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

Summary

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.

Abstract

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.

Introduction

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 regulation^2,3,4,5,6,7. 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 aggregation^8,9. 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 generated^13,14. 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 measurement^15,16. In comparison, using small unilamellar vesicles (SUVs, diameter <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 accuracy^18,19,20.

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 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.

Protocol

1. Cleaning of nanochip

1. 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).
2. Carefully add 1 mL of 98% sulfuric acid to the beaker, and ensure the acid fully covers the front and backside of the chip.
   NOTE: 98% sulfuric acid is extremely corrosive and can cause rapid tissue destruction and serious chemical burns upon contact with the skin or eyes. Use it in the fume hood with proper protection.
3. Slowly rotate the beaker and add 200 µL 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 nanochip¹⁷. There are alternative techniques for generating the SLB on clean hydrophilic surfaces, such as UV light and ozone exposure as described earlier²³.
   NOTE: The reaction is extremely exothermic and can cause the solution to boil, so add hydrogen peroxide to the sulfuric acid drop by drop and keep rotating.
4. Place the beaker in a secondary glass container and keep the nanochip immersed in the piranha solution overnight to clean the impurities thoroughly.
   NOTE: Place the beaker in the fume hood without any cover in case the reaction can generate gas.
5. Take the beaker out and carefully pipette the piranha solution into an acid waste container.
6. Load 5 mL of deionized water into the beaker to dilute the residual acid and discard it into the acid waste. Repeat this step five times and use 5 M NaOH to neutralize the acid waste.
7. 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 formation²².

2. Generation of small unilamellar vesicles (SUVs)

1. Add 100 µL of chloroform into an amber vial.
   NOTE: Chloroform is a highly volatile, colorless liquid, and it can be toxic if inhaled or swallowed. Use it in the fume hood with the mask and gloves on.
2. Dissolve 500 µg of the lipid mixture in chloroform. The composition of the lipid mixture used here is 89.5 mol% of egg phosphatidylcholines (PC), 10 mol% of brain phosphatidylserine (PS), and 0.5 mol% of Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (Texas Red DHPE).
3. Blow-dry the lipid mixture in the vial with 99.9% nitrogen gas until the chloroform is volatilized and the lipid mixture is dry.
   NOTE: This step should be performed in the fume hood. Avoid liquid spatter when blow-drying.
4. Place the lipid mixture present in the amber vial without the lid in the aluminum foil-covered vacuum desiccator. Then vacuum dry the sample with a pump for 3 h to completely volatilize the chloroform.
5. Add 250 µL of phosphate-buffered saline (PBS) buffer to the vial. Vortex the solution until it is homogeneous.
   NOTE: PBS buffer consists of 150 mM NaCl, without calcium, magnesium, and phenol red; the pH is adjusted to 7.2 for making the PC and PS lipid bilayer.
6. Sonicate the lipid mixture for 30 min at a frequency of 50 kHz in a bath sonicator. Then transfer the lipid mixture into a 1.5 mL centrifuge tube and seal with parafilm.
7. Freeze the lipid mixture in liquid nitrogen for 20 s and then thaw at 42 °C for 2 min in a water bath. Repeat the freeze-thaw cycles 30 times. After that, the lipid mixture looks like a clear liquid.
   CAUTION: Liquid nitrogen has a boiling point of about −195.8 ˚C. It can cause frostbite or cryogenic burns. Use it in the unconfined space with heat insulation gloves on.
8. Rinse two glass gas-tight syringes and the connector of the mini-extruder with ethanol, chloroform, and methanol respectively. Repeat the rinsing sequence five times to pre-clean the apparatus. Leave them in the fume hood until the organic solvent is completely volatilized.
9. Assemble the mini-extruder apparatus and pass 500 µL of PBS buffer through the connector to pre-wet the apparatus, then discard the buffer from another syringe. This step removes impurities and facilitates extrusion.
10. Assemble the mini-extruder apparatus with a 100 nm pore-sized polycarbonate filter membrane.
    NOTE: The pore size of the filter membrane determines the diameter of liposomes.
11. Take 500 µL of PBS buffer with one of the syringes and gently push it through the filter to fill the second empty syringe at the other end. Repeat the extrusion back and forth three times to check the apparatus leakage and discard the buffer from the second syringe.
12. Pass the lipid mixture through the mini extruder to replace the PBS buffer. Then extrude back and forth 20 times to form SUVs. The multilamellar character of the vesicles may be retained with insufficient number of extrusion times, which will affect SLB formation²³.
13. Collect the SUVs from the second syringe to reduce the contamination with larger particles and transfer it into a 1.5 mL centrifuge tube.
    NOTE: Remove the syringe straight out of the connector in case the syringe breaks off.
14. Wrap the tube with aluminum foil to prevent bleaching of fluorescent-labeled lipids and store them at 4 ˚C. Usually, the SUVs can be stored for up to 7 days.

