生物膜荧光探针力：受体 - 配体动力学并行和定量结合诱导细胞内信号作用于单细胞
1Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences,Georgia Institute of Technology, 2Coulter Department of Biomedical Engineering,Georgia Institute of Technology, 3Charles Perkins Centre,The University of Sydney, 4Institute of Biophysics, Laboratory of RNA Biology,Chinese Academy of Sciences, 5University of Chinese Academy of Sciences, 6School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases,Zhejiang University
Summary
We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.
Abstract
膜受体 – 配体相互作用介导许多细胞功能。结合动力学和由这些分子之间的相互作用引起的下游信号很可能受到机械环境中结合和信号发生。最近的一项研究表明，机械力可以调节抗原识别由与触发T细胞受体（TCR）的。这成为可能，我们开发了一种新技术，荧光地称为生物膜力探针（fBFP），它结合了单分子力谱荧光显微镜。使用超软人类红细胞作为敏感的力传感器，一个高速摄像机和实时成像跟踪技术，该fBFP是〜1 PN（10 -12 N）〜3纳米和〜0.5毫秒力，空间和时间分辨率。随着fBFP，可以精确地测量力下单调节受体 – 配体结合动力学，同时结合图像触发细胞内的CALcium信令在单个活细胞。这种新技术可以用来研究其他膜受体 – 配体相互作用和信号下机械调节的其它细胞。
Introduction
细胞-细胞和细胞-细胞外基质（ECM）的粘附是由细胞表面受体，细胞外基质蛋白和/或脂 质1之间的结合介导的。结合允许细胞以形成功能结构1，以及认识，沟通，并做出反应的环境1-3。不像可溶性蛋白（ 例如 ，细胞因子和生长因子）从一个三维（3D），其结合流体相到细胞表面受体，细胞粘附受体形成具有跨越窄交界间隙及其配体桥接两个相对的表面，限制分子键扩散在二维（2D）接口4-7。与此相反，以三维动力学是由传统的结合分析（ 例如 ，表面等离子体共振或SPR）常用的测量，2D动力学已与诸如原子力显微镜（AFM）8-10专门技术来量化，流室11,12，微管13,14，光镊子15和生物膜力探针（BFP）16-21。
以上仅仅提供物理连锁蜂窝内聚力，粘附分子是信令机械为细胞与其周围环境进行通信的主要组成部分。出现了在理解越来越大的兴趣如何粘附分子的配体接合启动细胞内信号传导，以及如何在初始信号被转导的细胞内。直观地，受体 – 配体的性质的结合可影响它诱导的信号。然而，很难使用，因为它们的许多限制， 例如 ，一个差时间分辨率和完全缺乏空间分辨率的生物化学分析的传统合奏解剖外相互作用和细胞内信号事件之间的机械关系。现有的方法，使生物物理（2D受体 – 配体结合动力学）和生化（信令）观察活细胞包括可调刚性22的荧光能力24-26掺入底物，弹性体柱阵列23和流动室/微流体装置。然而，信令和受体 – 配体结合的读数必须（通过不同的方法最多）分别求出，从而难以解剖的粘结性能与信号事件的时间和空间关系。
传统的BFP是一个超灵敏光谱力高时空分辨率17。它使用一个挠性红细胞（RBC），为力传感器，可实现单分子2D动力学，机械性能和构象变化14,16,19-21,27-29的测量。荧光成像基于BFP（fBFP）相关与结合触发的细胞信号在单分子尺度的受体 – 配体的结合动力学。采用这种设置， 原位细胞信号活动表面mechani的上下文卡尔刺激中观察到的T细胞27。该fBFP是通用的，可以用于细胞粘附和信号传导由在其他小区的其他分子介导的研究。
Protocol
该协议遵循的指导方针，并已获得由佐治亚理工学院的人类研究伦理委员会。 1.人类红细胞分离，生物素和渗透压调节注：步骤1.1应该由受过训练的医疗专业人员如护士进行，与机构审查委员会批准的协议。 得到的血液8-10微升（一滴）从手指刺，并添加至1ml的碳酸盐/碳酸氢盐缓冲液（ 表1和2）。轻轻涡旋或吸取的混合?…
Representative Results
的BFP技术是在1995年17率先由埃文斯实验室，这picoforce工具已被广泛用来测量固定化在表面上的蛋白质的相互作用，从而分析二维粘附分子动力学与它们的配体16,19,20相互作用， 30，测量分子的弹性21,29，并确定蛋白的构象变化21。对于fBFP，一组额外的与相应的软件系统（ 表1）加落射荧光关联装置（ 图1A – C）。 <p cla…
Discussion
一个成功的实验fBFP限嗣继承了一些关键的考虑因素。首先，对于力计算是可靠的，微量，红细胞，和探针珠应该对齐接近同轴越好。红细胞的吸移管内的投影应约一个探针移液器直径，使得红细胞和吸管之间的摩擦是微不足道的。对于一个典型的人RBC，最优吸管直径为2.0-2.4微米，这将产生等式1 17,30的最佳拟合。第二，以确保在力钳测定及热波动分析测量大多为单键，也可以保持在20％以…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Research related to this paper and the development of the fBFP technology in the Zhu lab were supported by NIH grants AI044902, AI077343, AI038282, HL093723, HL091020, GM096187, and TW008753. We thank Evan Evans for inventing this empowering experimental tool, and members of the Evans lab, Andrew Leung, Koji Kinoshita, Wesley Wong, and Ken Halvorsen, for helping us to build the BFP. We also thank other Zhu lab members, Fang Kong, Chenghao Ge and Kaitao Li, for their helps in the instrumentation development.
