在氧化应激期间细胞粘附和纤维蛋白扩散动力学
Summary
该方法可用于量化细胞粘附的早期动力学，并将锚固依赖细胞扩散到纤维质。此外，此测定可用于研究改变的氧化还原平衡对细胞扩散和/或细胞粘附相关细胞内信号通路的影响。
Abstract
细胞粘附和扩散到细胞外基质（ECM）是有机体发育和成人组织平衡过程中必不可少的细胞过程。有趣的是，氧化应激可以改变这些过程，从而促进转移性癌症等疾病的病理生理学。因此，了解在氧化还原状态扰动期间细胞如何附着和在ECM上传播的机制，可以深入了解正常和疾病状态。下面描述是一个循序式方案，它利用基于免疫荧光的测定来具体量化细胞粘附和在体外纤维素（FN）上传播不朽的成纤维细胞。简单地说，锚固依赖细胞被悬浮，并暴露于ATM激酶抑制剂Ku55933诱导氧化应激。然后，在 FN 涂层表面上镀层，并允许在预定的时间段内附着。仍然附着的细胞是固定的，并标有荧光基抗体标记的附着力（例如，白辛）和扩散（例如，F-actin）。数据采集和分析使用常用的实验室设备进行，包括荧光显微镜和免费提供的斐济软件。此过程用途广泛，可针对各种细胞系、ECM 蛋白或抑制剂进行修改，以检查广泛的生物学问题。
Introduction
细胞基质粘附（即焦点粘附）是大而动态的多分子蛋白复合物，可调节细胞粘附和扩散。这些过程对于组织发育、维护和生理功能至关重要。焦点粘附由膜结合受体（如整数）以及将细胞骨骼功能素与细胞外基质（ECM）1联系起来的脚手架蛋白组成。这些复合物能够通过激活各种信号转导途径来响应细胞外环境中的物理化学线索。因此，焦点粘附作为信号中心，将细胞外机械线索传播到许多细胞过程，包括定向迁移、细胞周期调节、分化和存活1、2。一组调节焦点粘附并与之相互作用的信号分子包括小GTPases的Rho家族的成员。Rho GTPass是关键的蛋白质，通过它们特定的时空活化3调节细胞迁移和粘附动力学。毫不奇怪，Rho蛋白功能调节障碍与一些人类病理有关，如转移、血管生成等。特别令人感兴趣的是，细胞氧化还原状态在细胞迁移和粘附的调制中起着主要作用。氧化还原平衡的改变，如活性氧物种（ROS）的增加，已被证明可以调节许多细胞类型和人类疾病中的Rho蛋白活性和附着力4、5、6 ，7，8.例如，患有神经紊乱（A-T），这是由DNA损伤修复丝氨酸/三甲氨酸激酶A-T突变（ATM）的突变引起的，转移性癌症的风险增加9， 10.这些患者和细胞系的ATM激酶活性丧失，无论是通过基因突变还是化学抑制，导致由于磷酸苯甲酸酯通路7、11的功能障碍而产生高水平的氧化应激。 12.此外，实验室最近的研究通过改变细胞骨骼动力学（即粘附和扩散）在A-T中对ROS的病理生理学作用，直接导致在体外激活Rho家族GTPases。最终，由Rho家族活化引起的细胞骨骼动力学的这些改变可能导致A-T患者5、13中注意到的转移性癌症风险增加。因此，了解氧化应激期间细胞-基质相互作用之间的相互作用，可以深入了解粘附和扩散的调节。这些研究还可以为进一步调查 Rho 家族 GTPass 在这些信号过程中可能扮演的角色设置舞台。
本文所述是研究ATM激酶活性抑制引起的氧化应激期间粘附组装和扩散的早期细胞动力学的一种方案。此测定基于锚固依赖细胞粘附于 ECM 蛋白纤维蛋白 （FN） 的良好特征机制。当悬浮细胞被镀在FN上时，几个Rho GTPass协调对细胞骨骼重塑的控制3，14。当细胞从外观的圆形和圆形向扁平和膨胀移动时，形态变化被观察。伴随这些观察的是与 ECM 一起开发许多矩阵粘附。这些变化归因于当细胞粘附并扩散15、16时，在1小时内用Rac1对RhoA进行双相激活。
已采用各种方法检查粘附形态和动力学以及细胞扩散。然而，这些方法依赖于复杂的长期实时成像全内反射荧光（TIRF）或共聚焦显微镜系统。因此，用户必须有权访问专用设备和软件。此外，这些生物成像系统所需的设置时间使得捕获早期粘附事件具有挑战性，尤其是在同时测试多种抑制剂或治疗条件时。
本文详细的方法提供了一种简单、经济、定量的方法来评估控制粘附组件和体外扩散的参数。该协议使用常用的实验室设备执行，如荧光显微镜和CCD相机。这种测定涉及在ATM激酶活性化学抑制引起一段时间的氧化应激后，将锚固依赖细胞应用于FN涂层表面，这在之前已经证明了这一点。电镀后，允许电池在指定的时间长度上附加和粘附。未附着的细胞被冲走，而附着的细胞被固定，并标有荧光基抗体的附着力（例如，帕西林）和扩散（例如，F-actin）2，5 。然后，这些蛋白质使用荧光显微镜进行可视化和记录。随后的数据分析使用免费提供的斐济软件进行。此外，该方法可以适应，以检查粘附动力学在广泛的条件下，包括不同的ECM蛋白，处理各种氧化剂/细胞培养条件或各种锚固依赖细胞系，以解决广泛的范围生物问题。
Protocol
Representative Results
Discussion
此处描述的协议是一种通用且经济的方法，可快速筛选多个与锚固相关的细胞类型，以便在细胞扩散期间进行动态细胞骨架重塑。特别是，当细胞粘附在FN上时，这种方法定量地检查氧化应激期间的应力纤维和焦点粘附形成（图1A）。此外，这些细胞表型可能建议一个监管作用的Rho家族的小GTPases的成员，因为他们有记录的作用，在细胞附件和传播15，16，22。</sup…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢斯科特·赫顿博士和梅根·布莱克利奇博士对手稿的批判性评论。这项工作由高点大学的研究和赞助项目（MCS）和北卡罗来纳州立大学生物技术项目资助。
Materials
| 0.05% Trypsin-EDTA (1x) | Gibco by Life Technologies | 25300-054 | cell dissociation |
| 10 cm2 dishes | Cell Treat | 229620 | sterile, tissue culture treated |
| 15 mL conical tubes | Fisher Scientific | 05-539-5 | sterile |
| 1X Phosphate Buffered Saline | Corning Cellgro | 21-031-CV | PBS, sterile, free of Mg2+ and Ca2+ |
| 24-well cell culture treated plates | Fisher Scientific | 07-200-740 | sterile, tissue culture treated |
