Although the ciliate Tetrahymena is a good model for the study of chemotaxis, its profound motility makes it difficult to monitor intracellular calcium (Ca2+) 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 Ca2+ 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 Ca2+ in the ciliate increased up to three-fold, and its distribution was skewed in the direction of NMDA stimulation. However, the Ca2+ 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 Ca2+ in T. pyriformis during chemotaxis.
Bibliographical noteFunding Information:
This research was equally supported by the Nano/Bioscience & Technology Program (M10536090002-05N3609-00210) of the Ministry of Education, Science, and Technology (MEST), the SRC program of the Korea Science and Engineering Foundation (KOSEF) through the Center for Intelligent Nano-Bio Materials at Ewha Womans University (Grant R11-2005-008-02003-0), and the Korean Research Foundation Grant (MOEHRD, Basic Research Promotion Fund) (KRF-2006-005-J04002).
- Ca signaling
- NMDA receptor