Patterned Substrate of Mobile and Immobile Ligands to Probe EphA2 Receptor Clustering

Materials and Reagents

1. Round bottom flask (25 mL)

2. Glass coverslips (Thorlabs, catalog number: CG15XH)

3. PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-biotin(50%) (Susos, https://susos.com/shop/pll20-g3-5-peg2peg3-4-biotin50-2/)

4. PLL(20)-g[3.5]-PEG(3.4)-NTA (Susos, https://susos.com/shop/pll20-g3-5-peg3-4-nta-2/)

5. PLL(20)-g[3.5]-PEG(2) (Susos, https://susos.com/shop/pll20-g3-5-peg2/)

6. 18:1 (Δ9-Cis) PC (DOPC) (Avanti, catalog number: 850375)

7. 18:1 DGS-NTA(Ni) (Avanti, catalog number:790404)

8. NeutrAvidin (ThermoFisher, catalog number:22831)

9. RGD-biotin (Vivitide, catalog number: PCI-3697-PI)

10. Tris (ThermoFisher, catalog number: 15504020)

11. NaCl

12. KCl

13. CaCl₂

14. MgCl₂

15. D-glucose

16. EphrinA1 protein (purified in the Groves’ lab, plasmid available upon request)

17. Alexa Fluor^TM 680 dye (ThermoFisher, catalog number: A37574)

18. Chromium-quartz photomask

    We use a 5-inch photomask that was fabricated in the Mechanobiology Institute Core facility, at the National University of Singapore. The photomask can also be purchased from other manufacturing companies.

19. TBS buffer (see Recipes)

20. Imaging buffer (see Recipes)

Equipment

1. Imaging chamber (ThermoFisher, Attofluor^TM cell imaging chamber, catalog number: A7816)

2. Water bath

3. UVO cleaner (Jelight Company INC, hbUVO Cleaner, model 342)

4. Rotary evaporator (with a standard dry ice condenser, and equipped with a vacuum pump, https://www.asynt.com/product/ika-dry-ice-rotary-evaporator-range/, or https://www.coleparmer.co.uk/i/buchi-23012c000-rotary-evaporator-dry-ice-condenser-220v/2301220)

5. Tip sonicator (https://www.sonicator.com/collections/sonicators/products/q125-sonicator)

Procedure

This technique requires preparation of (A) small unilamellar vesicles (SUVs), (B) micropatterned polymer surface on a glass substrate, and (C) assembly of membrane arrays on the micropatterned substrate and protein functionalization (Figure 1). The SUVs can be prepared in advance and stored at 4°C for up to 2 weeks. Steps (B) and (C) take 2–3 days, depending on the flexible incubation times.

Briefly, PLL-(g)-PEG scaffold polymers are first coated on a glass coverslip, followed by selective deep UV etching with a photomask and lipid vesicle deposition, to generate regions of immobile polymers and mobile lipid membranes. The ephrinA1 ligands are then functionalized to the substrate in mobile and immobile configurations, to probe EphA2 receptor clustering in cells.

Figure 1. Procedures to Fabricate Micropatterned Substrate of Mobile and Immobile Ligands.

1. Vesicle preparation

   For this step, the audiences can refer to a more detailed protocol (Lin et al., 2009).

   1. Prepare lipid films, by mixing required amounts of various lipid molecules [96% DOPC + 4% DGS-NTA(Ni), with a total mass of 2 mg], which are dissolved in chloroform in a round bottom flask, followed by evaporation of chloroform using a rotary evaporator under vacuum pumping and 50°C water bath, leading to the formation of a thin lipid film in the flask. During this procedure, slowly lower the round bottom flask to the water bath, to avoid chloroform boiling.

   2. Resuspend the lipid film in 2 mL of deionized water by pipetting, allowing the formation of large and often multilamellar vesicles (1 mg/mL).

   3. Sonicate the vesicle suspensions in ice using a probe tip sonicator, to generate SUVs. Typically, use a program of '10 s on, and 5 s off’ for 8–10 cycles for sonication. Transfer the SUV suspension to a fresh tube, and centrifuge at 20,000 × g and 4°C for 2 h, to remove any debris. Transfer the supernatant to a fresh tube, and store at 4°C until further use. The SUVs can be stored for up to 2 weeks, to form a good lipid bilayer.




2. Microfabrication

   Micropatterned surfaces are prepared on glass substrates, using the deep ultraviolet (UV) etching method.

   1. Clean glass coverslips by sonication in a 1:1 mixture of isopropanol and water for 15–30 min, followed by overnight (or longer) incubation in 50% H₂SO₄ solution. Before usage, take the coverslips out of H₂SO₄, rinse with water, and expose the glass coverslips to UV light in an enclosed UVO cleaner for 10 min. Rinse with water, and dry by N₂ jet.

