Using light to shape chemical gradients for parallel and automated analysis of chemotaxis.
Using light to shape chemical gradients for parallel and automated analysis of chemotaxis.
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DOI：
10.15252/msb.20156027
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发表时间：
2015-04-23
影响因子：
9.9
通讯作者：
Meyer T
中科院分区：
文献类型：
--
作者：
Collins SR;Yang HW;Bonger KM;Guignet EG;Wandless TJ;Meyer T
大量的分子成分已经被确定，它们调节真核细胞向化学诱导剂来源的定向迁移。然而，这个系统的组成部分是如何连接在一起以协调反应的多个方面，如方向性、速度和对刺激的敏感性，仍然知之甚少。在这里，我们开发了一种方法，通过测量速度变化和定向精度，光学地塑造化学吸引梯度，并分析96孔格式中每孔数百个活细胞的细胞趋化反应。然后，我们系统地表征了285个siRNA扰动的迁移和趋化表型。一个关键的发现是，G蛋白GIα亚单位选择性地控制迁移方向，而受体和Gβ亚单位成比例地控制速度和方向。此外，我们证明中性粒细胞对FMLF的梯度有持久的趋化反应，但对ATP的梯度的反应是短暂的。我们介绍的方法适用于各种化学线索和系统扰动，可用于测量多个细胞迁移和信号参数，并与低分辨率和高分辨率荧光显微镜兼容。
Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.