鈣離子在諸多細胞生理活動扮演重要次級傳訊者，例如:細胞增生與死亡、肌肉收縮、基因調控或者是癌症轉移與侵入。然而，現今研究尚無法在時間及空間上精準調控細胞內鈣離子。故此研究運用光遺傳學方法調控鈣離子依賴性相關轉錄因子(NFAT、NFκB及CREB)之活化以及對G1/S與G2/M過渡期中細胞週期之運轉。本研究建立客製化的光遺傳系統平台用以控制在活生物體細胞的刺激。藉由調控相關光參數(如光能量、頻率、作用時間)給予470 nm藍光刺激作用於人類骨肉瘤細胞(U2OS)表達之光敏感通道(calcium translocating channelrhodopsin, CatCh)而引發細胞外不同的鈣離子波流入細胞內部。本研究調變光遺傳學系統中的不同能量，進而產生一系列的鈣離子振盪波。研究結果顯示光遺傳學技術充分定義出鈣離子訊息的空間、時間和幅度特性，發現NFAT活化需要大量鈣離子，反之NFκB則需要少量的鈣離子，對於大量的鈣離子反而不反應，另一方面，不管是大量或少量的鈣離子都可活化CREB。此外，我們的研究發現鈣離子主要參與細胞週期中G1/S過渡期而非G2/M過渡期的運轉，並且不同類型的鈣離子振盪波會決定如何調控細胞週期運轉。本研究解碼動態鈣離子振盪波如何進行訊息傳遞且有發展相關鈣離子訊息研究之潛力。

Calcium ion (Ca2+) plays an one of the major second messengers in many physio-logical processes, such as cell proliferation, death, muscle contraction, gene regulation, and cancer migration and invasion. However, control of intracellular Ca2+ with spatial and temporal precision is a limitation in the current studies on these topics. Here, we present an optogenetic method to manipulate Ca2+-dependent gene transcription using three tran-scription factors, including NFAT (nuclear factor of activated T-cells), CREB (cAMP re-sponse element-binding protein), and NFκB (nuclear factor-κB) and the regulation of cell cycle progression in the transition of G1/S and G2/M. The customized optogenetics plat-form was setup for the purpose of controlling stimulations in living cells. U2OS cells overexpressing calcium translocating channelrhodopsin (CatCh) can be used to mediate Ca2+ influx by illumination with 470 nm blue light. The profiles of a Ca2+ wave can be regulated by using various parameters of blue light illumination, such as the power, fre-quency, duty cycle, and duration. By manipulating optogenetic induced Ca2+ influx, a se-ries of the patterns of Ca2+ oscillation with different parameters of power can be generated. The results show that optogenetic technology defines the spatial, temporal and amplitude features of Ca2+ signals. The finding indicates that NFAT activation requires large amounts of Ca2+, whereas NFκB only needs a small amount of Ca2+. On the other hand, CREB can be activated with not only large amounts of Ca2+ but also with small amounts of Ca2+. In addition, it is understood that Ca2+ participates in the G1/S transition of the cell cycle rather in the G2/M transition. And, the patterns of Ca2+ oscillation determine how the cell cycle progression is driven. This study resolves dynamic components of Ca2+ os-cillation with regard to decoding Ca2+ signals and has the potential to development Ca2+ signalling related research.

Berridge MJ, Bootman MD, Roderick HL. (2003) Calcium signalling: dynamics, ho-meostasis and remodelling. Nat Rev Mol Cell Biol 4(7):517-29.
Berridge MJ. (2012) Calcium signalling remodelling and disease. Biochem Soc Trans 40(2):297-309.
Bertoli C, Skotheim JM, de Bruin RA. (2013) Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 14(8):518-28.
Bito H, Deisseroth K, Tsien RW. (1996) CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87(7):1203-14.
Boulware MJ, Marchant JS. (2008) Timing in cellular Ca2+ signaling. Curr Biol 18(17):R769-R776.
Chamero P, Villalobos C, Alonso MT, García-Sancho J. (2002) Dampening of cytosolic Ca2+ oscillations on propagation to nucleus. J Biol Chem 277(52):50226-9.