3. Formation of the SLB on a nanochip

1. Take out the cleaned nanochip from deionized water carefully with a pair of tweezer and blow-dry with 99.9% nitrogen gas.
2. Perform surface cleaning of the nanochip with air plasma treatment for 1 h.
3. Assemble the nanochip in a polydimethylsiloxane (PDMS) chamber, which consists of two pieces of PDMS-a middle PDMS and a top PDMS (Figure 1A). The middle PDMS is thin (~0.5 mm) with a large oval-shaped opening in the center to ensure sufficient exposure to the nanobar pattern. The top PDMS is thick (~4.0 mm) with two small holes within the large oval opening as an inlet and an outlet for liquid handling.
   1. Place the chip on a clean surface with the pattern facing up.
   2. Gently cover the middle PDMS with the chip, and make sure that the whole pattern is exposed to the central area of the large oval-shaped opening in the middle PDMS.
   3. 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.
      NOTE: PDMS is the most widely used silicon-based organic polymer. It is biocompatible, transparent, as well as deformable to be designed as a customized shape for chip assembly and imaging under the microscope. More importantly, it can be covalently stuck to another PDMS layer or glass tightly after plasma treatment, making it suitable as the chamber to prepare the SLB. This step ensures that each layer of PDMS is tightly fitted to avoid gaps and leakage.
4. Load the SUVs into the PDMS chamber from one of the two small holes in the top PDMS with a pipette and incubate for 15 min at room temperature to form the SLB.
5. Gently pipette the PBS buffer into the PDMS chamber 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 nanochip (the quality of the SLB is tested by fluorescence recovery after photobleaching (FRAP) as shown in step 4.4 and discussed in the representative results section).
   ​NOTE: When pipetting the PBS buffer into the chamber, the pipette tip should be full of the buffer and in contact with the liquid surface to avoid injecting any bubbles.

4. Imaging the SLB and the curvature sensing protein binding on the nanochip

NOTE: This section will depend on the microscope system available for the experiment. Here, an overall guideline on how to perform the imaging will be described. The detailed settings can be changed between the different microscope setups.

1. Set up the laser scanning confocal microscopy using a 100x (N.A.1.4) 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 > Smart Setup > Texas Red DHPE / EGFP.
2. Adjust the focus with the focus knob to locate the nanobars on the chip until the nanobar edges are sharp under the lipid channel.
3. Set the scanning parameters as below to obtain a control image of the lipid channel before adding protein: Frame Size = 512 px x 512 px (512 pixels x 512 pixels, a pixel size of 124 nm), Bits per Pixel = 16 (16-bit depth), Scan speed = 5, and Averaging = 4 (averaging mode of four times/line).
4. Conduct the FRAP assay by bleaching the fluorescent-labeled lipid bilayer on a random single nanobar area.
   1. Select the Experiment Regions and Bleaching checkboxes. Draw a circular area of 5 µm diameter which can include the whole nanobar at the center (nanobar size is 2 µm) and add to the Experiment Regions. Input time-lapse imaging parameters as the following example: choose Scan speed = 9 and Averaging = 1. Choose Time Series > Duration = 100 cycles and Interval = 2 s. Input bleaching parameters as the following example: select the Start after # images checkbox and choose three images.
   2. Select the laser checkboxes that perform FRAP and change the power to 100.0%. Click Start Experiment for the FRAP experiment.
      NOTE: FRAP is a common method to characterize the mobility of cellular molecules. If the SLB has good fluidity, the fluorescence in the bleached area will gradually recover as bleached fluorophores diffuse out and unbleached fluorophores from other areas diffuse in.
5. Load the protein solution into the PDMS chamber and incubate for 5 min at room temperature to allow the binding of the protein on the SLB.
   NOTE: The proteins used in Figure 2G,H to demonstrate lipid composition facilitate protein binding on nanobar-curved SLBs are 74 µM GFP and 79 µM GFP-His. These two proteins are green fluorescence proteins without or with His-tag. The proteins used for curvature sensing study in Figure 4 and Figure 5 are 16 µM F-BAR, 16 µM IDR^FBP17, and 16 µM FBAR+IDR^FBP17. These three proteins are the domains from FBP17, which is a typical BAR protein that is intuitively assumed as both curvature generators and sensors. Each protein volume is 20 µL.
6. Re-focus the nanobars and repeat step 4.3 to take images of both lipid and protein channels for protein curvature sensing detection.
7. Repeat step 4.4 to conduct the FRAP assay on both lipid and protein channels and perform time-lapse imaging to characterize the mobility of curvature sensing protein.

