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Aug 2021

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Patterned Substrate of Mobile and Immobile Ligands to Probe EphA2 Receptor Clustering
用于探测 EphA2 受体聚类的移动和固定配体的图案化底物   

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Abstract

A multitude of membrane-localized receptors are utilized by cells to integrate both biochemical and physical signals from their microenvironment. The clustering of membrane receptors is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we describe a method to fabricate multicomponent, ligand-functionalized microarrays, for spatially segregated and simultaneous monitoring of receptor activation and signaling in individual living cells. While existing micropatterning techniques allow for the display of fixed ligands, this protocol uniquely allows for functionalization of both mobile membrane corrals and immobile polymers with selective ligands, as well as microscopic monitoring of cognate receptor activation at the cell membrane interface. This protocol has been developed to study the effects of clustering on EphA2 signaling transduction. It is potentially applicable to multiple cell signaling systems, or microbe/host interactions.


Graphical abstract:



A side-by-side comparison of clustered or non-clustered EphA2 receptor signaling in a single cell.


Keywords: Supported lipid bilayer (支持的脂质双层), Micropatterning (微图案), EphA2 receptor (EphA2受体), Receptor clustering (受体聚类), Signaling transduction (信号转导)

Background

Cells engage membrane-localized receptors to sense various signals present in their local environment, and respond to these signals appropriately to survive, organize, and proliferate. The signals presented to the cells include both soluble ligands, as well as ligands that are present in the extracellular matrix (ECM), or displayed on the membranes of other cells (also referred to as juxtacrine signaling systems). Membrane receptor interactions of the latter types of ligands enable sensing of the spatial organization of receptor-ligand complexes at the cell-cell interface, and ECM rigidity (Groves and Kuriyan, 2010; Manz and Groves, 2010). A number of these signaling systems have been reconstituted on synthetic supported lipid bilayers in a hybrid format, wherein a live cell interacts with the supported lipid bilayer, recapitulating many of the features of individual receptor types (Biswas and Groves, 2019). These include both reconstitution of the individual receptor signaling system, or a combination of two different receptor signaling systems, to recapitulate the cellular exposure to multiple signals simultaneously, and ensuing signaling crosstalk between the receptors (Chen et al., 2018).


Assembly of cell surface receptors into clusters or organized arrays is a common feature of cell membranes, and has long been implicated as an important factor for modulating signaling activity. However, it is not straightforward to deconvolve the contribution of receptor clustering on signaling itself. A major reason for this is that chemical perturbation of assemblies, such as those achieved with pharmacological agents or mutations (Davis et al., 1994; Seiradake et al., 2013; Bugaj et al., 2013, 2015; Schaupp et al., 2014; Wu et al., 2015; Su et al., 2016), are likely to produce side effects on the cell, other than modulating molecular assembly. Therefore, we seek to modulate spatial organization of receptors by controlling ligand mobility, instead of perturbing intracellular components. In the current protocol, we developed a technique wherein a ligand of interest is displayed in both mobile and immobile configurations, and spatially juxtaposed on length scales small enough to enable a side-by-side comparison within an individual living cell. The immobile ligands are displayed on a functionalized poly L-Lysine-poly ethylene glycol [PLL-(g)-PEG] scaffold, while the mobile ligands are displayed on supported lipid membranes, which allow cluster formation. This method has been applied to the study of EphA2 receptor signaling (Chen et al., 2021), and is potentially applicable to other cell signaling systems or microbe/host interactions (Wong et al., 2021).

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 FluorTM 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, AttofluorTM 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 FluorTM 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.

