通过粘附足迹测定法对细胞滚动中的分子粘附进行成像
Summary
该协议提供了执行粘附足迹测定的实验程序，以在快速细胞滚动粘合期间对粘附事件进行成像。
Abstract
由选择素介导的相互作用促进的滚动粘附是将白细胞募集到炎症部位的高度动态，被动的运动。这种现象发生在毛细血管后静脉中，其中血流在内皮细胞上滚动运动推动白细胞。稳定的轧制需要在粘附键形成与其机械驱动的解离之间取得微妙的平衡，使细胞在沿流动方向滚动时保持附着在表面上。与在相对静态环境中发生的其他粘附过程不同，滚动附着力是高度动态的，因为滚动电池以每秒数十微米的速度行进数千微米。因此，由于所需的时间尺度短且灵敏度高，传统的机械生物学方法（如牵引力显微镜）不适合测量单个粘附事件和相关分子力。在这里，我们描述了我们最新的粘附足迹测定方法，以对P-选择素进行成像：PSGL-1在分子水平上滚动粘附中的相互作用。该方法利用不可逆的基于DNA的张力计系绳，以荧光轨迹的形式产生分子粘附事件的永久历史。这些轨迹可以通过两种方式进行成像：（1）将数千张衍射极限图像拼接在一起以产生大视场，从而能够提取长度为数千微米的每个滚动细胞的粘附足迹，（2）执行DNA-PAINT以在小视场内重建荧光轨迹的超分辨率图像。在这项研究中，粘附足迹测定用于研究HL-60细胞在不同剪切应力下滚动。在此过程中，我们能够对P-选择蛋白的空间分布进行成像：PSGL-1相互作用，并通过荧光强度深入了解它们的分子力。因此，该方法为定量研究分子水平上滚动粘附所涉及的各种细胞 – 表面相互作用提供了基础。
Introduction
滚动粘附级联描述了循环细胞如何拴住并沿着血管壁滚动1。被动滚动主要由selectin介导，selectins是一类主要的细胞粘附分子（CAM）1。在血液的剪切流下，表达P-选择素糖蛋白配体-1（PSGL-1）的白细胞与P-选择素形成高度瞬态键，其可能在发炎的内皮细胞表面表达。这个过程对于白细胞迁移到炎症部位至关重要2。此外，PSGL-1也是一种机械敏感受体，能够在与P-selectin3接触时触发滚动粘附级联的后续牢固粘附阶段。
影响CAM功能的基因突变会严重影响免疫系统，例如在罕见的白细胞粘附缺乏症（LAD）中，介导滚动的粘附分子的故障导致严重免疫功能低下的个体4，5，6。此外，循环肿瘤细胞已被证明在类似的滚动过程之后迁移，导致转移7，8。然而，由于细胞滚动是快速和动态的，传统的实验机械生物学方法不适合研究细胞滚动过程中的分子相互作用。虽然原子力显微镜和光学镊子等单细胞和单分子操作方法能够在单分子水平上研究分子相互作用，例如P-selectin与PSGL-1的力依赖性相互作用9，但它们不适合研究细胞滚动过程中的活粘附事件。此外，体 外 表征的相互作用不能直接回答有关 体内分子粘附的问题。例如，当细胞在其天然环境中起作用时，什么分子张力范围与生物学相关？诸如粘合剂动力学模拟10 或简单稳态模型11 之类的计算方法已经捕获了某些分子细节以及它们如何影响滚动行为，但高度依赖于建模参数和假设的准确性。其他技术，如牵引力显微镜，可以检测细胞迁移过程中的力，但不能提供足够的空间分辨率或分子张力的定量信息。这些技术都不能提供对分子力的时间动力学，空间分布和幅度异质性的直接实验观察，这些与细胞在其天然环境中的功能和行为直接相关。
因此，实施能够准确测量选择介导相互作用的分子力传感器对于提高我们对滚动附着力的理解至关重要。在这里，我们描述了附着足迹测定12 的方案，其中PSGL-1涂层珠子滚动在呈现p-selectin功能化张力计系绳（TGT）13的表面上。这些TGT是基于DNA的不可逆的力传感器，以荧光读数的形式导致永久的破裂事件历史。这是通过TGT（dsDNA）的破裂，然后用荧光标记的互补链随后标记破裂的TGT（ssDNA）来实现的。该系统的一个主要优点是它与衍射极限和超分辨率成像兼容。荧光标记的互补链可以永久结合（>12 bp）用于衍射极限成像，或者瞬态结合（7-9 bp）用于通过DNA PAINT进行超分辨率成像。这是研究TGT在主动轧制过程中破裂时滚动附着力的理想系统，但在轧制后分析荧光读数。这两种成像方法还为用户提供了更大的自由度来研究滚动附着力。通常，衍射极限成像可用于通过荧光强度提取分子断裂力13，而超分辨率成像允许对受体密度进行定量分析。由于能够研究轧制粘附的这些特性，该方法为理解轧制电池在剪切流下分子粘附的力调节机制提供了一个有前途的平台。
Protocol
Representative Results
Discussion
粘附足迹测定可以可视化细胞滚动粘附过程中PSGL-1和P-选择素之间的分子粘附事件。该过程由P-选择素介导的捕获开始，然后在流体剪切应力下滚动。实验过程中的潜在问题通常涉及细胞滚动不良或缺少荧光轨迹，即使细胞滚动良好。这些问题通常是由于在协议中的关键步骤中进行质量控制而引起的，如故障排除表（表1）中所列。
生物分子和缓冲液需要过滤并储…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了加拿大创新基金会（CFI 35492），加拿大自然科学和工程研究委员会发现补助金（RGPIN-2017-04407），研究基金新前沿（NFRFE-2018-00969），迈克尔史密斯健康研究基金会（SCH-2020-0559）和不列颠哥伦比亚大学杰出基金的支持。
