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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
자동 번역
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생물학

结合3D磁力致动器和多功能荧光成像来研究细胞核力学生物学

Published: July 05, 2022
doi:
10.3791/64098
, , , , , , , , ,
1Department of Mechanical and Aerospace Engineering, Herbert Wertheim College of Engineering,University of Florida, 2Department of Materials Science and Engineering,University of Florida, 3Department of Biostatistics,University of Florida, 4Department of Biomedical Engineering, College of Engineering (COE),University of Delaware (UD), 5UF Health Cancer Center,University of Florida, 6Department of Physics, College of Liberal Arts and Sciences,University of Florida, 7Department of Anatomy and Cell Biology, College of Medicine,University of Florida, 8J. Crayton Pruitt Family Department of Biomedical Engineering,University of Florida, 9Department of Physiology and Functional Genomics,University of Florida

Summary

本研究提出了一种新的方案，通过传递到细胞质中的磁性微珠直接对细胞核施加机械力，并同时进行活细胞荧光成像。

Abstract

机械生物学的一个基本问题是活细胞如何在细胞生理学和病理学的背景下感知细胞外机械刺激。细胞外机械刺激的细胞机械感觉被认为是通过膜受体、相关的蛋白质复合物和细胞骨架。机械生物学的最新进展表明，细胞质本身的细胞核可以同时独立地感知机械刺激。然而，缺乏对细胞核如何感知、转导和响应机械刺激的机制理解，主要是因为通过传统工具访问和量化细胞核力学的技术挑战。本文描述了一种新型磁力致动器的设计、制造和实现，该致动器应用精确和非侵入性的 3D 机械刺激直接使细胞核变形。使用CRISPR / Cas9工程细胞，这项研究表明，该工具与高分辨率共聚焦荧光成像相结合，能够揭示单细胞中机械敏感yes相关蛋白（YAP）的实时动力学作为细胞核变形的函数。这种简单的方法有可能弥合机械生物学界当前的技术差距，并为细胞核机械转导与细胞功能之间关系中存在的知识差距提供答案。

Introduction

本研究旨在通过将直接施加机械力的磁致动器和同时成像结构和功能亚细胞变化的共聚焦荧光显微镜相结合，开发和应用一种新技术来阐明细胞核力学生物学。细胞感知细胞外生物物理信号，包括组织硬度1，2，3，4、间质流体压力和剪切应力5、6、7、表面拓扑/几何形状8、9、10、11、12 和拉伸/压缩应力 13，14，15，16.生物物理信号被转化为生化信号，并触发基因表达和细胞行为的潜在下游变化 – 这一过程称为机械转导17，18，19，20，21，22，23，24，25，26，27.为了研究机械转导过程，已经开发了无数技术来对细胞施加机械力，例如原子力显微镜28，细胞拉伸装置29，bio-MEMS（微机电系统）力传感器15，30，31，剪切流变学32和立体视觉系统33.最近的一篇综述总结了应用细胞外机械线索和干扰机械传感的方法34。迄今为止，这些方法中的大多数都对细胞质膜施加力，细胞通过膜受体（如整合素、钙粘蛋白、离子通道和 G 蛋白偶联受体）直接接收这些细胞外生物物理信号。随后，它们将信号传输到细胞内细胞骨架和细胞核。例如，使用yes相关蛋白（YAP）易位作为机械传感的指标，显示细胞从细胞膜感知底物刚度和细胞外张力的机械信号，并通过细胞骨架将它们传递到细胞核中以诱导YAP细胞质到细胞核易位28，35。

最近的证据表明，细胞核本身是一个独立的机械传感器8，36，37。对从细胞中收获的分离细胞核进行的实验证明了这一点，其中发现细胞核响应直接施加在它们上的机械力而自适应地改变其刚度36。在许多生理条件下，肿瘤和健康细胞中的细胞核都能感知细胞外生物物理信号并改变其机械性能和组装38，39，40。例如，在外渗时，肿瘤细胞的核硬度降低并保持柔软度超过24小时38。在通过密闭间质空间迁移的过程中，肿瘤细胞的细胞核经常失去并恢复其结构完整性39。然而，细胞核感知生物物理信号的方式尚不清楚，尽管已发现涉及几种核包膜蛋白和蛋白质家族，例如核粘连蛋白A / C和核骨架和细胞骨架（LINC）复合物38，41的接头。因此，可以直接向细胞核施加力的新型非侵入性方法将使力传递的效果与细胞 – 质膜和细胞骨架解耦，并将有助于阐明以前无法进入的核机械传感分子机制。