   2. Incubate the glass coverslips with PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-biotin(50%) at a concentration of 0.1 mg/mL at room temperature for 2 h, or overnight. To reduce the usage of reagents, drop ~30 μL of the incubating solution in parafilm, and lay the glass coverslips on top of it.

   3. Rinse the substrates with water to remove excess polymer, and dry by N₂ jet.

   4. Drop ~1.5 μL of water on the micropatterned area (1 cm × 1 cm square) of a chromium-quartz photomask, and lay the polymer-coated glass substrate on its top, to ensure close contact. Expose the substrates to deep UV light for 9–12 min in a UVO cleaner. The UV light-exposed polymer will be degraded. Rinse the substrates with excess water to remove any degraded polymer.

   5. Incubate the same glass substrates with PLL(20)-g[3.5]-PEG(3.4)-NTA at a concentration of 0.1 mg/mL at room temperature for 2 h, and repeat the deep UV etching and washing procedures. After the second etching, the glass substrates should be used for lipid bilayer deposition immediately.




3. Bilayer deposition and protein functionalization

   1. Before usage, mix SUVs with TBS buffer in 1:1 ratio.

   2. Incubate the micropatterned substrates with SUV solution for 5 min, to allow the self-assembly of lipid bilayers in regions of the substrates that were UV etched in the previous step.

   3. Rinse the substrates with excess TBS buffer, and assemble the glass coverslips into a cell culture imaging chamber, under the aqueous environment.

   4. Block the substrates with bovine serum albumin (BSA) solution (0.05–0.1 mg/mL in TBS buffer) at room temperature for 2h, or at 4°C overnight.

   5. Wash the substrates with TBS buffer, and incubate with NeutrAvidin (1.5 µg/mL) for 15 min, to bind surface biotin groups.

   6. Wash excess NeutrAvidin with TBS buffer, and incubate with ephrinA1-His 10 (purified ephrinA1 protein with a 10-histidine tail in the C-terminus, labeled with Alexa Fluor^TM 680), and RGD-biotin, for an additional 60–90 min. The ephrinA1-His 10 will bind to both DGS-NTA(Ni) lipids on mobile regions, and PLL(20)-g[3.5]-PEG(3.4)-NTA polymers on immobile regions, while RGD-biotin will bind to NeutrAvidin. The RGD peptides can bind integrins at the cell membrane, to allow cell spreading, during which cells dynamically interact with multiple ephrinA1-functionalized mobile or immobile regions.

   7. Remove unbound proteins by washing with imaging buffer.

   8. Check fluorescence intensity of mobile and immobile ephrinA1 under a microscope.

   9. The substrates are ready for use in a live cell experiment. Prepare cells in the imaging buffer, and seed a low density of cells into the substrate chamber at 37°C for live imaging. The cellular EphA2 receptors will form clusters only after binding with mobile ephrinA1 (Figure 2).

   
   

   
   Figure 2. Images of a cell spreading on the micropatterned substrate. Left: The RIC (reflective interference contrast) image showing the cell surface that is adhered to the substrate; Right: The fluorescent image of micropatterned ephrinA1, with the yellow line marking the contour of the cell.

Data analysis

The substrates were observed under a standard fluorescence microscope to compare the fluorescence intensity of mobile and immobile ephrinA1 regions. The mobility of ephrinA1 on lipid bilayers was checked by fluorescence recovery after photobleaching (FRAP).

Notes

1. The mobile and immobile regions are aligned randomly in two independent UV-etch steps. Usually, the two regions are overlapped in some of the areas, but it is easy to find non-overlapped regions in a centimeter-sized substrate.

2. The mobile and immobile ephrinA1 intensity may not be the same. Titrate PLL(20)-g[3.5]-PEG(3.4)-NTA with non-reactive PLL(20)-g[3.5]-PEG(2), to modify surface ephrinA1 intensity on immobile regions, or change the lipids molar ratio of DOPC and DGS-NTA(Ni), to modify ephrinA1 intensity on mobile regions.

Recipes

1. TBS buffer

   25 mM Tris

   150 mM NaCl

   3 mM KCl

2. Imaging buffer

   25 mM Tris

   140 mM NaCl

   3 mM KCl

   2 mM CaCl₂

   1 mM MgCl₂

   5.5 mM D-glucose

Acknowledgments

This work was supported by the National Institutes of Health, National Cancer Institute Physical Sciences in Oncology Network Project 1-U01CA202241, and Shanghai Pujiang Program (20PJ1400800). This protocol is derived from published papers (Chen et al., 2018, 2021).

Competing interests

The authors declare no conflicts of interest or competing interests.

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