活體影像研究異位表達ATP合成酶的運輸過程

Abstract

三磷酸腺苷合成酶(ATP synthase)位於粒線體內膜上，用來生產三磷酸腺苷(ATP)供給細胞內多種反應途徑使用。先前的研究指出三磷酸腺苷合成酶出現在不同種癌細胞的細胞膜上，我們將之稱做異位表達三磷酸腺苷合成酶(ectopic ATP synthase)。異位表達三磷酸腺苷合成酶在細胞膜上面的確切作用已經在之前被證實過。因此我們想探討異位表達三磷酸腺苷合成酶在細胞內的運輸途徑。首先透過活體細胞影像，我們將綠色螢光標記在三磷酸腺苷合成酶上，在拍攝活體影像前另外染了細胞膜，而在我們所拍攝的影像中，可以觀察到隨著時間的變化，三磷酸腺苷合成酶漸漸往細胞膜移動，最後與細胞膜結合形成異位表達三磷酸腺苷合成酶。確認異位表達三磷酸腺苷合成酶由細胞質而來，進一步我們想探討異位表達三磷酸腺苷合成酶的運輸路徑。根據實驗室之前由基因集富集分析的結果，顯示異位表達三磷酸腺苷合成酶表現量高的細胞，粒線體運輸路徑和以細胞骨架當作運送介質高度參與其中，所以我們假設三磷酸腺苷合成酶以完整複合體的形式鑲嵌於粒腺體膜上，隨著粒腺體沿著細胞骨架被運輸到細胞膜上，進一步表現在細胞膜上。進一步針對前人在粒腺體上的研究，知道粒腺體分裂的情形下較易被運輸，為了證實我們的推測，分別在肺癌細胞A549跟神經母細胞瘤SK-N-BE(2)C加入抑制粒線體分裂的藥物Mdivi-1，加藥之後進行活體影像拍攝。在經由一連串的影像分析，我們觀察到在粒線體分裂被抑制後，三磷酸腺苷合成酶的移動路徑變短，移動的速率也明顯下降。進一步觀察細胞膜跟三磷酸腺苷合成酶的共定位，可以發現在抑制粒線體分裂後，膜上的異位表達三磷酸腺苷合成酶跟細胞膜的共定位隨之減少，代表異位表達三磷酸腺苷合成酶的表現量下降。根據上面結果我們推斷在粒線體融合的情況下，粒線體的運輸過程因此減少，影響了三磷酸腺苷合成酶的運輸，進一步影響異位表達三磷酸腺苷合成酶在細胞膜上的表現量。確認粒線體的型態會影響三磷酸腺苷合成酶的運送，我們再來觀察細胞骨架對三磷酸腺苷合成酶的作用。因此我們使用抑制細胞骨架據合的藥物-諾考達唑(Nocodazole)，同樣用活體影像拍攝記錄下加藥後對細胞的影響，我們分析細胞影像的結果也發現在抑制細胞骨架的聚合後，三磷酸腺苷合成酶的行動能力下降許多，在共定位的分析下也可發現，膜上的異位表達三磷酸腺苷合成酶表現量也被抑制，因此我們推測細胞骨架在三磷酸腺苷合成酶的運送途徑扮演重要的角色。根據上述的實驗結果，我們推測異位表達三磷酸腺苷合成酶在細胞裡面的運送是需要透過粒線體分裂的型態，並且運送的途徑是需要藉由細胞骨架來進行移動，最後藉由粒線體與細胞膜的融合將異位表達三磷酸腺苷合成酶表現在細胞膜上。

Adenosine triphosphate (ATP) synthase is the most commonly used as 'energy currency' of cells for all organisms. So far, several studies have shown that ATP synthase not only exists on mitochondrial inner membrane but also translocates to the plasma membrane. ATP synthase which ectopically expresses on the cell surface is known as ectopic ATP synthase (eATP synthase) and found in various cells. Our previous studies revealed that eATP synthase on plasma membrane may come from divided mitochondria which are transported along with microtubule. Here, we aim to verify this hypothesis by using the approach of time-lapse live cell image to monitor the intracellular trafficking of ATP synthase. Fluorescent protein tags, such as GFP, YFP, and mCherry, are generally used for tracing cellular components in living cells, however, most fluorescent proteins easily get photobleach which results in the dilemma under long-period monitor. Therefore, we manipulated photo-activatable-GFP (pa-GFP) which displays a 100-fold increase in green fluorescent intensity, and the narrow spectra achieved purified signal relative to GFP. In addition, through the special photoactivation characteristic, pa-GFP is a suitable material for observing the moving track of target proteins which have been excited by ~400 nm wavelength specifically rather than new synthetic target proteins. At first, we introduced the sequence of ATP5B, a subunit of ATP synthase complex, into a pa-GFP vector and transfected this recombinant construction into cancer cells for the observation of ATP synthase movement. After stimulated with 413 nm light, ATP5B-paGFP was recorded every 4 secs for 30 min. The time-lapse video showed that ATP5B-paGFP moved from the perinuclear region to the plasma membrane. Consistently, the colocalization analysis also indicated that approximately 80% ATP5B-paGFP colocalized with the cell plasma membrane labeling, Cell Mask Deep Red. To further determine whether the mitochondrial dynamic is related to ATP synthase transportation, we captured the movement of ATP5B-paGFP in cancer cells treated with Mdivi-1, an inhibitor of mitochondrial fission. The live image analysis suggested that Mdivi1 treatment reduced significantly movement distance and velocity of ATP5B-paGFP compared to control. Moreover, we also investigated whether microtubule plays the critical role in ATP synthase transportation. The results showed that the movement ability of ATP synthase was significantly slowed down after treatment with the microtubule-depolymerizing agent. In conclusion, our time-lapse imaging analysis provides evidence that mitochondria dynamic and microtubule network play important roles in ectopic ATP synthase trafficking.