使用光镊操纵细胞器42 和注射到细胞43 中的微珠的研究表明，直接对细胞核施加力的技术能力。然而，光学镊子技术有几个局限性：（1）低通量光学镊子通常一次只能操作一个细胞或微珠;（2）核的潜在光损伤和温度伪影变形需要数十pN36，相应的必要激光功率约为每pN10 mW 44，45。这样的激光强度足以在实验过程中触发细胞中的光损伤并扰乱细胞功能46。

通过活细胞内的微珠施加的磁力显示出直接对细胞核施加力的潜力，并克服了光镊的局限性。一旦微珠被输送到细胞质中，磁场就可以以高通量的方式同时对多个微珠施加磁力。磁场不影响细胞功能47，但产生从pN到nN的力，足以诱发核变形36，48，49。迄今为止，磁性微珠的操作已应用于细胞质膜48，细胞质50内部，F-肌动蛋白51，细胞核内47和分离的细胞核36。然而，微珠的磁操作从未被用于在核包膜上施加直接的机械力来研究原子核中的机械转导。

在本文中，开发了一种简单的技术，将磁性微珠无创地递送到细胞质中，并使用这些微珠在细胞核上施加机械力（图1）。在这里，使用CRISPR / Cas9工程的内源性表达mNeonGreen21-10/11标记的YAP的人正常B2B细胞系来验证该方法。YAP是一种机械敏感蛋白，YAP的易位受核机械传感14，28的调节。选择CRISPR / Cas9调控的敲入方法用荧光蛋白（FP）mNeonGreen21-10/11标记内源性YAP。尽管已知CRISPR编辑具有不完全效率和脱靶效应，但先前出版物中的协议集成了荧光分选以选择正确的开放阅读框插入52，53，54。通过这种额外的选择层，在先前产生的20+细胞系中未观察到脱靶标记事件52，53，54，55。这是一种分裂的荧光蛋白构建体，但原则上，任何可表达的荧光标签都可以使用。这种标记方法优于转基因或抗体方法。首先，与转基因表达不同，标记蛋白保持单拷贝基因剂量并在天然基因调控网络的生理背景下表达，从而限制了蛋白质浓度、定位和相互作用的偏差。本研究中使用的标记方法比全FP标记实现了超过数量级的吞吐量和效率。它还避免了由于固定伪影和高质量、高特异性抗体的可用性有限而导致的与免疫荧光相关的挑战。其次，本文中使用的方法对细胞生理学的干扰最小，并且能够真实地实时揭示所有内源性YAPs。相比之下，其他常见的转基因方法通常会导致YAP的过表达。由此产生的人工分布可能引起细胞毒性并影响细胞的机械传感56，57，58。

本研究提出了一种方案，通过传递到细胞质中的磁性微珠直接对细胞核施加力，并同时进行活细胞荧光成像。总之，这里介绍的方案演示了如何（1）在细胞核外将磁性微珠递送到细胞中，（2）操纵微珠以在细胞核上施加磁力，（3）在操作过程中对细胞进行共聚焦荧光成像，以及（4）在整个力施加过程中定量分析YAP核/细胞质（N / C）比率。结果表明：（1）通过内吞作用，磁性微珠可以在7 h内无创递送到B2B细胞的细胞质中（图2和图3）;（2）直接施加在细胞核上的量化磁力（图4，图5和图6）可以触发CRISPR / Cas9工程B2B细胞中YAP N / C比率的各种变化（图7和图8）。

Protocol

1. 维持 CRISPR/Cas9 工程化的 B2B 细胞 在补充有10%胎牛血清和1%青霉素链霉素的RPMI-1640的T25烧瓶中培养B2B细胞。 将B2B细胞保持在37°C的加湿培养箱中，并含有5%CO2。 当汇合度达到70%至80%时传代培养B2B细胞。 将B2B细胞系储存在RPMI-1640培养基中，在-80°C冰箱中含有10%（v / v）DMSO。 在实验中使用传代数小于10的B2B细胞。 <stron…

