- 學位論文
粒線體自噬與融合修復機制之交互作用
Study of Reciprocal Interactions between Mitophagy and Fusion-based Recovery of Mitochondrial Membrane Potential
摘要
粒線體在細胞中同時扮演能量供給與調控細胞凋亡的角色，其內膜上的電子傳遞鏈促使內膜兩側形成氫離子之濃度差並藉此推動三磷酸腺苷(ATP)合成酶合成ATP維持細胞能量供給。然而電子自傳遞鏈洩漏所產生的活性氧化物質(ROS)則會對細胞內的各種組成造成傷害，受損的粒線體更會進一步產生更多的ROS。當粒線體的品質失去控制時會導致許多不同類型的疾病，在遺傳早發性帕金森氏症中發現並命名為Parkin的E3泛素連接脢揭開了細胞如何控制受損粒線體被細胞自噬來清除的機制，又稱為粒線體自噬。為了控制粒線體的品質與效率，因應不同的環境或細胞狀態，細胞能藉由融合、分裂、移動來改變粒線體的形狀、大小與分佈，稱為粒線體動態。粒線體的動態失去控制的其中一個例子是造成胰島細胞無法受外在葡萄糖濃度的刺激進而產生胰島素，甚至會造成胰島細胞死亡而形成糖尿病。先前的研究指出粒線體自噬大多是利用氫離子去耦合劑來破壞粒線體內的膜電位來產生，然而此時細胞中所有的粒線體都已經受到傷害，與正常情況下單一粒線體的破壞的情形不論在細胞的能量供給或整體粒線體動態都不盡相同。在我們設計的實驗中，利用只能夠在特定波長雷射激發時才會釋放氧化壓力的重組蛋白表現到粒線體中，並追蹤受損的粒線體與其他健康粒線體的互動，發現當受損的粒線體與周遭的健康粒線體融合後再度恢復其膜電位，其氧化壓力也沒有繼續提升，相比於沒有融合的粒線體，這些粒線體逃過了被細胞自噬的命運。更進一步我們藉由抑制與粒線體移動相關的蛋白質(TRAK1)的表現，當粒線體的移動被抑制時，受損粒線體與健康粒線體融合的機會降低，此時粒線體被細胞自噬系統辨認的比例提高，顯示出細胞動態跟細胞自噬之間存在交互影響，同時我們也將這樣的實驗設計應用於神經細胞跟胰島細胞中，希望能進一步研究當細胞面臨來自內或外的環境改變時，如何調整其控制粒線體品質的策略。
並列摘要
Mitochondria play crucial roles in metabolic and apoptotic pathway within the cell. Dysfunction of mitochondria has been implicated in the pathogenesis of many neurodegenerative diseases. To ensure mitochondrial quality, cells can degrade aged and damaged mitochondria through PINK1/Parkin-mediated mitophagy. Defects in ubiquitin ligase Parkin are linked to early-onset cases of Parkinson's disease. On the other side, cell control mitochondria shape and distribution by fusion, fission and motility of mitochondria to face external or enteral cell stresses. However, the coordination of mitophagy and mitochondrial dynamic still unclear because most study of mitophagy impairs mitochondria through proton uncoupler such as CCCP (Carbonyl cyanide m-chlorophenyl hydrazone). In response to CCCP's attack, the potential of restore mitochondrial function by fusion with healthy mitochondria has been blocked. Because entire mitochondrial network has been depolarized and fragmented. Here we used a protein-based photosensitizer Killer-Red to damage selected mitochondria and traced their fate with PAGFP, a photoactivatable fluorescent protein. We found that fusion with nearby mitochondrial network can promote the recovery of a damaged mitochondrion’s function and escape from the fate of mitophagy. Inhibition of mitochondrial motility by knockdown mitochondrial motor-adaptor protein TRAK1 lead to increase the ratio of mitophagy in KillerRed damage mitochondria but didn’t shift mitophagy activity in CCCP-induced entire cell damaging.