| 4°C refrigerator | Fisher Scientific | ||
| Mouse IgG anti-paxillin primary antibody (clone 165) | BD Transduction Laboratories | 610620 | marker of focal adhesions |
| Aspirator | Argos | EV310 | |
| Biosafety cabinet | Nuair | NU-477-400 | Class II, Type A, series 5 |
| Delipidated Bovine Serum Albumin (Fatty Acid Free) Powder | Fisher Scientific | BP9704-100 | dlBSA |
| Dimethyl Sulfoxide | Fisher Scientific | BP231-100 | organic solvent to dissolve Ku55933 |
| Dulbecco's Modified Eagle Media, High Glucose | Fisher Scientific | 11965092 | REF52 base cell culture medium |
| Fetal bovine serum | Fisher Scientific | 16000044 | certified, cell culture medium supplement |
| Fiji | National Institutes of Health | http://fiji.sc/ | image analysis program |
| Filter syringe | Fisher Scientific | 6900-2502 | 0.2 µM, sterile |
| Glass coverslips (12-Cir-1.5) | Fisher Scientific | 12-545-81 | autoclave in foil to sterilize |
| Goat anti-mouse IgG secondary antibody Alexa Fluor 488 | Invitrogen | A11001 | fluorescent secondary antibody, light sensitive |
| Goat Serum | Gibco by Life Technologies | 16210-064 | component of blocking solution for immunofluorescence |
| Hemocytometer | Fisher Scientific | 22-600-107 | for cell counting |
| Human Plasma Fibronectin | Gibco by Life Technologies | 33016-015 | FN |
| IX73 Fluorescence Inverted Microscope | Olympus | microscope to visualize fluorescence, cell morphology, counting and dissociation | |
| Ku55933 | Sigma-Aldrich | SML1109-25MG | ATM kinase inhibitor, inducer of reactive oxygen species |
| L-glutamine | Fisher Scientific | 25-030-081 | cell culture medium supplement |
| Monochrome CMOS 16 bit camera | Optimos | ||
| Paraformaldehyde | Sigma-Aldrich | P6148-500G | PFA, fixative for immunofluorescence |
| Penicillin-streptomycin | Fisher Scientific | 15-140-122 | P/S, antibiotic solution for culture medium |
| Alexa Fluor 594 phalloidin (F-actin probe) | Invitrogen | A12381 | marker of F-actin, light sensitive |
| ProLong Gold Anti-fade reagent with DAPI | Invitrogen | P36941 | cover slip mounting media including nuclear dye DAPI, light sensitive |
| REF52 cells | Graham, D.M. et. al. Journal of Cell Biology 2018 | ||
| Stir plate with heat control | Corning Incorporated | PC-420D | |
| Syringe | BD Biosciences | 309653 | 60 mL syringe |
| Tissue culture incubator | Nuair | ||
| Triton X-100 | Fisher Scientific | BP151-500 | detergent used to permeabilize cell membranes |
| Trypan Blue Solution | Fisher Scientific | 15-250-061 | for cell counting |
| Trypsin Neutralizing Solution (1x) | Gibco by Life Technologies | R-002-100 | TNS, neutralizes trypsin instead of fetal bovine serum |
| tube rotator | Fisher Scientific | 11-676-341 | |
| water bath | Fisher Scientific | FSGPD02 |
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