Cheng KT, Liu X, Ong HL, Swaim W, Ambudkar IS. (2011) Local Ca2+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca2+ signals required for specific cell functions. PLoS Biol 9(3):e1001025.
Chen YW, Chen YF, Chen YT, Chiu WT, Shen MR. (2016) The STIM1-Orai1 pathway of store-operated Ca2+ entry controls the checkpoint in cell cycle G1/S transition. Sci Rep 6:22142.
Clapham DE. (2007) Calcium signaling. Cell 131(6):1047-58.
Dedkova EN, Sigova AA, Zinchenko VP. (2000) Mechanism of action of calcium iono-phores on intact cells: ionophore-resistant cells. Membr Cell Biol 13(3):357-68.
Deisseroth K. (2011) Optogenetics. Nat Methods 8(1):26-9.
Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. (1997) Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386(6627):855-8.
Feske S. (2007) Calcium signalling in lymphocyte activation and disease. Nat Rev Im-munol. 7(9):690-702.
Foutz TJ, Arlow RL, McIntyre CC. (2012) Theoretical principles underlying optical stimulation of a channelrhodopsin-2 positive pyramidal neuron. J Neurophysiol 107(12):3235-45.
Fric J, Zelante T, Wong AY, Mertes A, Yu HB, Ricciardi-Castagnoli P. (2012) NFAT control of innate immunity. Blood 120(7):1380-9.
Fric J, Lim CX, Mertes A, Lee BT, Viganò E, Chen J, Zolezzi F, Poidinger M, Larbi A, Strobl H, Zelante T, Ricciardi-Castagnoli P. (2014) Calcium and calcineurin-NFAT signaling regulate granulocyte-monocyte progenitor cell cycle via Flt3-L. Stem Cells 32(12):3232-44.
Gilmore TD. (2006) Introduction to NF-kappaB: players, pathways, perspectives. Onco-gene 25(51):6680-4.
Hannanta-Anan P, Chow BY. (2016) Optogenetic Control of Calcium Oscillation Wave-form Defines NFAT as an Integrator of Calcium Load. Cell Syst 2(4):283-8.
Hayden MS, Ghosh S. (2008) Shared principles in NF-kappaB signaling. Cell 132(3):344-62.
Hoesel B, Schmid JA. (2013) The complexity of NF-κB signaling in inflammation and cancer. Mol Cancer 2;12:86.
Takuwa N, Zhou W, Takuwa Y. (1995) Calcium, calmodulin and cell cycle progression. Cell Signal 7(2):93-104.
Hogan PG, Chen L, Nardone J, Rao A. (2003) Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 17(18):2205-32.
Hogan PG, Lewis RS, Rao A. (2010) Molecular basis of calcium signaling in lympho-cytes: STIM and ORAI. Annu Rev Immunol 28:491-533.
John PC, Mews M, Moore R. (2001) Cyclin/Cdk complexes: their involvement in cell cycle progression and mitotic division. Protoplasma 216(3-4):119-42.
Kahl CR, Means AR. (2003) Regulation of cell cycle progression by calci-um/calmodulin-dependent pathways. Endocr Rev 24(6):719-36.
Kapur N, Mignery GA, Banach K. (2007) Cell cycle-dependent calcium oscillations in mouse embryonic stem cells. Am J Physiol Cell Physiol 292(4):C1510-8.
Kar P, Nelson C, Parekh AB. (2012) CRAC channels drive digital activation and provide analog control and synergy to Ca2+-dependent gene regulation. Curr Biol 22(3):242-7.
Kar P, Parekh AB. (2015) Distinct spatial Ca2+ signatures selectively activate different NFAT transcription factor isoforms. Mol Cell 58(2):232-43.
Kaufman RJ, Malhotra JD. (2014) Calcium trafficking integrates endoplasmic reticu-lum function with mitochondrial bioenergetics. Biochim Biophys Acta 1843(10):2233-9.
Khan I, Tang E, Arany P. (2015) Molecular pathway of near-infrared laser phototoxicity involves ATF-4 orchestrated ER stress. Sci Rep 5:10581.
Kito H, Yamamura H, Suzuki Y, Yamamura H, Ohya S, Asai K, Imaizumi Y. (2015) Regulation of store-operated Ca2+ entry activity by cell cycle dependent up-regulation of Orai2 in brain capillary endothelial cells. Biochem Biophys Res Commun 459(3):457-62.