5. Reuse of nanochip

1. Grab the nanochip with tweezers and carefully detach it from the PDMS chamber in a 50 mL beaker filled with deionized water.
   NOTE: This is a critical step. Prevent the tweezers from touching the nanostructure, and do not force the chip to separate to avoid cracks.
2. Wash the nanochip thoroughly with anhydrous ethanol to remove organic residues attached on the surface.
   NOTE: Use clean tissue paper to remove water on the surface before washing with anhydrous ethanol.
   CAUTION: Anhydrous ethanol is a highly volatile and slightly hazardous chemical. Avoid contact with the skin and eyes. Use it in the fume hood with the mask and gloves on.
3. Wash the chip thoroughly with plenty of deionized water to remove residual ethanol. Then blow-dry the chip with 99.9% nitrogen gas.
   NOTE: It is important to remove all traces of ethanol on the chip before proceeding to the next step because the piranha acid can react violently with organic solvent and may cause explosions.
4. Place the chip in a clean 10 mL beaker with the pattern side facing up and clean the chip with piranha solution for reuse which follows the same steps as 'cleaning of nanochip'.

6. Image quantification

1. Rotate the images acquired by a microscope so that the nanobar array is in a vertical position for subsequent analysis in Fiji software.
2. Use a custom-written MATLAB code 'c_pillarmask_averaging' to locate the individual nanobar by a square mask (51 pixels x 51 pixels) both in the lipid channel and protein channel.
   NOTE: This MATLAB code is specially designed for the nanochip. It can be obtained on GitHub via the link: https://github.com/wtzhaolab/GNB_SLB_MX.
3. Subtract the background signals of each image with the three ROIs (5 pixels x 5 pixels) at the corners of the image by the meshgrid function in MATLAB code 'BarGra_avg'. This operation aims to correct potential uneven background noises caused by the microscope setup or chip leveling on the microscope stage.
4. Generate the average images of the same-sized nanobars using MATLAB code 'BarGra_avg', where each point is the mean of the values at that point in all the individual nanobar square masks. Generate 3D surface plots of the average images in Fiji software to show the average signal distribution around the nanobars intuitively. Choose Analyze > Surface Plot > Input 100 for Polygon Multiplier, and select Shade, Draw Axis, and Smooth checkboxes.
5. Segment each nanobar into three areas, which include two nanobar-end areas and one nanobar-center area to extract their fluorescence intensities respectively by MATLAB code 'avg_nanobar_quantification'. The sizes of the nanobar ROIs are adjusted according to the dimension of the nanobars using the 'position' file.
6. Divide the protein intensities by lipid intensities extracted from the MATLAB code 'avg_nanobar_quantification' at the bar-end area to get the nanobar-end density which excludes the surface area effect.
7. Plot the nanobar-end density with different concentrations in GraphPad Prism to get the binding curve. Open the Analyze menu. Choose Nonlinear regression (curve fit) > Binding - Saturation > Specific binding with Hill slope function to fit the curve with the Hill equation and then calculate the KD and Hill coefficient value.
8. The lipid and protein intensities are normalized to their corresponding intensities at the 600 nm nanobars center acquired in the same image using the MATLAB code 'avg_nanobar_quantification'.
9. Use the brightest nine pixels of normalized protein intensities at the nanobar-end area divided by the bar-center area to quantify the protein curvature sensing with same-sized nanobars, the value is named as "end-to-center ratio". Use the ratio of normalized protein intensity to normalize lipid intensity to quantify the protein curvature sensing range with different-sized nanobars, the value is named as "Normalized Nanobar-End Density". These values are calculated by the MATLAB code 'avg_nanobar_quantification'.
10. Load the FRAP stack image in Fiji software. Choose the FRAP area and generate an ROI. Choose the More > Multi measure function to measure the intensity of the FRAP area for each time. Plot the intensity in GraphPad Prism to generate the FRAP recovery curve.

Results

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 ...

Discussion

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 nanobars^8,29. It also offers a wide range of precisely controlled curvature for simultaneous comparison as th...

Materials

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 μm	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