Representative Results

磁力移动装置的设计及磁力的应用为了通过磁微珠对原子核施加力，设计并建造了一个磁铁移动装置来控制磁铁的空间位置。磁铁移动设备包含一个中央框架、三个旋钮和导轨，用于以每个周期 1.59 mm 的空间分辨率在 x、y 和 z 方向上独立移动连接的磁铁（图 1A）。一旦磁体靠近输送到细胞中的7μm微珠（图1B），它就会磁力吸引微珠并在…

Discussion

磁性微珠的内化（第2.2节）至关重要，因为细胞外微珠不能直接对细胞核施加力。力施加和成像（第5.3节）是本实验中的关键步骤，使细胞核变形并引起有意义的生物学后果所需的力可能取决于样品。该实验中的力大小（0.8 nN和1.4 nN）可以进一步增加，以触发不太敏感的细胞中的核机械传感。

为了以高通量定量方式施加磁力，单个微珠的内化是一种理想的方法。在这项研究?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

该项目由UF佳得乐奖启动包（X.T.），UFHCC飞行员奖（X.T.和Dietmar Siemann博士），UF机会种子基金（X.T.）和UFHCC大学学者计划（H.Y.Wang）资助。我们衷心感谢Jonathan Licht博士（UFHCC），Rolf Renne博士（UFHCC），Christopher Vulpe博士（UFHCC），Blanka Sharma博士（BME），Mark Sheplak博士（MAE和ECE），Daniel Ferris博士（BME），Malisa Sarntinoranont博士（MAE），Ashok Kumar博士（MAE），Benjamin Keselowsky博士（BME），Brent Gila博士（RSC），Philip Feng博士（ECE）的智力讨论和技术支持， Gregory A. Hudalla博士（BME），Steven Ghivizzani博士（OSSM），Yenisel Cruz-Almeida博士（CDBS），Roger Fillingim博士（CD-BS），Robert Caudle博士（OMS），John Neubert博士（DN-OR），Justin Hiliard博士（神经外科），田和博士（哈佛大学），谭友华博士（香港理工大学），Jessie L-S Au博士（定量系统药理学研究所），David Hahn博士（亚利桑那大学）， 尼康支持团队（Jose Serrano-Velez博士、Larry Kordon博士和Jon Ekman博士）。我们非常感谢Tang’s，Yamaguchi’s，Sharma’s，Au’s，Siemann’s和Guan研究实验室的所有成员以及UF MAE部门的所有工作人员的有效支持。

Materials

0.05 % Trypsin Corning 25-051-CI
25 cm2 flask Corning 156340
7-µm mean diameter carbonyl iron microbeads N/A N/A
A1R confocal system Nikon
Carbonyl Iron Powder CM BASF 30042253 Magnetic microbead
Culture medium (RPMI-1640) Gibco 11875093
Desktop Computer Dell with Windows 10 operating system
Environmental chamber TIZB Tokai Hit TIZB
Fetal bovine serum (FBS) Gibco 26140
Fiji ImageJ National Institutes of Health and the Laboratory for Optical and Computational Instrumentation
Glass-bottom petri dish MatTek P35G-1.5-14-C
Magnet K&J Magnetics, Inc. D99-N52
Monochrome Camera FLIR BFS-U3-70S7M-C
NIS-Elements software platform Nikon software platform
Nucleus mask ImageJ macro https://github.com/KOLIUG/Nuclear mask
NucSpot Live 650 Biotium #40082 Nuclear stain
Penicillin-streptomycin Gibco 15140122
Phosphate buffered saline (PBS) Gibco 10010023
Ti2-E inverted microscope Nikon
XYZ mover (CAD files) https://github.com/KOLIUG/XYZ-mover
DOWNLOAD MATERIALS LIST

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Tags

3D Magnetic Force ActuatorMulti-functional Fluorescence ImagingNucleus MechanobiologyMagnetic MicrobeadsCell InternalizationConfocal Fluorescence ImagingLive-cell ImagingNuclear StainYES-associated Protein (YAP)Cell BoundaryCytoskeletonOptical TweezersCell FunctionsHigh Throughput
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Huang, M., Wang, H., Delgado, A. A., Reid, T. A., Long, J., Wang, S., Sussman, H., Guan, J., Yamaguchi, H., Tang, X. Combining 3D Magnetic Force Actuator and Multi-Functional Fluorescence Imaging to Study Nucleus Mechanobiology. J. Vis. Exp. (185), e64098, doi:10.3791/64098 (2022).
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