: A) Cleaning and assembly of the chip with a microfluidic chamber; B) Preparation of SUVs&#160;with defined lipid composition; C) Formation of the SLB on a nanochip and binding with curvature sensitive proteins; D) Imaging and characterization of the SLB and curvature sensitive proteins under fluorescence microscopy; E) Cleaning the chip for reuse; F) Image processing for quantitative analysis of protein curvature sensing ability. The detailed protocol is described step-by-step below.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anhydrous Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>100983</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Diamond</td>
			<td>RN0879999FU</td>
			<td></td>
		</tr>
		<tr>
			<td>Amber Vial</td>
			<td>Sigma-Aldrich</td>
			<td>27115-U</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain PS: L-&#945;-phosphatidylserine (Brain, Porcine) (sodium salt)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>840032</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Beaker</td>
			<td>Schott-Duran</td>
			<td>SCOT211060804</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Beaker</td>
			<td>Schott-Duran</td>
			<td>SCOT211061706</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Beaker</td>
			<td>Schott-Duran</td>
			<td>SCOT211065408</td>
			<td>The second container&#160;</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>V800117</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton buds</td>
			<td>Watsons</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18:1 DGS-NTA(Ni): 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>790404</td>
			<td></td>
		</tr>
		<tr>
			<td>Egg PC: L-&#945;-phosphatidylcholine (Egg, Chicken)</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>840051</td>
			<td></td>
		</tr>
		<tr>
			<td>F-BAR</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>F-BAR+IDR</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>GFP</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>GFP-His</td>
			<td>Protein Production Plaftorm, School of Biological Sciences, Nanyang Technological University, Singapore</td>
			<td></td>
			<td>Proteins and peptide</td>
		</tr>
		<tr>
			<td>GraphPad Prism</td>
			<td>GraphPad</td>
			<td>V9.0.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide, 30% (Certified ACS)</td>
			<td>Thermo Scientific</td>
			<td>H325-500</td>
			<td></td>
		</tr>
		<tr>
			<td>IDR from human FBP17</td>
			<td>Sangon Biotech (Shanghai) Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td>1.50d</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal Microscopy</td>
			<td>Zeiss&#160;</td>
			<td>LSM 800 with Airyscan</td>
			<td>100x (N.A.1.4) oil objective.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher scientific</td>
			<td>10010240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extuder&#160;</td>
			<td>Avanti Polar Lipids, Inc.</td>
			<td>610000-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microtubes</td>
			<td>Greiner</td>
			<td>616201</td>
			<td></td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>Mathworks</td>
			<td>R2018b</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclepore Hydrophilic Membrane,0.1 &#956;m</td>
			<td>Whatman</td>
			<td>800309</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Bufferen Saline (PBS)</td>
			<td>Life Technologies Holdings Pte Ltd.</td>
			<td>70013</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) Base</td>
			<td>Dow Corning Corporation</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS) Crosslinker</td>
			<td>Dow Corning Corporation</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>HARRICK PLASMA</td>
			<td>PDC-002-HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz Nanochip</td>
			<td>Donghai County Alfa Quartz Products CO., LTD</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>795429</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red DHPE: Texas Red 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium Salt</td>
			<td>Life Technologies Holdings Pte Ltd.</td>
			<td>T1395MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezer</td>
			<td>Gooi</td>
			<td>PDC-002-HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic Cleaners</td>
			<td>Elma</td>
			<td>D-78224</td>
			<td></td>
		</tr>
		<tr>
			<td>Voterx</td>
			<td>Scientific Industries</td>
			<td>G560E</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Desiccator</td>
			<td>NUCERITE</td>
			<td>5312</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Julabo</td>
			<td>TW8</td>
			<td></td>
		</tr>
	</tbody>
</table>

a nanobar-supported lipid bilayer system for the study of membrane curvature sensing proteins in vitro

Membrane curvature plays important roles&#160;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&#160;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.

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 ...

Watch this Scientific Journal Video about A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro at JoVE.com

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

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.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Membrane curvature plays important roles&#160;in various essential processes of cells, such as cell migration, cell division, and vesicle trafficking. It ...

