JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Tadução automática
Please note that all translations are automatically generated. Click here for the English version.
Bioquímica

纳米巴支持的脂质双层系统，用于体外研究膜曲率传感蛋白

Published: November 30, 2022
doi:
10.3791/64340
, ,
1School of Chemistry, Chemical Engineering, and Biotechnology,Nanyang Technological University, 2State Key Laboratory Breeding Base of Green Chemistry Synthesis Technology, College of Chemical Engineering,Zhejiang University of Technology, 3Institute for Digital Molecular Analytics and Science,Nanyang Technological University

Summary

在这里，开发了一种纳米巴支持的脂质双层系统，以提供具有定义曲率的合成膜，从而能够在 体外表征具有曲率传感能力的蛋白质。

Abstract

膜曲率在细胞的各种基本过程中起着重要作用，例如细胞迁移、细胞分裂和囊泡运输。它不仅由细胞活动被动产生，而且还主动调节蛋白质相互作用并参与许多细胞内信号传导。因此，研究膜曲率在调节蛋白质和脂质的分布和动力学中的作用具有重要价值。最近，已经开发了许多技术来研究体 外弯曲膜和蛋白质之间的关系。与传统技术相比，新开发的纳米巴支持的脂质双层（SLB）通过在具有预定义膜曲率和局部平坦控制的纳米条图案阵列上形成连续的脂质双层，在膜曲率生成方面提供了高通量和更高的精度。脂质流动性和蛋白质对弯曲膜的敏感性都可以使用荧光显微镜成像进行定量表征。本文介绍了如何在含有纳米棒阵列的人造玻璃表面上形成SLB的详细程序，以及如何在这种SLB上表征曲率敏感蛋白。此外，还涵盖了纳米芯片重用和图像处理的协议。除了纳米巴-SLB，该协议还适用于用于曲率传感研究的所有类型的纳米结构玻璃芯片。

Introduction

膜曲率是细胞的关键物理参数，发生在各种细胞过程中，例如形态发生、细胞分裂和细胞迁移1.现在人们普遍认为，膜曲率不仅仅是细胞事件的简单结果;相反，它已成为蛋白质相互作用和细胞内信号传导的有效调节剂。例如，发现参与网格蛋白介导的内吞作用的几种蛋白质优先与弯曲膜结合，导致内吞作用2的热点形成。膜变形有许多不同的原因，例如细胞骨架力拉动膜，存在不同大小头基的脂质不对称性，存在锥形跨膜蛋白，膜成形蛋白如BAR结构域蛋白（以Bin，两栖蛋白和Rvs蛋白命名）的积累，以及两亲性螺旋结构域插入膜1.有趣的是，这些蛋白质和脂质不仅使膜变形，而且还可以感知膜曲率并表现出优先积累1。因此，研究具有不同曲率的膜是否以及如何改变附着在其上的蛋白质和脂质的分布和动力学以及对相关细胞内过程的潜在影响至关重要。

已经开发了许多技术来分析活细胞和体外系统中弯曲膜和蛋白质之间的相互作用。活细胞系统提供了一个真实的细胞环境，具有丰富的脂质多样性和动态蛋白质信号传导调节2，3，4，5，6，7。然而，由于细胞内环境的不确定性和波动性，这种复杂的系统很难研究。因此，使用由已知脂质种类和纯化蛋白质组成的人造膜的体外测定已成为表征蛋白质和弯曲膜之间关系的强大重构系统。传统上，通过挤出产生不同直径的脂质体，以通过使用离心力的共沉降测定或具有密度梯度的共浮选测定来检测曲率敏感的蛋白质，以避免蛋白质聚集8，9。然而，挤出脂质体的曲率受到挤出机10中使用的膜过滤器的可用孔径的限制。单脂质体曲率（SLiC）测定已被证明可以克服这一限制，其中不同直径的脂质体被荧光标记并固定在表面上，以便曲率可以用荧光强度11标记。然而，在小囊泡中观察到脂质组成的强烈变化，这影响了曲率测量的准确性12。系绳拉动实验使用光学镊子更准确地测量从巨型单层囊泡 （GUV） 拉出的瞬态系绳上的曲率，其中曲率可以通过产生的膜张力很好地控制13，14。该方法适用于研究正曲率或负曲率传感蛋白，但受到管生成10的通量的限制。支持的膜管 （SMrT） 测定可同时生成多个膜管，这些膜管通过微流体流从同一脂质储层挤出。然而，膜曲率沿纳米管固有地变化，这损害了基于荧光强度的曲率测量的准确性15，16。相比之下，使用小的单层囊泡（SUV，直径<100nm17）在含有设计形貌的表面上形成支持的脂质双层（SLB），产生单个双层膜，其曲率由纳米制造或纳米材料预先确定，精度为18，19，20。

