Nam Seong-Won, Kim Sang-Tae, Lee Kang-Mu, Kim So Hyun, Kou Songzi, Lim Jeesun, Hwang Hyejin, Joo Min Kyung, Jeong Byeongmoon, Yoo Seung Hyun, Park Sungsu
Department of Chemistry and Nano Sciences (BK21), Ewha Womans University, Daehyundong, Seodaemungu, Seoul 120-750, Korea.
Protist. 2009 May;160(2):331-42. doi: 10.1016/j.protis.2008.10.005. Epub 2009 Feb 11.
Although the ciliate Tetrahymena is a good model for the study of chemotaxis, its profound motility makes it difficult to monitor intracellular calcium (Ca(2+)) changes induced by chemotactic stimuli. In this study, we report a microfluidic-based chemotaxis system generating directional chemotactic gradients under highly viscous conditions, suppressing T. pyriformis motility, and allowing for the stable confocal imaging of changes in intracellular Ca(2+) in the ciliate. Once the viscous condition was achieved, directional chemical gradients were formed inside the center chamber via the release of N-methyl-d-aspartate (NMDA), a known chemoattractant, from the surrounding chemical reservoirs into the center chamber. As a result, intracellular Ca(2+) in the ciliate increased up to three-fold, and its distribution was skewed in the direction of NMDA stimulation. However, the Ca(2+) in ciliates pretreated with phospholipase C (PLC) or phosphatidylinositol-3-kinase (PI3K) blockers did not increase even after stimulation. Additionally, the PI3K blocker induced the secretion of granules, the size of which was dependent on the concentration of the blocker. Collectively, the results indicate that both PLC and PI3K perform pivotal roles in controlling the levels of intracellular Ca(2+) in T. pyriformis during chemotaxis.
尽管纤毛虫四膜虫是研究趋化性的良好模型，但其强烈的运动性使得监测趋化性刺激诱导的细胞内钙（Ca(2+)）变化变得困难。在本研究中，我们报告了一种基于微流控的趋化性系统，该系统在高粘性条件下产生定向趋化梯度，抑制梨形四膜虫的运动，并允许对纤毛虫细胞内Ca(2+)变化进行稳定的共聚焦成像。一旦达到粘性条件，通过将已知的化学引诱剂N-甲基-D-天冬氨酸（NMDA）从周围的化学储库释放到中心腔室中，在中心腔室内形成定向化学梯度。结果，纤毛虫细胞内的Ca(2+)增加了三倍，并且其分布在NMDA刺激的方向上发生了偏移。然而，用磷脂酶C（PLC）或磷脂酰肌醇-3-激酶（PI3K）阻滞剂预处理的纤毛虫中的Ca(2+)即使在刺激后也没有增加。此外，PI3K阻滞剂诱导颗粒分泌，其大小取决于阻滞剂的浓度。总体而言，结果表明PLC和PI3K在趋化性过程中控制梨形四膜虫细胞内Ca(2+)水平方面都起着关键作用。
