集成张力传感器在亚微米分辨率下的成像整合和细胞力
Summary
整合素张力在各种细胞功能中起着重要作用。利用集成张力传感器, 用 picoNewton (pN) 灵敏度校准整合素张力, 并以亚微米分辨率进行成像。
Abstract
整合素配体键传递的分子张力是整合素通路中的基本机械信号, 在许多细胞功能和行为中发挥着重要作用。为了对高力灵敏度和空间分辨率的图像整合子张力进行校准和图像整合, 我们开发了一种基于 dna 的集成张力传感器 (ITS)。如果维持分子张力, ITS 被激活以荧光, 从而在分子水平上将力转化为荧光信号。ITS 激活的张力阈值可在 10-60 pN 范围内进行调谐, 很好地覆盖了细胞内整合蛋白张力的动态范围。在用 ITS 接枝的基板上, 用荧光显示粘附细胞的整合素张力, 并以亚微米分辨率进行成像。ITS 还与活细胞和固定细胞的细胞结构成像兼容。ITS 已成功地应用于血小板收缩和细胞迁移的研究。本文详细介绍了智能交通系统的合成和应用在整合物传递细胞力研究中的应用。
Introduction
细胞依靠整合素来粘附和施加细胞外基质的细胞力。整合素介导的细胞粘附和力传递是细胞扩散1,2、迁移3,4 和生存5, 6,7的关键。从长期来看, 整合素生物力学信号也会影响细胞增殖8、9、10 和分化11、12.研究人员已经开发出了各种方法来测量和映射细胞矩阵界面上的整合蛋白传递的细胞力。这些方法是基于弹性底层13, 阵列的微柱 14, 或原子力显微镜 (afm)15,16。弹性底层和微柱方法依靠基板的变形来报告细胞应力, 在空间分辨率和力敏感性方面存在局限性。AFM 具有较高的力灵敏度, 但不能同时检测多个点的力, 因此很难绘制整合物传递的细胞力图。
近年来, 在分子水平上研究细胞力的几种技术已经发展起来。开发了一套基于聚乙二醇17、18、蜘蛛丝肽 19和 dna20、21、22 、23的分子张力传感器。可视化和监测分子蛋白传递的张力。在这些技术中, dna 首先被用作张力量系系 (tgt) 的合成材料, tgt 是一种可破坏的链接器, 可调节活细胞22,24中整合素张力的上限。后来, DNA 和荧光共振转移技术结合起来, 首先由陈氏23组和萨拉伊塔第20组创建了基于发夹 dna 的荧光张力传感器。基于发夹 dna 的张力传感器实时报告整合蛋白张力, 已成功地应用于一系列细胞功能21的研究。随后, 王的实验室将 TGT 与氟淬火器对结合起来, 报告整合素张力。这个传感器被命名为 its25,26。ITS 基于双链 DNA (dsDNA), 具有更广泛的动态范围 (10-60 pN) 的整合素张力校准。与基于发夹 dna 的传感器不同的是, ITS 不实时地报告蜂窝力, 而是将所有历史整合事件记录为细胞力的足迹;这种信号积累过程提高了细胞力成像的灵敏度, 即使使用低端荧光显微镜也可以对细胞力进行成像。ITS 的合成相对更方便, 因为它是通过杂交两个单链 Dna (ssDNA) 而产生的。
ITS 是一个18基配对 dsDNA 与生物素、荧光蛋白、淬火剂 (黑洞淬火 2 [BHQ2])27和环状精氨酰糖基环氨酸 (rgd) 肽28作为整合素肽配体 (图 1)结合而成。较低的链与荧光体结合 (Cy3 在本手稿中使用, 而其他染料, 如 Cy5 或亚历克莎系列, 也已被证明是可行的, 在我们的实验室) 和生物素标签, its 被固定在基板上的生物素-阿维丁键。上链与 rgd 肽和黑洞淬火结合, 淬火 cy3 约98% 淬火效率 26,27。根据本文提出的协议, ITS 在基板上的涂层密度约为 1100/μm 2.这是我们先前通过遵循相同的涂层协议29在中和功能化基板上包覆的 18 bp 生物基化 dsdna 的密度。当细胞粘附在涂有 ITS 的基板上时, 整合蛋白通过 RGD 将 its 结合起来, 并将张力传递到 ITS。ITS 有一个特定的张力耐受性 (ttol), 它被定义为在 dsDNA 内机械分离 Its dsDNA 的张力阈值。ITS 被整合蛋白张力断裂导致淬火器与随后发出荧光的染料分离。因此, 将不可见的整合素张力转化为荧光信号, 并通过荧光成像映射细胞力。
为了证明 its 的应用, 我们在这里使用鱼类角化细胞, 这是一种广泛使用的细胞迁移模型,用于细胞迁移研究 30,31, 32, chok1 细胞, 一种常用的非运动细胞系, 和 nih 3t3 成纤维细胞。还对整合素张力和细胞结构进行了成像。
Protocol
Representative Results
Discussion
ITS 是一种高度可访问但功能强大的细胞力映射技术, 在合成和应用方面都是有效的。在所有材料准备好的情况下, ITS 可以在1天内合成。在实验过程中, 在细胞电镀之前只需要三个步骤的表面涂层。最近, 我们进一步简化了涂层程序, 将 ITS 与牛血清白蛋白直接连接, 从而使 ITS 能够直接物理吸附到玻璃或聚苯乙烯表面 33.ITS 将细胞力信号的荧光强度提升到可比的细胞结构成像水平。?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了爱荷华州立大学和国家普通医学研究所 (R35GM128747) 提供的启动基金的支持。
Materials
| BSA-biotin | Sigma-Aldrich | A8549 | |
| Neutravidin | Thermo Fisher Scientific | 31000 | |
| Streptavidin | Thermo Fisher Scientific | 434301 | |
| upper strand DNA | Integrated DNA Technologies | N/A | Customer designed. DNA sequence is shown in PROTOCOL section |
| lower strand DNA | Integrated DNA Technologies | N/A | Customer designed. DNA sequences are shown in PROTOCOL section. |
| sulfo-SMCC | Thermo Fisher Scientific | A39268 | |
| Cyclic peptide RGD with an amine group | Peptides International | PCI-3696-PI | |
| IMDM | ATCC | 62996227 | |
| FBS | ATCC | 302020 | |
| Penicillin | gibco | 15140122 | |
| TCEP | Sigma-Aldrich | C4706 | |
| 200 uL petri dish | Cellvis | D29-14-1.5-N | |
| NanoDrop 2000 | Thermo Scientific | N/A | spectrometer |
| SE410 Tall Air-Cooled Vertical Protein Electrophoresis Unit | Hoefer | SE410-15-1.5 | Device for electroporesis |
| CHO-K1 cell line | ATCC | CCL-61 | |
| NIH/3T3 cell line | ATCC | CRL-1658 | |
| Anti-Vinculin Antibody | EMD Millipore | 90227 | Primary antibody for vinculin immunostaining |
| Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 | Invitrogen | A28175 | Secondary antibody for vinculin immunostaining |
| Alexa Fluor 647 Phalloidin | Invitrogen | A22287 | |
| Eclipse Ti | Nikon | N/A | microscope |
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