References

  1. Biswas, K. H. and Groves, J. T. (2019). Hybrid Live Cell-Supported Membrane Interfaces for Signaling Studies. Annu Rev Biophys 48: 537-562.
  2. Bugaj, L. J., Choksi, A. T., Mesuda, C. K., Kane, R. S. and Schaffer, D. V. (2013). Optogenetic protein clustering and signaling activation in mammalian cells. Nat Methods 10(3): 249-252.
  3. Bugaj, L. J., Spelke, D. P., Mesuda, C. K., Varedi, M., Kane, R. S. and Schaffer, D. V. (2015). Regulation of endogenous transmembrane receptors through optogenetic Cry2 clustering. Nat Commun 6: 6898.
  4. Chen, Z., Oh, D., Biswas, K. H., Yu, C. H., Zaidel-Bar, R. and Groves, J. T. (2018). Spatially modulated ephrinA1:EphA2 signaling increases local contractility and global focal adhesion dynamics to promote cell motility. Proc Natl Acad Sci U S A 115(25): E5696-E5705.
  5. Chen, Z., Oh, D., Biswas, K. H., Zaidel-Bar, R. and Groves, J. T. (2021). Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility. Elife 10: e67379.
  6. Davis, S., Gale, N. W., Aldrich, T. H., Maisonpierre, P. C., Lhotak, V., Pawson, T., Goldfarb, M. and Yancopoulos, G. D. (1994). Ligands for EPH-related receptor tyrosine kinases that require membrane attachment or clustering for activity. Science 266(5186): 816-819.
  7. Groves, J. T. and Kuriyan, J. (2010). Molecular mechanisms in signal transduction at the membrane. Nat Struct Mol Biol 17(6): 659-665.
  8. Lin, W. C., Yu, C. H., Triffo, S. and Groves, J. T. (2010). Supported membrane formation, characterization, functionalization, and patterning for application in biological science and technology. Curr Protoc Chem Biol 2(4): 235-269.
  9. Manz, B. N. and Groves, J. T. (2010). Spatial organization and signal transduction at intercellular junctions. Nat Rev Mol Cell Biol 11(5): 342-352.
  10. Schaupp, A., Sabet, O., Dudanova, I., Ponserre, M., Bastiaens, P. and Klein, R. (2014). The composition of EphB2 clusters determines the strength in the cellular repulsion response. J Cell Biol 204(3): 409-422.
  11. Seiradake, E., Schaupp, A., del Toro Ruiz, D., Kaufmann, R., Mitakidis, N., Harlos, K., Aricescu, A. R., Klein, R. and Jones, E. Y. (2013). Structurally encoded intraclass differences in EphA clusters drive distinct cell responses. Nat Struct Mol Biol 20(8): 958-964.
  12. Su, X., Ditlev, J. A., Hui, E., Xing, W., Banjade, S., Okrut, J., King, D. S., Taunton, J., Rosen, M. K. and Vale, R. D. (2016). Phase separation of signaling molecules promotes T cell receptor signal transduction. Science 352(6285): 595-599.
  13. Wong, J. J., Chen, Z., Chung, J. K., Groves, J. T. and Jardetzky, T. S. (2021). EphrinB2 clustering by Nipah virus G is required to activate and trap F intermediates at supported lipid bilayer-cell interfaces. Sci Adv 7(5): eabe1235.
  14. Wu, Y., Kanchanawong, P. and Zaidel-Bar, R. (2015). Actin-delimited adhesion-independent clustering of E-cadherin forms the nanoscale building blocks of adherens junctions. Dev Cell 32(2): 139-154.

简介

[摘要] 细胞利用多种膜定位受体来整合来自其微环境的生化和物理信号。膜受体的聚集被广泛认为对随后的信号转导具有功能性后果。然而,在不改变细胞系统的其他生化方面的情况下选择性地操纵受体聚类在实验上具有挑战性。在这里,我们描述了一种制造多组分、配体功能化微阵列的方法,用于空间隔离和同时监测单个活细胞中的受体激活和信号传导。虽然现有的微图案技术允许显示固定配体,但该协议独特地允许使用选择性配体对移动膜围栏和固定聚合物进行功能化,以及在细胞膜界面上对同源受体激活进行微观监测。该协议旨在研究集群对 EphA2 信号转导的影响。它可能适用于多细胞信号系统或微生物/宿主相互作用。