在这里，我们提出了一种在具有纳米棒阵列的制造纳米芯片表面上形成SLB的协议，以及如何使用它来探测 体外蛋白质的曲率敏感性。如图 1所示，该测定有六个基本组成部分：A）用微流体室清洁和组装芯片;B）制备具有确定脂质组成的SUV;C）在纳米芯片上形成SLB并与曲率敏感蛋白结合;D）在荧光显微镜下对SLB和曲率敏感蛋白进行成像和表征;e） 清洁芯片以便重复使用;F）用于定量分析蛋白质曲率传感能力的图像处理。下面将逐步介绍详细的协议。

Protocol

1. 纳米芯片的清洗 将纳米芯片放入 10 mL 烧杯中，图案面朝上。注意：该石英纳米芯片已通过电子束光刻 技术制造 ，如前所述 21.芯片上纳米结构的几何形状和排列可以定制设计。这里使用的梯度纳米棒的尺寸为长度为2000 nm，高度为600 nm，宽度为100至1000 nm（步长为100 nm）。 小心地向烧杯中加入 1 mL 98% 硫酸，并确保酸完全覆盖芯片的正面和?…

Representative Results

建议使用纳米棒设计来探测正曲率传感蛋白，其两端包含一个半圆，曲率由纳米条宽度定义，中心局部有一个平坦/零曲率控制（图 2A，B）。在纳米棒上成功形成SLB会导致脂质标记信号均匀分布在整个纳米棒表面上，如图 2C所示。来自多个纳米棒的信号可以通过平均单个纳米棒图像来组合（图2D），以便可以最小化不?…

Discussion

这里描述的纳米棒-SLB系统提供了几种现有体外测定中优势的独特组合。作为脂质体漂浮或沉降测定，它有效地揭示了蛋白质与高度弯曲的膜的优先结合，但需要的样品要少得多，并且在单个纳米棒8，29上提供更精确定义的曲率。它还提供广泛的精确控制曲率，可与SLiC测定同时进行比较，较少关注不同曲率下的脂质组成变化，以及随着尺寸低于光…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢南洋理工大学（NTU）南洋纳米制造中心（N2FC）和颠覆性光子技术中心（CDPT）支持纳米结构制造和SEM成像，感谢南洋理工大学生物科学学院蛋白质生产平台（PPP）用于蛋白质纯化，感谢南洋理工大学化学与生物医学工程学院的共聚焦显微镜。这项工作由新加坡教育部（W. Zhao，RG112/20，RG95/21和MOE-T2EP30220-0009），数字分子分析与科学研究所（IDMxS）资助，由教育部资助的卓越研究中心计划（W. Zhao），人类前沿科学计划基金会（W. Zhao，RGY0088 / 2021），NTU启动基金（W. Zhao）， 南洋理工大学化学与生物医学工程学院研究奖学金（苗X.）和中国国家留学基金委研究奖学金（吴杰）。

