Revealing Ca2+ oscillations in regulation of mitosis using optogenetics

• Main Authors: Chi-SianYu, 余紀賢
• Other Authors: Wen-Tai Chiu
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/twny4d

碩士 === 國立成功大學 === 生物醫學工程學系 === 106 === Calcium (Ca2+) is a vital signal transduction element involved in the modulation of many cellular functions. In particular, intracellular Ca2+ is crucial for orderly cell cycle progression and plays an essential role in regulation of cell proliferation. Recently, in vivo and in vitro studies have demonstrated that Ca2+ signaling is associated with cell growth. In this study, we propose an investigation of Ca2+ signaling in mitotic progression. However, it is difficult to control biological processes with high spatial and temporal precision. Optogenetic technologies, a new approach to spatial and temporal precision, have been developed and applied to control cell activity in many biomedical studies. Therefore, we set up optogenetic platforms that can be placed in an incubator or on a microscope with 470 nm blue light illumination where the optical parameters (power, frequency, duty cycle) can be modulated using a functional generator. The stable U2OS cell line overexpressing Ca2+ translocating channelrhodopsin (CatCh) is used to control Ca2+ influx using an illuminating 470 nm blue light. Then, we demonstrate a reversible synchronization treatment with RO-3306 in order to provide the late G2 arrest and make sure that cells would enter mitosis immediately after release. Our results showed that high frequency Ca2+ oscillations disturbed mitotic progression. However, some patterns of low frequency Ca2+ oscillations promoted mitosis at specific time points. It is thus possible to clarify the effect of different Ca2+ signals in mitotic progression.