Gradient sensing in defined chemotactic fields
Gradient sensing in defined chemotactic fields
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DOI：
10.1039/c0ib00033g
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发表时间：
2010-01-01
影响因子：
2.5
通讯作者：
Rappel, Wouter-Jan
中科院分区：
文献类型：
--
作者：
Skoge, Monica;Adler, Micha;Rappel, Wouter-Jan
细胞通过改变它们的生理、生长模式和行为来对各种分泌的分子作出反应。运动细菌和真核细胞可以感受到细胞外的化学引诱物和化学排斥物，并改变它们的运动。通过这种方式，成纤维细胞和白细胞可以找到它们的方式到达损伤部位，癌细胞可以在释放生长因子的部位归巢。社会性变形虫如网骨藻对cAMP具有趋化性，它们在开始发育后数小时分泌cAMP。已知这些真核细胞能够感知极浅的梯度，但其高度敏感性背后的过程仍在很大程度上未知。在这项研究中，我们确定了发展中的细胞Dictyosteelium discoideum稳定的线性梯度的cAMP的不同陡度产生的2 μ m深的梯度室的微流控装置的响应。通过两个80 μ m深的流通通道之间的分子扩散产生梯度，其中一个通道灌注cAMP溶液，另一个通道灌注缓冲液，作为连续补充的源和汇。这些低天花板梯度室在垂直维度上限制细胞，促进共聚焦成像，使得荧光标记蛋白质的亚细胞定位可以被跟踪长达30分钟而没有明显的光毒性。趋化细胞通过变平和伸长进入这些低顶室，然后几乎像不受约束的细胞一样迅速移动。通过使用融合到绿色荧光蛋白（RBD-GFP）的Ras结合结构域（RasGTP）定位激活的Ras，我们观察到在伪足尖端快速出现膜相关斑块。这些补丁仍然与伪足，而他们继续延伸，但迅速解体时，伪足停滞和细胞移动过去。同样，当梯度关闭时，与局部RasGTP相关的荧光迅速消失。RasGTP补丁的大小和持久性与扩展的伪足的相关性可以设置规则，了解信号转导机制如何将弱的外部信号转换为强的方向性偏差。
Cells respond to a variety of secreted molecules by modifying their physiology, growth patterns, and behavior. Motile bacteria and eukaryotic cells can sense extracellular chemoattractants and chemorepellents and alter their movement. In this way fibroblasts and leukocytes can find their way to sites of injury and cancer cells can home in on sites that are releasing growth factors. Social amoebae such as Dictyostelium are chemotactic to cAMP which they secrete several hours after they have initiated development. These eukaryotic cells are known to be able to sense extremely shallow gradients but the processes underlying their exquisite sensitivity are still largely unknown. In this study we determine the responses of developed cells of Dictyostelium discoideum to stable linear gradients of cAMP of varying steepness generated in 2 mu m deep gradient chambers of microfluidic devices. The gradients are generated by molecular diffusion between two 80 mu m deep flow-through channels, one of which is perfused with a solution of cAMP and the other with buffer, serving as continuously replenished source and sink. These low ceiling gradient chambers constrained the cells in the vertical dimension, facilitating confocal imaging, such that subcellular localization of fluorescently tagged proteins could be followed for up to 30 min without noticeable phototoxicity. Chemotactic cells enter these low ceiling chambers by flattening and elongating and then move almost as rapidly as unconstrained cells. By following the localization of activated Ras (RasGTP) using a Ras Binding Domain fused to Green Fluorescent Protein (RBD-GFP), we observed the rapid appearance of membrane associated patches at the tips of pseudopods. These patches remained associated with pseudopods while they continued to extend but were rapidly disassembled when pseudopods stalled and the cell moved past them. Likewise, fluorescence associated with localized RasGTP rapidly disappeared when the gradient was turned off. Correlation of the size and persistence of RasGTP patches with extension of pseudopods may set the rules for understanding how the signal transduction mechanisms convert a weak external signal to a strong directional bias.