Materials
| 4-channel drill guide | Custom made | 3D printed with ABS filament | |
| 4-holes slide | Custom made | Drill clean microscope slide using a Dremel with diamond coated drill bits on a 4-channels drill guide which has a layout that matches with the centers of the 8-32 threaded holes on the aluminum clamp. | |
| Acetone | VWR | BDH1101-4LP | |
| Acrylic spacer | Custom made | Cut two blocks of acrylic sheets with the dimension of 40 mm x 30 mm x 2.5 mm. On each block, drill two 3 mm holes that are precisely aligned with the 4-40 holes on the aluminium holder. | |
| Aluminium chip holder | Custom made | Machine anodized aluminium block into a C-shaped holder with the outer dimension of 640 mm x 500 mm x 65 mm and the opening dimension of 400 mm x 380 mm x 65 mm. Inlets and outlets are tapped with 8-32 thread. | |
| Aminosilane | AlfaAesar | L14043 | CAS 1760-24-3 |
| Antibiotic/antimycotic solution | Cytiva HyClone | SV3007901 | Pen/Strep/Fungiezone |
| Beads, ProtG coated polystyrene | Spherotech | PGP-60-5 | |
| Bovine serum albumin | VWR | 332 | |
| Buffer, DNA PAINT | 0.05% Tween-20, 5 mM Tris, 75 mM MgCl2, 1 mM EDTA | ||
| Buffer, T50M5 | 10mM Tris, 50 mM NaCl, 5 mM MgCl2 | ||
| Buffer, Rolling | HBSS with 2mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 0.1% BSA | ||
| Buffer, Wash | 10 mM Tris, 50 mM NaCl, 5 mM MgCl2 and 2 mM CaCl2, 0.05% Tween 20 | ||
| Calcium chloride | VWR | BDH9224 | |
| Cell culture flasks | VWR | 10062-868 | |
| Concentrated sulfuric acid | VWR | BDH3072-2.5LG | 95-98% |
| Coverslip holding tweezers | Techni-Tool | 758TW150 | |
| Diamond-coated drill bits | Abrasive technology | C5250510 | 0.75 mm diamond drill |
| DNA, amine-ssDNA (top strand) | IDT DNA | Custom oligo | CCGGGCGACGCAGGAGGG /3AmMO/ |
| DNA, biotin-ssDNA (bottom strand) | IDT DNA | Custom oligo | /5BiotinTEG/ TTTTT CCCTCCTGCGTCGCCCGG |
| DNA, imager strand for DNA-PAINT | IDT DNA | Custom oligo | GAGGGAAA TT/3Cy3Sp/ |
| DNA, imager strand for permanent labelling | IDT DNA | Custom oligo | CCGGGCGACGCAGG /3Cy3Sp/ |
| Double-sided tape | Scotch | 237 | 3/4 inch width, permanent double-sided tape |
| EDTA | Thermofisher | 15575020 | 0.5 M EDTA, pH 8.0 |
