Department of Mechanical Engineering, Stanford University, Stanford, CA 94305-4045, USA.
Integr Biol (Camb). 2013 Aug;5(8):1076-85. doi: 10.1039/c3ib40025e.
Besides its cooperating effects on stem cell proliferation and survival, Kit ligand (KL) is a potent chemotactic protein. While transwell assays permit studies of the frequency of migrating cells, the lack of direct visualization precludes dynamic chemotaxis studies. In response, we utilize microfluidic chambers that enable direct observation of murine bone marrow-derived mast cells (BMMC) within stable KL gradients. Using this system, individual Kit+ BMMC were quantitatively analyzed for migration speed and directionality during KL-induced chemotaxis. Our results indicated a minimum activating threshold of ~3 ng ml(-1) for chemoattraction. Analysis of cells at KL concentrations below 3 ng ml(-1) revealed a paradoxical chemorepulsion, which has not been described previously. Unlike chemoattraction, which occurred continuously after an initial time lag, chemorepulsion occurred only during the first 90 minutes of observation. Both chemoattraction and chemorepulsion required the action of G-protein coupled receptors (GPCR), as treatment with pertussis toxin abrogated directed migration. These results differ from previous studies of GPCR-mediated chemotaxis, where chemorepulsion occurred at high ligand concentrations. These data indicate that Kit-mediated chemotaxis is more complex than previously understood, with the involvement of GPCRs in addition to the Kit receptor tyrosine kinase and the presence of both chemoattractive and chemorepellent phases.
除了对干细胞增殖和存活的协同作用外，Kit 配体（KL）还是一种有效的趋化蛋白。虽然 Transwell 测定允许研究迁移细胞的频率，但缺乏直接可视化会妨碍动态趋化性研究。为了解决这个问题，我们使用微流控室，使我们能够在稳定的 KL 梯度内直接观察到来自鼠骨髓的肥大细胞（BMMC）。使用该系统，我们对 Kit+BMMC 进行了定量分析，以研究 KL 诱导的趋化作用过程中的迁移速度和方向性。我们的结果表明，趋化吸引的最小激活阈值约为 3ng/ml。对 KL 浓度低于 3ng/ml 的细胞进行分析表明，存在以前未描述过的悖论性趋化排斥现象。与初始滞后后连续发生的趋化吸引不同，趋化排斥仅在观察的前 90 分钟内发生。趋化吸引和趋化排斥都需要 G 蛋白偶联受体（GPCR）的作用，因为百日咳毒素处理消除了定向迁移。这些结果与先前关于 GPCR 介导的趋化性的研究不同，在先前的研究中，趋化排斥发生在高配体浓度下。这些数据表明，Kit 介导的趋化性比以前理解的更为复杂，涉及 GPCR 以及 Kit 受体酪氨酸激酶，并且存在趋化吸引和趋化排斥两个阶段。
