鈣離子是一個重要的訊息傳遞因子調控著許多細胞內的功能與作用，在細胞週期的各個階段中扮演著決定性的重要角色。由於近期的研究指出了一些鈣離子和細胞分裂生長之間的關聯，在本研究中我們要探討鈣離子波對於細胞有絲分裂期的影響。然而要掌握時間和空間的精確性去調控生理反應是很困難的，因此我們會利用光遺傳學這個近期被發展且應用在許多生物醫學研究中的方法來克服這一點。在本研究中我們應用了具有470奈米波長藍光的光遺傳學系統，藉由示波器來調控不同的光波參數在培養箱內外進行觀察。並且使用了穩定表達CatCh蛋白的U2OS人類骨肉瘤細胞可以藉由藍光的激發來引發細胞外不同的鈣離子波流進入細胞。然後再利用RO-3306這個可以有效同步化細胞週期的藥物來將細胞週期停滯在G2晚期，藉此來觀察藥物釋放後進入M期的細胞。本篇的研究發現了在比較高頻率波段的鈣離子波會對細胞造成損壞並使細胞無法進入M期，而在一些特定的低頻率波段則可以促進在M期某些階段的進展。本研究釐清了鈣離子波在細胞週期M期各個階段造成的影響。

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.

Abstract…………………………………………………………………………………….i
中文摘要…………………………………………………………………………………iii
Acknowledgement………………………………………………………………………iv
Contents……………………………………………………………………………………v
Figure contents…………………………………………………………………………viii
Chapter 1. Introduction…………………………………………………………………1
1.1 The versatility and universality of Ca2+ signaling……………………………………1
1.1.1 Ca2+ oscillation……………………………………………………………………2
1.2 Cell cycle progression………………………………………………………………3
1.2.1 G2/M checkpoint………………………………………………………………4
1.2.2 Mitotic progression……………………………………………………………6
1.2.3 Cytokinesis……………………………………………………………………7
1.3 Ca2+ signals in mitosis………………………………………………………………8
1.3.1 CaMKII regulation in mitosis…………………………………………………10
1.3.2 Ca2+ regulation in cytokinesis…………………………………………………10
1.4 Optical tools of Ca2+ ion……………………………………………………………11
1.5 The specific aim of this study………………………………………………………12
Chapter 2. Materials and Methods……………………………………………………13
2.1 Cell culture…………………………………………………………………………13
2.2 Plasmid transfection…………………………………………………………………13
2.3 Optogenetic platform………………………………………………………………13
2.4 Microscopic image…………………………………………………………………14
2.5 Flow cytometry……………………………………………………………………14
2.6 Immunofluorescence staining………………………………………………………15
2.7 Fluorescence staining………………………………………………………………15
2.8 Western blotting……………………………………………………………………..16
2.9 Statistical analysis…………………………………………………………………...16
Chapter 3. Results………………………………………………………………………18
3.1 Mitotic cells were collected using a synchronization process via an RO-3306 treatment…………………………………………………………………………………18
3.2 High-frequency Ca2+ oscillations induced defect in mitotic progression………..….19
3.3 Low-frequency Ca2+ oscillations promoted the critical step of G2/M transition……20
3.4 Optogenetic-engineered Ca2+ oscillations affect the progression of metaphase/anaphase transition and cytokinesis but do not affect the progression of nuclear envelope breakdown…………………………………………………………21
Chapter 4. Discussion……………………………………………………………………24
References………………………………………………………………………………28
Figures……………………………………………………………………………………36
Supplemental figures……………………………………………………………………59

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