Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding.
Visualizing Spatiotemporal Dynamics of Intercellular Mechanotransmission upon Wounding.
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DOI：
10.1021/acsphotonics.8b00383
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发表时间：
2018-08
期刊：
影响因子：
7
通讯作者：
Pengzhi Wang;Jing Liang;Linda Z. Shi;Yi Wang;Ping Zhang;Mingxing Ouyang;D. Preece;Qin Peng;Lunan Shao;Jason Fan;Jie Sun;Shawn S. Li;M. Berns;Huimin Zhao;Yingxiao Wang
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文献类型：
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作者：
Pengzhi Wang;Jing Liang;Linda Z. Shi;Yi Wang;Ping Zhang;Mingxing Ouyang;D. Preece;Qin Peng;Lunan Shao;Jason Fan;Jie Sun;Shawn S. Li;M. Berns;Huimin Zhao;Yingxiao Wang
在细胞与细胞之间的通信中，物理和生化信号之间的相互作用对于信息交换和功能协调至关重要，特别是在多细胞生物中。然而，在单个活细胞中可视化细胞间信号传导动力学仍然是一个挑战。在这里，我们报告了一个光子的方法，激光显微剪刀和Förster共振能量转移（FRET）显微镜的基础上，研究细胞间的信号传递。首先，使用我们的高通量筛选平台，我们开发了一种高灵敏度的基于FRET的Src激酶生物传感器（SCAGE），Src激酶是细胞间相互作用和信号级联的关键调节因子。值得注意的是，SCAGE在活的哺乳动物细胞中显示出比原始生物传感器高出40倍以上的灵敏度增强。接下来，在飞秒激光脉冲局部切断细胞间的物理连接后，SCAGE能够可视化相邻细胞间的瞬时Src激活。最后，我们发现细胞与细胞接触丧失后观察到的短暂Src激活取决于细胞骨架的被动结构支持，而不是主动肌动球蛋白收缩性。因此，通过精确地引入局部物理扰动并直接可视化随后的信号事件的时空传输，我们的综合方法可以广泛地应用于以单细胞分辨率模拟和研究创伤过程。这种集成的方法与高度敏感的FRET为基础的生物传感器提供了一个独特的系统，以促进我们深入了解的分子机制的物理-生化基础的细胞间耦合和创伤过程。
During cell-to-cell communications, the interplay between physical and biochemical cues is essential for informational exchange and functional coordination, especially in multicellular organisms. However, it remains a challenge to visualize intercellular signaling dynamics in single live cells. Here, we report a photonic approach, based on laser microscissors and Förster resonance energy transfer (FRET) microscopy, to study intercellular signaling transmission. First, using our high-throughput screening platform, we developed a highly sensitive FRET-based biosensor (SCAGE) for Src kinase, a key regulator of intercellular interactions and signaling cascades. Notably, SCAGE showed a more than 40-fold sensitivity enhancement than the original biosensor in live mammalian cells. Next, upon local severance of physical intercellular connections by femtosecond laser pulses, SCAGE enabled the visualization of a transient Src activation across neighboring cells. Lastly, we found that this observed transient Src activation following the loss of cell-cell contacts depends on the passive structural support of cytoskeleton but not on the active actomyosin contractility. Hence, by precisely introducing local physical perturbations and directly visualizing spatiotemporal transmission of ensuing signaling events, our integrated approach could be broadly applied to mimic and investigate the wounding process at single-cell resolutions. This integrated approach with highly sensitive FRET-based biosensors provides a unique system to advance our in-depth understanding of molecular mechanisms underlying the physical-biochemical basis of intercellular coupling and wounding processes.