| Epoxy | Gorilla | 42001 | 5 minute curing time |
| Fetal Bovine Serum (FBS) | Avantor | 97068-085 | |
| GelGreen | Biotium | 41005 | |
| Glacial acetic acid | VWR | BDH3094-2.5LG | |
| Glass, Coverslips | Fisher Scientific | 12-548-5P | |
| Glass, Microscope slide | VWR | 48300-026 | 75 mm x 25 mm x 1 mm |
| Glass, Staining jar | VWR | 74830-150 | Wheaton Staining Jar (900620) |
| Hanks' Balanced Salt solution (HBSS) | Lonza | 04-315Q | |
| Hemocytometer | Sigma-Aldrich | Z359629-1EA | |
| HL-60 cells | ATCC | CCL-240 | |
| Humidity chamber slide support | Custom made | 3D printed with ABS filament | |
| Hydrogen peroxide | VWR | BDH7690-1 | 30% |
| Imidazole | Sigma-Aldrich | I2399 | |
| Inlets/outlets | Custom made | Drill through eight 8-32 set screws using cobalt drill bits. Insert 1.5 cm polyethylene tubing (Tygon, I.D. 1/32” O.D. 3/32”) into each hollow setscrew | |
| Iscove Modified Dulbecco Media (IMDM) | Lonza | 12-722F | |
| Magnesium chloride | VWR | BDH9244 | |
| Magnetic Ni-NTA beads | Invitrogen | 10103D | |
| Mailer tubes | EMS | EMS71406-10 | |
| Methanol | VWR | BDH1135-4LP | |
| Micro Bio-Spin P-6 Gel Columns | Biorad | 7326200 | In SSC Buffer |
| PEG | Laysan Bio | MPEG-SVA-5000 | |
| PEG-biotin | Laysan Bio | Biotin-PEG-SVA-5000 | |
| Potassium hydroxide | VWR | 470302-132 | |
| Protein, Protein G | Abcam | ab155724 | N-terminal His-Tag and C-terminal cysteine |
| Protein, P-selectin-Fc | R&D System | 137-PS | Recombinant Human P-Selectin/CD62P Fc Chimera Protein, CF |
| Protein, Streptavidin | Cedarlane | CL1005-01-5MG | |
| Pump Syringe | Harvard Apparatus | 704801 | |
| Sodium bicarbonate | Ward’s Science | 470302-444 | |
| Sodium chloride | VWR | 97061-274 | |
| Sulfo-SMCC | Thermofisher | 22322 | |
| Syringe | Hamilton | 81520 | Syringes with PTFE luer lock, 5 mL |
| Syringe needles | BD | 305115 | Precision Glide 26 G, 5/8 Inch Length |
| TCEP | Sigma-Aldrich | C4706-2G | |
| Tris | VWR | BDH4502-500GP | |
| Tubing, Adaptor | Tygon | ABW00001 | Formulation 3350, I.D. 1/32”; O.D. 3/32” |
| Tubing, Polyethylene | BD Intramedic | 427406 | Intramedic (PE20) I.D. 0.38mm; O.D. 1.09mm |
| Tween-20 | Sigma-Aldrich | 93773 |
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