Materials

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
DOWNLOAD MATERIALS LIST

Referências

  1. McMahon, H. T., Gallop, J. L. Membrane curvature and mechanisms of dynamic cell membrane remodelling. Nature. 438 (7068), 590-596 (2005).
  2. Zhao, W. et al. Nanoscale manipulation of membrane curvature for probing endocytosis in live cells. Nature Nanotechnology. 12 (8), 750-756 (2017).
  3. Galic, M. et al. External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane. Nature Cell Biology. 14 (8), 874-881 (2012).
  4. Rosholm, K. R. et al. Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells. Nature Chemical Biology. 13 (7), 724-729 (2017).
  5. Lou, H. Y. et al. Membrane curvature underlies actin reorganization in response to nanoscale surface topography. Proceedings of the National Academy of Sciences. 116 (46), 23143-23151 (2019).
  6. Cail, R. C., Shirazinejad, C. R., Drubin, D. G. Induced nanoscale membrane curvature bypasses the essential endocytic function of clathrin. Journal of Cell Biology. 221 (7), e202109013 (2022).
  7. Mu, H. et al. Patterning of oncogenic ras clustering in live cells using vertically aligned nanostructure arrays. Nano Letter. 22 (3), 1007-1016 (2022).
  8. Peter, B. J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science. 303 (5657), 495-499 (2004).
  9. Bigay, J., Casella, J. F., Drin, G., Mesmin, B., Antonny, B. ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif. The EMBO Journal. 24 (13), 2244-2253 (2005).
  10. Ebrahimkutty, M. P., Galic, M. Receptor-free signaling at curved cellular membranes. Bioessays. 41 (10), e1900068 (2019).
  11. Bhatia, V. K. et al. Amphipathic motifs in BAR domains are essential for membrane curvature sensing. The EMBO Journal. 28 (21), 3303-3314 (2009).
  12. Larsen, J., Hatzakis, N. S., Stamou, D. Observation of inhomogeneity in the lipid composition of individual nanoscale liposomes. Journal of the American Chemical Society. 133 (28), 10685-10687 (2011).
  13. Prevost, C. et al. IRSp53 senses negative membrane curvature and phase separates along membrane tubules. Nature Communications. 6, 8529 (2015).
  14. Simunovic, M. et al. How curvature-generating proteins build scaffolds on membrane nanotubes. Proceedings of the National Academy of Sciences. 113 (40), 11226-11231 (2016).
  15. Holkar, S. S., Kamerkar, S. C., Pucadyil, T. J. Spatial control of epsin-induced clathrin assembly by membrane curvature. Journal of Biological Chemistry. 290 (23), 14267-14276 (2015).
  16. Dar, S., Kamerkar, S. C., Pucadyil, T. J. Use of the supported membrane tube assay system for real-time analysis of membrane fission reactions. Nature Protocols. 12 (2), 390-400 (2017).
  17. Nair, P. M., Salaita, K., Petit, R. S., Groves, J. T. Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling. Nature Protocols. 6 (4), 523-539 (2011).
  18. Lee, I. H., Kai, H., Carlson, L. A., Groves, J. T., Hurley, J. H. Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly. Proceedings of the National Academy of Sciences. 112 (52), 15892-15897 (2015).
  19. Beber, A. et al. Membrane reshaping by micrometric curvature sensitive septin filaments. Nature Communications. 10 (1), 420 (2019).
  20. Bridges, A. A., Jentzsch, M. S., Oakes, P. W., Occhipinti, P., Gladfelter, A. S. Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton. Journal of Cell Biology. 213 (1), 23-32 (2016).
  21. Li, X. et al. A nanostructure platform for live-cell manipulation of membrane curvature. Nature Protocols. 14 (6), 1772-1802 (2019).
  22. Su, M. et al. Comparative study of curvature sensing mediated by F-BAR and an intrinsically disordered region of FBP17. iScience. 23 (11), 101712 (2020).
  23. Mayer, L. D., Hope, M. J., Cullis, P. R. Vesicles of variable sizes produced by a rapid extrusion procedure. Biochimica et Biophysica Acta. 858 (1), 161-168 (1986).
  24. Santoro, F. et al. Revealing the cell-material interface with nanometer resolution by focused ion beam/scanning electron microscopy. ACS Nano. 11 (8), 8320-8328 (2017).
  25. Platt, V. et al. Influence of multivalent nitrilotriacetic acid lipid-ligand affinity on the circulation half-life in mice of a liposome-attached His6-protein. Bioconjugate Chemistry. 21 (5), 892-902 (2010).