Kleinlogel S, Feldbauer K, Dempski RE, Fotis H, Wood PG, Bamann C, Bamberg E. (2011) Ultra light-sensitive and fast neuronal activation with the Ca2+-permeable chan-nelrhodopsin CatCh. Nat Neurosci 14(4):513-8.
Kuklina EM, Shirshev SV. (2001) Role of transcription factor NFAT in the immune re-sponse. Biochemistry (Mosc) 66(5):467-75.
Kushibiki T, Okawa S, Hirasawa T, Ishihara M. (2015) Optogenetic control of insulin secretion by pancreatic β-cells in vitro and in vivo. Gene Ther 22(7):553-9.
Kyung T, Lee S, Kim JE, Cho T, Park H, Jeong YM, Kim D, Shin A, Kim S, Baek J, Kim J, Kim NY, Woo D, Chae S, Kim CH, Shin HS, Han YM, Kim D, Heo WD. (2015) Optogenetic control of endogenous Ca2+ channels in vivo. Nat Biotech-nol 33(10):1092-6.
Lieben L, Carmeliet G. (2012) The Involvement of TRP Channels in Bone Homeostasis. Front Endocrinol (Lausanne) 3:99.
Madsen CP, Klausen TK, Fabian A, Hansen BJ, Pedersen SF, Hoffmann EK. (2012) On the role of TRPC1 in control of Ca2+ influx, cell volume, and cell cycle. Am J Physiol Cell Physiol 303(6):C625-34.
Marks AR. (1997) Intracellular calcium-release channels: regulators of cell life and death. Am J Physiol 272(2 Pt 2):597-605.
Martorana F, Guidotti G, Brambilla L, Rossi D. (2015) Withaferin A Inhibits Nuclear Factor-κB-Dependent Pro-Inflammatory and Stress Response Pathways in the Astrocytes. Neural Plast 2015:381964.
Mayr B, Montminy M. (2001) Transcriptional regulation by the phosphoryla-tion-dependent factor CREB. Nat Rev Mol Cell Biol 2(8):599-609.
Mohanty SK, Lakshminarayananan V. (2015) Optical Techniques in Optogenetics. J Mod Opt 62(12):949-970.
Müller MR, Rao A. (2010) NFAT, immunity and cancer: a transcription factor comes of age. Nat Rev Immunol 10(9):645-656.
Müller M, Cárdenas C, Mei L, Cheung KH, Foskett JK. (2011) Constitutive cAMP re-sponse element binding protein (CREB) activation by Alzheimer's disease prese-nilin-driven inositol trisphosphate receptor (InsP3R) Ca2+ signaling. Proc Natl Acad Sci U S A 108(32):13293-8.
Nankova B, Hiremagalur B, Menezes A, Zeman R, Sabban E. (1996) Promoter ele-ments and second messenger pathways involved in transcriptional activation of tyrosine hydroxylase by ionomycin. Brain Res Mol Brain Res 35(1-2):164-72.
Oheim M, van 't Hoff M, Feltz A, Zamaleeva A, Mallet JM, Collot M. (2014) New red-fluorescent calcium indicators for optogenetics, photoactivation and multi-color imaging. Biochim Biophys Acta 1843(10):2284-306.
O'Neill PR, Gautam N. (2014) Subcellu-lar optogenetic inhibition of G proteins generates signaling gradients and cell migration. Mol Biol Cell 25(15):2305-2314.
Ong HL, Jang SI, Ambudkar IS. (2012) Distinct contributions of Orai1 and TRPC1 to agonist-induced [Ca2+]i signals determine specificity of Ca2+-dependent gene expression. PLoS ONE 7(10):e47146.
Parkash J, Asotra K. (2010) Calcium wave signaling in cancer cells. Life Sci 87(19-22):587-95.
Parekh AB. (2011) Decoding cytosolic Ca2+ oscillations. Trends Biochem Sci 36(2):78-87.
Reinbothe TM, Safi F, Axelsson AS, Mollet IG, Rosengren AH. (2014) Optogenetic control of insulin secretion in intact pancreatic islets with β-cell-specific expression of Channelrhodopsin-2. Islets 6(1):e28095.