Materials
| Table 1: Reagents/Equipment | |||
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Sodium Phosphate Monobasic Monohydrate (NaH2PO4•H2O) | Sigma-Aldrich | S9638 | Phosphate buffer preparation |
| Anhy. Sodium Phosphate Dibasic (Na2HPO4) | Sigma-Aldrich | S7907 | Phosphate buffer preparation |
| Sodium Carbonate (Na2CO3) | Sigma-Aldrich | S2127 | Carbonate/bicarbonate buffer preparation |
| Sodium Bicarbonate (NaHCO3) | Sigma-Aldrich | S5761 | Carbonate/bicarbonate buffer preparation |
| Sodium chloride (NaCl) | Sigma-Aldrich | S7653 | N2-5% buffer preparation |
| Potassium chloride (KCl) | Sigma-Aldrich | P9541 | N2-5% buffer preparation |
| Potassium phosphate monobasic (KH2PO4) | Sigma-Aldrich | P5655 | N2-5% buffer preparation |
| Sucrose | Sigma-Aldrich | S0389 | N2-5% buffer preparation |
| MAL-PEG3500-NHS | JenKem | A5002-1 | Bead functionalization |
| Biotin-PEG3500-NHS | JenKem | A5026-1 | RBC biotinylation |
| Nystatin | Sigma-Aldrich | N6261 | RBC osmolarity adjustment |
| Ammonium Hydroxide (NH4OH) | Sigma-Aldrich | A-6899 | Glass bead silanization |
| Methanol | BDH | 67-56-1 | Glass bead silanization |
| 30% Hydrogen Peroxide (H2O2) | J. T. Barker | Jan-86 | Glass bead silanization |
| Acetic Acid (Glacial) | Sigma-Aldrich | ARK2183 | Glass bead silanization |
| 3-MERCAPTOPROPYLTRIMETHOXYSILANE(MPTMS) | Uct Specialties, llc | 4420-74-0 | Glass bead functionalization |
| Borosilicate Glass beads | Distrilab Particle Technology | 9002 | Glass bead functionalization |
| Streptavidin−Maleimide | Sigma-Aldrich | S9415 | Glass bead functionalization |
| BSA | Sigma-Aldrich | A0336 | Ligand functionalizing |
| Fura2-AM | Life Technologies | F-1201 | Intracellular calcium fluorescence dye loading |
| Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D2650 | Intracellular calcium fluorescence dye loading |
| Quantibrite PE Beads | BD Biosciences | 340495 | Density quantification |
| Flow Cytometer | BD Biosciences | BD LSR II | Density quantification |
| Capillary Tube 0.7-1.0mm x 30" | Kimble Chase | 46485-1 | Micropipette making |
| Flaming/Brown Micropipette Puller | sutter instrument | P-97 | Micropipette making |
| Pipette microforce | Narishige | MF-900 | Micropipette making |
