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系統識別號 U0026-1106201922440300
論文名稱(中文) 光遺傳學引發的鈣離子振盪波通過活化PKCα與Rac1促進侵襲偽足之再組裝
論文名稱(英文) Activation of PKCα and Rac1 promotes invadopodia reassembly through optogenetically engineered Ca2+ oscillations
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 107
學期 2
出版年 108
研究生(中文) 徐繼煊
研究生(英文) Ji-Xuan Xu
學號 P86053018
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 口試委員-湯銘哲
口試委員-葉儀君
口試委員-陳宜芳
指導教授-邱文泰
中文關鍵字 光遺傳學  侵襲偽足  鈣離子振盪波 
英文關鍵字 optogenetics  invadopodia  Ca2+ oscillation 
學科別分類
中文摘要 鈣離子在細胞的信息傳遞中扮演了非常重要的角色,調控著例如受精、細胞增殖、細胞收縮、細胞爬行和細胞凋亡等等細胞的生理活動。侵襲偽足(invadopodia)是一種細胞膜上的突起,它可以利用金屬蛋白酶來降解細胞外基質。侵襲偽足的對於癌細胞的侵襲與轉移扮演了重要的角色。鈣離子也被證明對於侵襲偽足的形成與癌細胞對細胞外基質的瓦解有重要的意義。然而,在先前的文獻中,並沒有精確的研究指出究竟是什麼樣的鈣離子信號會導致侵襲偽足的組裝。而光遺傳學技術是一種能夠提供高時間解析度與高空間解析度的研究方法,已經被開發與應用於許多精確的生物醫學研究中。因此,我們利用光遺傳學技術結合470 nm 的藍光活化表達與人類黑色素癌細胞(A375)上的光敏感通道(Ca2+ translocating channelrhodopsin, CatCh)創造出不同的鈣離子振盪波,使鈣離子信號能夠以不同的振盪波形式流入細胞內,以此來探究鈣離子信號如何對侵襲偽足的形成產生影響。我們的研究顯示,低頻率(0.01 Hz, 0.1 Hz)的鈣離子振盪波會促進侵襲偽足的再組裝,而高頻率(1 Hz)的鈣離子振盪則會造成侵襲偽足的瓦解。此外,我們還發現光刺激造成的鈣離子振盪是通過活化PKCα使得其下游的Rho family small GTPases 中的Rac1活化來促進侵襲偽足的形成,至於RhoA與Cdc42在這個過程中扮演了什麼樣的角色還需要進一步的研究證明。
英文摘要 Calcium (Ca2+) plays an important role in the signal transduction of cells, regulating the physiological activities of cells such as fertilization, cell proliferation, cell contraction, cell migration, and apoptosis. Invadopodia is a protrusion on the cell membrane that uses metalloproteinases to degrade the extracellular matrix. Invadopodia plays an important role in the invasion and metastasis of cancer cells. Ca2+ has also been shown to be important for the formation of invadopodia and the disintegration of extracellular matrices by cancer cells. However, in the previous literature on this topic, there has been no precise study to indicate what kind of Ca2+ signal causes invadopodia assembly. Optogenetic technology is a research method that provides high temporal and spatial resolution and has been developed and applied to many precise biological and medical studies. Therefore, we use optogenetic techniques combining 470 nm of blue light to activate light-sensitive channels (Ca2+ translocating channelrhodopsin, CatCh) expressed on human melanoma cancer cells (A375) to create different oscillating waves, so that Ca2+ signals can flow into cells in different oscillating waves. In this study, we explore how Ca2+ signals affect the formation of invadopodia. Our research showed that low frequency (0.01 Hz, 0.1 Hz) Ca2+ oscillations promoted reassembly of invadopodia, while high frequency (1 Hz) Ca2+ oscillations caused the collapse of invadopodia. In addition, we also found that low frequency Ca2+ oscillation caused by light stimulation promotes the reassembly of invadopodia by activating PKCα (protein kinase C alpha) and Rac1 (Ras-related C3 botulinum toxin substrate 1). As for the role of RhoA (Ras homolog gene family, member A) and Cdc42 (Cell division control protein 42 homolog) in this process, we need further research to prove it.
論文目次 Abstract i
中文摘要 ii
Acknowledgement iii
Contents iv
Figure contents vi
Chapter 1 Introduction 1
1.1 The role of Ca2+ in cells 1
1.2 Invadopodia 2
1.2.1 Structure of Invadopodia 2
1.2.2 Invadopodia and podosomes 3
1.2.3 The function of invadopodia in cells 5
1.3 Ca2+ signal in invadopodia 6
1.3.1 The role of the Rho family of GTPases in invadopodia 6
1.3.2 Effect of the Ca2+ signal on the Rho family of GTPases 7
1.4 Optogenetics 8
1.5 The specific aim of this study 9
Chapter 2 Materials and Methods 10
2.1 Cell Culture 10
2.2 Optogenetic system 10
2.3 Live and dead assay 10
2.4 Immunofluorescence staining 11
2.5 Inhibitor 11
2.6 RhoA/Rac1/Cdc42 Activation Assay 11
2.7 Western blotting 12
2.8 Statistical analysis 12
Chapter 3 Results 13
3.1 Preparation of the optogenetic tool 13
3.2 The lack of Ca2+ leads to the degradation of the podosome-like adhesion 14
3.3 Optogenetically-engineered Ca2+ oscillations regulate the degradation of invadopodia 14
3.4 Use of different frequencies of Ca2+ oscillation affects the reassembly of invadopodia 15
3.5 Light treatment promotes the reassembly of invadopodia by activating PKCα and Rac1 16
Chapter 4 Discussion 18
References 22
Figures 29

Figure 1. Podosome rosettes in Src-transformed fibroblasts 29
Figure 2. Live and dead analysis of the 3T3-cSrc-Y527F-CatCh-Venus cells after light treatment 31
Figure 3. The lack of Ca2+ leads to the degradation of podosomes and podosome rosettes 33
Figure 4. Invadopodia can be reassembled over time 35
Figure 5. Light treatment regulates the degradation of invadopodia 37
Figure 6. The 0.01 Hz light treatment regulates the reassembly of invadopodia 39
Figure 7. The 0. 1 Hz light treatment regulates the reassembly of invadopodia 42
Figure 8. The 1 Hz light treatment regulates the reassembly of invadopodia 45
Figure 9. The inhibition of Rac1 or PKCα blocked the reassembly of invadopodia regulated by light treatment 48
Figure 10. Light treatment promotes the reassembly of invadopodia by activating PKCα and Rac1 51
Figure 11. The schematic of Ca2+ regulated invadopodia dynamics 54
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