Resende RR, Adhikari A, da Costa JL, Lorençon E, Ladeira MS, Guatimosim S, Kihara AH, Ladeira LO. (2010) Influence of spontaneous calcium events on cell-cycle progression in embryonal carcinoma and adult stem cells. Biochim Biophys Acta 1803(2):246-60.
Resende RR, Andrade LM, Oliveira AG, Guimarães ES, Guatimosim S, Leite MF. (2013) Nucleoplasmic calcium signaling and cell proliferation: calcium signaling in the nucleus. Cell Commun Signal 11(1):14.
Roderick HL, Cook SJ. (2008) Ca2+ signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival. Nat Rev Cancer 8(5):361-75.
Russa AD, Maesawa C, Satoh Y. (2009) Spontaneous [Ca2+]i oscillations in G1/S phase-synchronized cells. J Electron Microsc (Tokyo) 58(5):321-9.
Santella L. (1998) The role of calcium in the cell cycle: facts and hypotheses. Biochem Biophys Res Commun 244(2):317-24.
Schwarz DS, Blower MD. (2016) The endoplasmic reticulum: structure, function and re-sponse to cellular signaling. Cell Mol Life Sci 73(1):79-94.
Sée V, Rajala NK, Spiller DG, White MR. (2004) Calcium-dependent regulation of the cell cycle via a novel MAPK-NFkB pathway in Swiss 3T3 cells. J Cell Biol 166(5):661-72.
Smedler E, Uhlén P. (2014) Frequency decoding of calcium oscillations. Biochim Bio-phys Acta 1840(3):964-9.
Song S, Li J, Zhu L, Cai L, Xu Q, Ling C, Su Y, Hu Q. (2012) Irregular Ca2+ oscilla-tions regulate transcription via cumulative spike duration and spike amplitude. J Biol Chem 287(48):40246-55.
Stroh A, Tsai HC, Wang LP, Zhang F, Kressel J, Aravanis A, Santhanam N, Deisseroth K, Konnerth A, Schneider MB. (2011) Tracking stem cell differentiation in the setting of automated optogenetic stimulation. Stem Cells 29(1):78-88.
Tomida T, Hirose K, Takizawa A, Shibasaki F, Iino M. (2003) NFAT functions as a working memory of Ca2+ signals in decoding Ca2+ oscillation. EMBO J 22(15):3825-32.
Uhlén P, Fritz N. (2010) Biochemistry of calcium oscillations. Biochem Biophys Res Commun 396(1):28-32.
Wang Y, He H, Li S, Liu D, Lan B, Hu M, Cao Y, Wang C. (2014) All-optical regula-tion of gene expression in targeted cells. Sci Rep 4:5346.
Wojtovich AP, Foster TH. (2014) Optogenetic control of ROS production. Redox Biol 2:368-76.
Wong J, Abilez OJ, Kuhl E. (2012) Computational Optogenetics: A Novel Continuum Framework for the Photoelectrochemistry of Living Systems. J Mech Phys Solids 60(6):1158-78.
Wu GY, Deisseroth K, Tsien RW. (2001) Activi-ty-dependent CREB phosphorylation: convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. Proc Natl Acad Sci USA 98(5):2808-13.
Xu N, Luo KQ, Chang DC. (2003) Ca2+ signal blockers can inhibit M/A transition in mammalian cells by interfering with the spindle checkpoint. Biochem Biophys Res Commun 306(3):737-45.
Yissachar N, Sharar Fischler T, Cohen AA, Reich-Zeliger S, Russ D, Shifrut E, Porat Z, Friedman N. (2013) Dynamic response diversity of NFAT isoforms in individual living cells. Mol Cell 49(2):322-30
Zarbin MA, Arlow T, Ritch R. (2013) Regenerative nanomedicine for vision restoration. Mayo Clin Proc 88(12):1480-90.
Zhu L, Song S, Pi Y, Yu Y, She W, Ye H, Su Y, Hu Q. (2011) Cumulated Ca2+ spike dura-tion underlies Ca2+ oscillation frequency-regulated NFκB transcriptional activity. J Cell Sci 124(Pt 15):2591-601.