| Mineral Oil | Fisher Scientific | BP2629-1 | Chamber assembly |
| Microscope Cover Glass | Fisher Scientific | 12-544-G | Chamber assembly |
| Micro-injector | World Precision Instruments | MF34G-5 | Chamber assembly |
| 1ml Syringe | BD | 309602 | Chamber assembly |
| Micropipette holder | Narishige | HI-7 | Chamber assembly |
| Home-designed mechanical parts and adaptors fabrications using CNC machining. | Biophysics Instrument | All parts are customized according to the CAD designs. | BFP system |
| Microscope (TiE inverted) | Nikon | MEA53100 | BFP system |
| Objective CFI Plan Fluor 40x (NA 0.75, WD 0.72mm, Spg) | Nikon | MRH00401 | BFP system |
| Camera, GE680, 640×480, GigE, 1/3" CCD, mono | Graftek Imaging | 02-2020C | BFP system |
| Prosilica GC1290 – ICX445, 1/3", C-Mount, 1280×960, Mono., CCD, 12 Bit ADC | Graftek Imaging | 02-2185A | BFP system |
| Manual submicron probehead with high resolution remote control | Karl Suss | PH400 | BFP system |
| Anti-vibration table (5’ x 3’) | TMC | 77049089 | BFP system |
| 3D manual translational stage | Newport | 462-XYZ-M | |
| SolidWorks 3D CAD software | SOLIDWORKS Corp. | Version 2012 SP5 | BFP system |
| LabVIEW software | National Instruments | Version 2009 | BFP system, BFP program |
| 3D piezo translational stage | Physik Instrumente | M-105.3P | BFP system |
| Linear piezo accuator | Physik Instrumente | P-753.1CD | BFP system |
| Micromanager software | Version 1.4 | fBFP system, fluorescence imaging program | |
| Dual Cam (DC-2) | Photometrics | 77054724 | fBFP system |
| Dual Cam emission filter (T565LPXR) | Photometrics | 77054725 | fBFP system |
| Fluorescence Camera | Hamamatsu | ORCA-R2 C10600-10B | fBFP system |
| Plastic paraffin film (Parafilm) | Bemis Company, Inc | PM996 | bottle sealing |
| Table 2: Buffer solutions | |||
| Carbonate/bicarbonate buffer (pH 8.5) | |||
| Sodium Carbonate (Na2CO3) | 8.4g/L | ||
| Sodium Bicarbonate (NaHCO3) | 10.6g/L | ||
| Phosphate buffer (pH 6.5-6.8) | |||
| NaPhosphate monobasic NaH2PO4•H2O | 27.6g/L | ||
| Anhy. NaPhosphate dibasic Na2HPO4 | 28.4g/L | ||
| N2-5% buffer (pH 7.2) | |||
| Potassium chloride (KCl) | 20.77g/L | ||
| Sodium chloride (NaCl) | 2.38g/L | ||
| Potassium phosphate monobasic (KH2PO4) | 0.13g/L | ||
| Anhy. Sodium Phosphate Dibasic (Na2HPO4) | 0.71g/L | ||
| Sucrose | 9.70g/L | ||
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Citer Cet Article
Chen, Y., Liu, B., Ju, L., Hong, J., Ji, Q., Chen, W., Zhu, C. Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell. J. Vis. Exp. (102), e52975, doi:10.3791/52975 (2015).