This nanobar-supported lipid bilayer system can be used to study the role of membrane curvature in regulating the dynamics and distribution of protein and lipids during cell activities. This technique offers both high sub root and bad in the membrane curvature generation by forming a continuous lipid bilayer on patent nanobar errors with membrane curvature predefined by high resolution nanofabrication. To begin, place the nano chip in a 10 milliliter beaker with the pattern side facing up.Carefully add one milliliter of 98%sulfuric acid to the beaker and ensure the acid fully covers the front and back side of the chip. Slowly rotate the beaker and add 200 microliters of 30%hydrogen peroxide drop by drop until the whole beaker becomes hot. Ensure that sulfuric acid and hydrogen peroxide are well-mixed to form Piranha solution for the removal of organic molecules from the nano chip.Place the beaker in a secondary glass container and keep the nano chip immersed in the Piranha solution overnight to clean the impurities thoroughly. Take the beaker out and carefully pipette the Piranha solution into an acid waste container. Load five milliliters of deionized water into the beaker to dilute the residual acid and discard it into the acid waste.Repeat this step five times. Grab the chip with tweezers and wash with a continuous stream of deionized water to remove residual acid thoroughly. Blow dry the chip with 99.9%nitrogen gas for SLB formation.Sonicate the lipid mixture for 30 minutes. Freeze the lipid mixture in liquid nitrogen for 20 seconds and then thaw at 42 degrees Celsius for two minutes in a water bath. Repeat the freeze/thaw cycles 30 times.After that, the lipid mixture looks like a clear liquid. Pass the lipid mixture through the mini extruder and extrude back and forth 20 times. Collect the SUVs from the syringe at the other side to reduce the contamination with larger particles or foreign material, and transfer it into a 1.5 milliliter centrifuge tube.Take out the cleaned nano chip from deionized water carefully with a pair of tweezers and blow dry with 99.9%nitrogen gas. Perform surface cleaning of the nano chip with air plasma treatment for one hour. Assemble the nano chip in a PDMS chamber.Start with placing the chip on a clean surface with the pattern facing up. Gently cover the middle PDMS with the chip and make sure the whole pattern is exposed to the central area of the large oval-shaped opening in the middle PDMS. Cover the top PDMS with the middle PDMS and keep its two small holes within the region of the large oval hole of the middle PDMS.Then, the PDMS chamber is assembled. Load the SUVs into the PDMS channel from one of the two small holes in the top PDMS with a pipette and incubate for 15 minutes at room temperature to form the SLB. Gently pipette the PBS buffer into the PDMS channel from one side of the small hole and remove the waste with a cotton bud from the other hole to wash away the unbound SUVs.Then acquire the SLB formed on the nano chip. Set up the laser scanning confocal microscopy using a 100 x oil objective. Open the Zen software to select the excitation laser power that can excite the fluorescence of the lipid and protein.Choose Acquisition Mode. Then click Smart Setup, followed by Texas Red. Adjust the focus with the focus knob to locate the nano bars on the chip until the nano bar edges are sharp under the lipid channel.Set the scanning parameters to obtain a control image of the lipid channel before adding protein. Conduct the FRAP assay by bleaching with fluorescent labeled lipid bilayer on a random single nanobar area. Select the Experiment Regions and Bleaching check boxes.Draw a circular area of five micrometers diameter which can include the whole nanobar at the center, and add to the experiment regions. Input TimeLapse imaging parameters. Choose Time Series.Then click Duration. Select 100 cycles and interval equal to two seconds. Input bleaching parameters.Select the laser check boxes that perform FRAP and change the power to 100%Click Start Experiment for the FRAP experiment. Load the protein solution into the PDMS channel and incubate for five minutes at room temperature to allow the binding of the protein on the SLB. Refocus the nanobars and set the scanning parameters to take images of both lipid and protein channels for protein curvature sensing detection.Select Experiment Regions and conduct the FRAP assay on both lipid and protein channels, and perform TimeLapse imaging to characterize the mobility of curvature sensing protein. Both show increased accumulation on the end of SLB coated individual nanobars with 300 nanometers width. Here the SLB contains 10%PS to electrostatically enhance the protein binding.Both proteins have a higher end to center ratio than lipids, which is around one. When comparing the IDR FBP17 and F-Bar, the higher end to center ratio of IDR FBP17 indicates stronger curvature sensing than F-Bar. All three proteins showed preferential accumulation at the nanobar end curved membrane sites when the curvature decreased below 400 nanometers in diameter.Among these three protein domains, IDR FBP17 gives the highest nano bar and density, indicating its strongest curvature sensing ability, while F-Bar shows the lowest value. Protein binding curve can be plotted by gradually increasing the protein concentration which shows that IDR FBP17 has a strong cooperative curvature sensing ability. Compared to the fast recovery of lipid signals on nanobars, the F-Bar cannot recover within two minutes, suggesting its significantly decreased membrane mobility and association dynamic at the curved membrane sites.Surprisingly, different from the behavior of F-Bar, IDR FBP17 signals showed obvious recovery within the same timeframe, indicating the dynamic nature of IDR FBP17 accumulated at the curved membranes. However, after washing away the unbound IDR FBP17 in the solution, the same recovery in the IDR FBP17 channel couldn't be observed as before. The SLB quality is very important when studying the interaction between curved membrane and protein.So the FRAP test is necessary in our experiment to validate the membrane mobility. This system will help the researchers to study various parameters affecting the protein interaction with the curved membrane, such as the curvature sensing domains, and the lipid composition as well as to conduct the dynamic studies on curved membrane such as face separation behaviors.

A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro

Abstract