图形概要:

的并排比较。


[背景] 细胞与膜定位受体结合以感知其局部环境中存在的各种信号,并对这些信号做出适当的反应以生存、组织和增殖。呈现给细胞的信号包括可溶性配体,以及存在于细胞外基质 (ECM) 中或显示在其他细胞膜上的配体(也称为juxtacrine信号系统)。后一类配体的膜受体相互作用能够感知细胞-细胞界面处受体-配体复合物的空间组织以及 ECM 刚性(Groves 和 Kuriyan,2010;Manz 和 Groves,2010) 。许多这些信号系统已在合成支持的脂质双层上以混合形式重建,其中活细胞与支持的脂质双层相互作用,概括了单个受体类型的许多特征(Biswas 和 Groves,2019) 。这些包括重建单个受体信号系统,或两种不同受体信号系统的组合,以概括细胞同时暴露于多个信号,以及随后的受体之间的信号串扰(Chen等,2018) 。
细胞表面受体组装成簇或有组织的阵列是细胞膜的共同特征,长期以来一直被认为是调节信号活性的重要因素。然而,去卷积受体聚集对信号本身的贡献并不简单。造成这种情况的一个主要原因是组件的化学扰动,例如通过药物或突变实现的那些(Davis et al ., 1994; Seiradake et al ., 2013; Bugaj et al ., 2013, 2015; Schaupp et al ., 2014;吴等人,2015;苏等人,2016) , 可能对细胞产生副作用,而不是调节分子组装。因此,我们寻求通过控制配体迁移率来调节受体的空间组织,而不是扰乱细胞内成分。在当前协议中,我们开发了一种技术,其中感兴趣的配体以移动和固定配置显示,并且在空间上并列在足够小的长度尺度上,以便在单个活细胞内进行并排比较。固定配体展示在功能化的聚 L-赖氨酸-聚乙二醇 [PLL-(g)-PEG] 支架上,而可移动配体展示在支持的脂质膜上,从而形成簇。该方法已应用于 EphA2 受体信号传导的研究(Chen et al ., 2021) ,并可能适用于其他细胞信号传导系统或微生物/宿主相互作用(Wong et al ., 2021) 。

关键字:支持的脂质双层, 微图案, EphA2受体, 受体聚类, 信号转导



材料和试剂


1.圆底烧瓶(25 mL)
2.玻璃盖玻片(Thorlabs,目录号:CG15XH)
3.PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-生物素(50%) ( Susos , https://susos.com/shop/pll20-g3-5-peg2peg3-4-biotin50- 2/ )
4.PLL(20)-g[3.5]-PEG(3.4)-NTA(苏索斯, 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,目录号:850375)
7.18:1 DGS-NTA(Ni)(Avanti,目录号:790404)
8.NeutrAvidin ( ThermoFisher ,目录号:22831)
9.RGD-生物素( Vivitide ,目录号:PCI-3697-PI)
10.Tris(ThermoFisher,目录号:15504020)
11.氯化钠
12.氯化钾
13.氯化钙2
14.氯化镁2
15.D-葡萄糖
16.EphrinA1 蛋白(在 Groves 实验室纯化,可根据要求提供质粒)
17.Alexa Fluor TM 680 染料( ThermoFisher ,目录号:A37574)
18.铬石英光掩模
我们使用在新加坡国立大学机械生物学研究所核心设施制造的 5 英寸光掩模。光掩模也可以从其他制造公司购买。
19.TBS 缓冲液(见配方)
20.成像缓冲液(见配方)


设备


1.成像室( ThermoFisher , Attofluor TM 细胞成像室,目录号:A7816)
2.水浴
3.UVO 清洁剂( Jelight Company INC, hbUVO清洁剂,342 型)
4.旋转蒸发器(带有标准干冰冷凝器,并配备真空泵, https : //www.asynt.com/product/ika-dry-ice-rotary-evaporator-range/或https://www. coleparmer.co.uk/i/buchi-23012c000-rotary-evaporator-dry-ice-condenser-220v/2301220 )
5.提示 sonicator ( https://www.sonicator.com/collections/sonicators/products/q125-sonicator )


