Molecular Tension Probes for Imaging Forces at the Cell Surface.
Molecular Tension Probes for Imaging Forces at the Cell Surface.
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DOI：
10.1021/acs.accounts.7b00305
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发表时间：
2017-12-19
影响因子：
18.3
通讯作者：
Salaita K
中科院分区：
文献类型：
--
作者：
Liu Y;Galior K;Ma VP;Salaita K
机械力对于从转录和翻译到细胞粘附、迁移和分化的各种生物过程至关重要。通过激活机械敏感信号通路，细胞感知并响应周围环境的物理刺激，这一过程被广泛称为机械转导。在细胞膜上，许多信号传导受体，例如整合素、钙粘蛋白和 T 或 B 细胞受体，与其相邻细胞或细胞外基质 (ECM) 表面上的配体结合，以介导机械转导。连接后，这些受体-配体键传递皮牛顿（pN）机械力，该机械力部分由细胞骨架产生。重要的是，这些力暴露了机械敏感蛋白内的神秘位点，并调节受体-配体复合物的结合动力学（开/关速率），以进一步微调机械转导和相应的细胞行为。在过去的三十年中，已经开发了两类方法来测量细胞受体力。第一类是牵引力显微镜 (TFM) 和微柱阵列探测器 (mPAD)。在这些方法中，细胞在弹性聚合物或在机械力下变形的微结构上培养。第二类技术是单分子力谱（SMFS），包括原子力显微镜（AFM）、光/磁镊子和生物膜力探针（BFP）。在 SMFS 中，实验者施加外力来连续探测单个细胞或单个受体-配体复合物的力学，一次一个键。尽管这些技术很强大，但 SMFS 的有限通量和 TFM 的 nN 力敏感性阻碍了力转导分子机制的进一步阐明。在本文中，我们介绍了最近出现的分子张力荧光显微镜（MTFM）作为活细胞受体力学分子成像的新兴工具。 MTFM 探针由可延伸的接头（例如聚合物、寡核苷酸或蛋白质）组成，两侧是荧光团和猝灭剂。通过测量固定化 MTFM 探针的荧光发射，可以推断出接头的延伸和外部施加的力。因此，MTFM 结合了 TFM 和 SMFS 的各个方面，以光学方式报告整个细胞表面的受体力，具有 pN 灵敏度。具体来说，我们对 MTFM 探针设计进行了深入的回顾，其中包括可伸缩的“弹簧”、光谱尺、表面固定化学和配体设计策略。我们还通过讨论涉及 EGFR、整合素和 T 细胞受体信号通路的 pN 力的案例研究，展示了不同版本 MTFM 探针的优点和缺点。最后，我们主要从化学家的角度对下一代 MTFM 探针设计的挑战和机遇进行了简要的展望。
Mechanical forces are essential for a variety of biological processes ranging from transcription and translation to cell adhesion, migration and differentiation. Through the activation of mechanosensitive signaling pathways, cells sense and respond to physical stimuli from the surrounding environment, a process widely known as mechanotransduction. At the cell membrane, many signaling receptors, such as integrins, cadherins and T or B-cell receptors, bind to their ligands on the surface of adjacent cells or the extracellular matrix (ECM) to mediate mechanotransduction. Upon ligation, these receptor-ligand bonds transmit piconewton (pN) mechanical forces that are generated, in part, by the cytoskeleton. Importantly, these forces expose cryptic sites within mechanosensitive proteins and modulate the binding kinetics (on/off rate) of receptor-ligand complexes to further fine-tune mechanotransduction and the corresponding cell behavior. Over the past three decades, two categories of methods have been developed to measure cell receptor forces. The first class is traction force microscopy (TFM) and micro-post array detectors (mPADs). In these methods, cells are cultured on elastic polymers or microstructures that deform under mechanical forces. The second category of techniques is single molecule force spectroscopy (SMFS) including atomic force microscopy (AFM), optical/magnetic tweezers and biomembrane force probe (BFP). In SMFS, experimenters apply external forces to probe the mechanics of individual cells or single receptor-ligand complexes, serially, one bond at a time. Although these techniques are powerful, the limited throughput of SMFS and the nN force sensitivity of TFM have hindered further elucidation of molecular mechanisms of mechanotransduction. In this account, we introduce the recent advent of molecular tension fluorescence microscopy (MTFM) as an emerging tool for molecular imaging of receptor mechanics in living cells. MTFM probes are composed of an extendable linker, such as polymer, oligonucleotide, or protein, and flanked by a fluorophore and quencher. By measuring the fluorescence emission of immobilized MTFM probes, one can infer the extension of the linker and the externally applied force. Thus, MTFM combines aspects of TFM and SMFS to optically report receptor forces across the entire cell surface with pN sensitivity. Specifically, we provide an in-depth review of MTFM probe design, which includes the extendable “spring”, spectroscopic ruler, surface immobilization chemistry, and ligand design strategies. We also demonstrate the strengths and weaknesses of different versions of MTFM probes by discussing case studies involving the pN forces involved in EGFR, integrin and T-cell receptor signaling pathways. Lastly, we present a brief future outlook, primarily from a chemists’ perspective, on the challenges and opportunities for the design of next generation MTFM probes.
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影响因子：
10.8
作者：
Choi CL;Koski KJ;Sivasankar S;Alivisatos AP
通讯作者：
Alivisatos AP
DOI：
10.1083/jcb.200810002
发表时间：
2009-06-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Kong F;García AJ;Mould AP;Humphries MJ;Zhu C
通讯作者：
Zhu C
DOI：
10.1073/pnas.1212429109
发表时间：
2012-11-13
影响因子：
11.1
作者：
Asally, Munehiro;Kittisopikul, Mark;Sueel, Guerol M.
通讯作者：
Sueel, Guerol M.
影响因子：
19.6
作者：
Brugués A;Anon E;Conte V;Veldhuis JH;Gupta M;Colombelli J;Muñoz JJ;Brodland GW;Ladoux B;Trepat X
通讯作者：
Trepat X
影响因子：
15
作者：
Chen, Hu;Yuan, Guohua;Winardhi, Ricksen S.;Yao, Mingxi;Popa, Ionel;Fernandez, Julio M.;Yan, Jie
通讯作者：
Yan, Jie