  26. Williams, D., Vicogne, J., Zaitseva, I., McLaughlin, S., Pessin, J. E. Evidence that electrostatic interactions between vesicle-associated membrane protein 2 and acidic phospholipids may modulate the fusion of transport vesicles with the plasma membrane. Molecular Biology of the Cell. 20 (23), 4910-4919 (2009).
  27. El Alaoui, F. et al. Structural organization and dynamics of FCHo2 docking on membranes. Elife. 11, e73156 (2022).
  28. Seu, K. J. et al. Effect of surface treatment on diffusion and domain formation in supported lipid bilayers. Biophysical Journal. 92 (7), 2445-2450 (2007).
  29. Hung, Y. F. et al. Amino terminal region of dengue virus NS4A cytosolic domain binds to highly curved liposomes. Viruses. 7 (7), 4119-4130 (2015).
  30. Hatzakis, N. S. et al. How curved membranes recruit amphipathic helices and protein anchoring motifs. Nature Chemical Biology. 5 (11), 835-841 (2009).
  31. Johnson, J. M., Ha, T., Chu, S., Boxer, S. G. Early steps of supported bilayer formation probed by single vesicle fluorescence assays. Biophysical Journal. 83 (6), 3371-3379 (2002).
  32. Jing, Y., Trefna, H., Persson, M., Kasemo, B., Svedhem, S. Formation of supported lipid bilayers on silica: relation to lipid phase transition temperature and liposome size. Soft Matter. 10 (1), 187-195 (2014).
  33. Cole, R. W., Jinadasa, T., Brown, C. M. Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nature Protocols. 6 (12), 1929-1941 (2011).
  34. Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Developmental Cell. 9 (6), 791-804 (2005).
  35. Florentsen, C. D. et al. Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles. Soft Matter. 17 (2), 308-318 (2021).
  36. Sarkar, Y., Majumder, R., Das, S., Ray, A., Parui, P. P. Detection of curvature-radius-dependent interfacial pH/polarity for amphiphilic self-assemblies: positive versus negative curvature. Langmuir. 34 (21), 6271-6284 (2018).
  37. Raiborg, C., Stenmark, H. The ESCRT machinery in endosomal sorting of ubiquitylated membrane proteins. Nature. 458 (7237), 445-452 (2009).
  38. Alqabandi, M. et al. The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization. BMC Biology. 19 (1), 66 (2021).
  39. Leitenberger, S. M., Reister-Gottfried, E., Seifert, U. Curvature coupling dependence of membrane protein diffusion coefficients. Langmuir. 24 (4), 1254-1261 (2008).
  40. Bozelli, J. C., Jr. et al. Membrane curvature allosterically regulates the phosphatidylinositol cycle, controlling its rate and acyl-chain composition of its lipid intermediates. Journal of Biological Chemistry. 293 (46), 17780-17791 (2018).
  41. Parthasarathy, R., Yu, C. H., Groves, J. T. Curvature-modulated phase separation in lipid bilayer membranes. Langmuir. 22 (11), 5095-5099 (2006).
  42. Yuan, F. et al. Membrane bending by protein phase separation. Proceedings of the National Academy of Sciences. 118 (11), e2017435118 (2021).
  43. London, E. Membrane structure-function insights from asymmetric lipid vesicles. Accounts of Chemical Research. 52 (8), 2382-2391 (2019).
  44. Rossetti, F. F., Textor, M., Reviakine, I. Asymmetric distribution of phosphatidyl serine in supported phospholipid bilayers on titanium dioxide. Langmuir. 22 (8), 3467-3473 (2006).
  45. Richter, R. P., Maury, N., Brisson, A. R. On the effect of the solid support on the interleaflet distribution of lipids in supported lipid bilayers. Langmuir. 21 (1), 299-304 (2005).
  46. Wacklin, H. P., Thomas, R. K. Spontaneous formation of asymmetric lipid bilayers by adsorption of vesicles. Langmuir. 23 (14), 7644-7651 (2007).
  47. Lin, W. C., Blanchette, C. D., Ratto, T. V., Longo, M. L. Lipid asymmetry in DLPC/DSPC-supported lipid bilayers: a combined AFM and fluorescence microscopy study. Biophysical Journal. 90 (1), 228-237 (2006).

Tags

Keywords: Nanobar-supported Lipid BilayerMembrane Curvature SensingProtein DynamicsNanofabricationPiranha SolutionLipid MixtureSUVsPDMS Chamber
check_url/pt/64340?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Miao, X., Wu, J., Zhao, W. A Nanobar-Supported Lipid Bilayer System for the Study of Membrane Curvature Sensing Proteins in vitro. J. Vis. Exp. (189), e64340, doi:10.3791/64340 (2022).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image