程序


该技术需要制备 (A) 小单层囊泡 (SUV)、(B) 玻璃基板上的微图案聚合物表面和 (C) 在微图案基板上组装膜阵列和蛋白质功能化 (图 1)。 SUV 可以提前准备好并在 4 ° C下储存长达 2 周。步骤 (B) 和 (C) 需要 2 - 3 天,具体取决于灵活的孵育时间。


简而言之,首先将 PLL-(g)-PEG 支架聚合物涂覆在玻璃盖玻片上,然后使用光掩模和脂质囊泡沉积进行选择性深紫外蚀刻,以生成固定聚合物区域和可移动脂质膜。然后将 ephrinA1 配体功能化为可移动和固定配置的底物,以探测 EphA2 受体在细胞中的聚集。




图 1. 制造移动和固定配体的微图案基板的程序。


A.囊泡制备
对于这一步,观众可以参考更详细的协议(Lin et al ., 2009) 。
1.制备脂质膜,通过混合所需量的各种脂质分子 [96% DOPC + 4% DGS-NTA(Ni),总质量为 2 mg],将其溶解在圆底烧瓶中的氯仿中,然后蒸发在真空泵和 50 °C水浴下使用旋转蒸发器使用氯仿,导致在烧瓶中形成薄脂质膜。在此过程中,将圆底烧瓶慢慢降低到水浴中,以避免氯仿沸腾。
2.通过移液将脂质膜重新悬浮在 2 mL 的去离子水中,从而形成大型且通常为多层的囊泡(1 mg/mL)。
3.使用探头尖端超声仪对冰中的囊泡悬浮液进行超声处理,以生成 SUV。通常,使用“开 10秒,关 5 秒”的程序进行 8-10次超声处理。将 SUV 悬架转移到新管中,并在 20,000 × g和 4 °C 下离心2 小时,以去除任何碎屑。将上清液转移到新管中,并储存在 4 °C直至进一步使用。 SUV 可以储存长达 2 周,以形成良好的脂质双层。


B.微细加工
使用深紫外 (UV) 蚀刻方法在玻璃基板上制备微图案表面。
1.通过在异丙醇和水的 1:1 混合物中超声清洗玻璃盖玻片 15 – 30 分钟,然后在 50% H 2 SO 4溶液中过夜(或更长时间)孵育。使用前,将盖玻片从 H 2 SO 4中取出,用水冲洗,然后在封闭的 UVO 清洁器中将玻璃盖玻片暴露在紫外线下 10 分钟。用水冲洗,用N 2喷射干燥。
2.用 PLL(20)-g[3.5]-PEG(2)/PEG(3.4)-生物素(50%) 在室温下以 0.1 毫克/毫升的浓度孵育玻璃盖玻片 2 小时或过夜。为了减少试剂的使用,将约 30 μL 的孵育溶液滴入封口膜中,并将玻璃盖玻片放在上面。
3.用水冲洗基材以去除多余的聚合物,并通过 N 2喷射干燥。
4.的微图案区域(1 厘米× 1 平方厘米)上滴 ±1.5 μL水,并将聚合物涂层玻璃基板放在其顶部,以确保紧密接触。在 UVO 清洁器中将基材暴露在深紫外光下 9-12分钟。暴露在紫外光下的聚合物会降解。用过量的水冲洗基材以去除任何降解的聚合物。
5.用 PLL(20)-g[3.5]-PEG(3.4)-NTA 在室温下以 0.1 毫克/毫升的浓度孵育相同的玻璃基板 2 小时,并重复深紫外蚀刻和洗涤程序。第二次蚀刻后,玻璃基板应立即用于脂质双层沉积。


C.双层沉积和蛋白质功能化
1.使用前,将 SUV 与 TBS 缓冲液以 1:1 的比例混合。
2.用 SUV 溶液孵育微图案基板 5 分钟,以允许在上一步中被紫外线蚀刻的基板区域中的脂质双层自组装。
3.用多余的 TBS 缓冲液冲洗基板,并将玻璃盖玻片组装到水环境下的细胞培养成像室中。
4.在室温下用牛血清白蛋白 (BSA) 溶液 (0.05 – 0.1 mg/mL 在 TBS 缓冲液中)封闭基板2 小时, 或在 4 °C过夜。
5.用 TBS 缓冲液清洗基板,并用NeutrAvidin (1.5 µg/mL) 孵育 15 分钟,以结合表面生物素基团。
6.用 TBS 缓冲液清洗多余的NeutrAvidin ,并与 ephrinA1-His 10(纯化的 ephrinA1 蛋白,C 末端有 10 个组氨酸尾,用Alexa Fluor TM 680标记)和 RGD-生物素孵育 60 – 90 分钟. ephrinA1-His 10 将与移动区域上的 DGS-NTA(Ni) 脂质和固定区域上的 PLL(20)-g[3.5]-PEG(3.4)-NTA 聚合物结合,而 RGD-生物素将与NeutrAvidin结合. RGD 肽可以结合细胞膜上的整合素,以允许细胞扩散,在此期间细胞与多个 ephrinA1 功能化的移动或固定区域动态相互作用。
7.通过用成像缓冲液洗涤去除未结合的蛋白质。
8.在显微镜下检查移动和固定 ephrinA1 的荧光强度。
9.底物已准备好用于活细胞实验。在成像缓冲液中准备细胞,并在 37 °C 下将低密度的细胞播种到基板室中以进行实时成像。细胞 EphA2 受体只有在与移动的 ephrinA1 结合后才会形成簇(图 2) 。




图 2. 细胞在微图案基板上扩散的图像。
左图:RIC(反射干涉对比)图像,显示粘附在基板上的细胞表面;右图:微图案 ephrinA1 的荧光图像,黄线标记了细胞的轮廓。


数据分析


在标准荧光显微镜下观察底物以比较移动和固定ephrinA1区域的荧光强度。通过光漂白后的荧光恢复(FRAP)检查 ephrinA1 在脂质双层上的流动性。


笔记


1.移动和固定区域在两个独立的 UV 蚀刻步骤中随机排列。通常,这两个区域在某些区域是重叠的,但在厘米大小的基板中很容易找到不重叠的区域。
2.移动和固定 ephrinA1 强度可能不同。用非反应性 PLL(20)-g[3.5]-PEG(2)滴定PLL(20)-g[3.5]-PEG(3.4)-NTA,以改变固定区域的表面 ephrinA1 强度,或改变脂质摩尔DOPC 和 DGS-NTA (Ni) 的比率,以修改移动区域的 ephrinA1 强度。


食谱


1.TBS 缓冲液
25 毫米三
150 毫米氯化钠
3 毫米氯化钾
2.成像缓冲器
25 毫米三
140 毫米氯化钠
3 毫米氯化钾
2 毫米氯化钙2
1 毫米氯化镁2
5.5 毫米 D-葡萄糖


致谢


这项工作得到了美国国立卫生研究院、国家癌症研究所物理科学研究肿瘤网络项目 1-U01CA202241 和上海浦江计划 (20PJ1400800) 的支持。该协议源自已发表的论文(Chen et al ., 2018, 2021) 。


利益争夺


作者声明没有利益冲突或竞争利益。


参考


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Copyright Chen et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Chen, Z., Biswas, K. H. and Groves, J. T. (2022). Patterned Substrate of Mobile and Immobile Ligands to Probe EphA2 Receptor Clustering. Bio-protocol 12(11): e4434. DOI: 10.21769/BioProtoc.4434.
  2. Chen, Z., Oh, D., Biswas, K. H., Zaidel-Bar, R. and Groves, J. T. (2021). Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility. Elife 10: e67379.
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