| 摘要: | 此計畫主要目標為發展單細胞生物物理研究平台，來探究細菌鞭毛的定位與自我組裝機制。主要分為三大目標: 1.即時細菌鞭毛成長量測。2.發展單細菌細胞能量量測。3.細菌鞭毛定位與組裝機制。單細胞生物物理是一個新興的重要實驗領域，從單分子技術擴展到單一活體細胞上的即時量測。此領域興起的主要兩個原因:一是在細胞上有著大量基因表達差異性，造成細胞間的差異；二是許多細胞內的運轉機制，必須要到單一細胞尺度才能將其釐清。我們將用在細菌上發展的一系列能量狀態量測工具，來研究細菌鞭毛生長與組裝的精細過程。細菌鞭毛馬達是一個複雜的天然分子馬達，是一個45奈米寬，嵌在細菌細胞膜上的步進馬達。許多細菌利用它來轉動體外的鞭毛在液體中游動。細菌鞭毛是一個巨大的胞外結構，長度可達數微米，但是寬度只有20奈米的中空螺旋管。它是細菌鞭毛馬達的螺旋槳，由鞭毛馬達帶動，可以在低雷諾數環境以每秒數百轉旋轉。細菌鞭毛由鞭毛蛋白所堆疊而成，總共由數萬個蛋白有系統地由鞭毛基底送出，透過細菌鞭毛本身的中空管空間，送到鞭毛末端組裝。送出的過程需要ATP能量消耗與質子驅動勢，但是在管內卻不需要能量消耗。這是一個超精美的一維奈米自我組裝的機械，但是我們對其組成機制與動態過程卻瞭解甚少。為了能進一步了解細菌鞭毛的生成機制，我們計劃以下的研究。第一: 我們發展了即時螢光標定的方法來研究細菌鞭毛的成長，是目前已知首次即時觀測單一鞭毛成長的實驗。我們將利用這一個新的技術，探索細菌鞭毛生長的機制。首先將建利高通量超高解析全自動顯微鏡系統，可以對鞭毛的成長作即時的量測。再透過量測不同細菌鞭毛型態的突變種鞭毛成長速度曲線，或是利用光驅動蛋白來增加細胞膜電位，我們將一窺此一從未有人能探索的一維奈米管線。第二: 我們將發展四項單細胞的能量狀態量測，一是利用細菌鞭毛馬達轉速量測PMF總和，二是量測細胞內pH值，三是發展新的膜電位感應分子，與四是改良量測細胞內ATP濃度。第三: 溶藻弧菌是一個單邊單鞭毛的細菌的鞭毛生長有特定位置，細胞分裂時需要製造新的鞭毛。透過追蹤其鞭毛馬達組成蛋白與定位蛋白，我們將可以一窺這一個精準定位系統的原理。透過這三個研究，我們將可以瞭解細菌鞭毛生長機制，鞭毛生長能量消耗，與細菌鞭毛定位原理。我們可以利用這一些新知，拓展與發展在奈米世界製作裝置的能力。同時藉由了解鞭毛的性質，我們將可發展新微生物偵測技術。 ;The aim of this project is to develop cutting edge single-cell biophysical experimental platforms and investigate the localization and self-assembly mechanisms of bacterial flagellar filaments. There are three objectives. 1. The real-time measurement of flagellar growth rate and the growth mechanisms. 2. Single cell energetic measurements. 3. The mechanism of flagellar motor polar localization and self assembly process.Single-Cell Biophysics is a developing leading-edge experimental field extended from single-molecular biophysics to real-time living single cell measurements. There are two main reasons to develop this field. Firstly, there is considerable large expression difference between cells causes the functional difference. Secondly, for many intracellular functions, it is necessary to go down to single cell level to understand the mechanisms. We will combine several single-cell energetical and optical measurements to investigate the growth and self-assembly process of bacterial flagella. Bacterial flagellar motor is a natural and complex molecular machine. It is a 45nm-wide stepping motor anchored in the bacterial cell membrane. Many species of bacterial use flagella to swim. Bacterial flagellum is a giant extracellular organelle, a hollow helical pipe of several micro meters long and only 20 nm wide. The flagellum is the propeller spinning at several hundred hertz at this low Reynolds number environment. 11 flagellar proteins, flagellins, form one round the flagellar filament. There are more than 10,000 flagellins be transported from the base of flagellar motor to form the whole flagellar filament. Flagellar filament is a one-dimensional self-assembly nano-machine. However, the true mechanism of the flagella formation remains unclear.In order to reveal the assembly mechanism of flagellar filament, we propose the following research. Firstly, we have developed real-time fluorescent labeling technique to probe the flagellar growth rate. This is the first report in the history of single flagellum real-time growth rate experiment. We will apply this cutting-edge technique to explore the mechanism of flagellar growth. We will build a high throughput super-resolution microscopy to measure the flagella length in real time. Through the comparison of growth rates of different phenotype mutants, we can reveal the growth mechanism of this one-dimensional nano-wire. Secondly, we will develop four single-cell energetic measurements. (1) Using bacterial flagellar motor rotation to measure the PMF. (2) Using pH sensitive protein, pHluorin to measure intracellular pH. (3) Developing new membrane potential probs. (4) Improving the expression of ATP sensing protein Queen in bacteria. Thirdly, Vibrio alginolyticus has single polar flagellum specifically localized in space and time during the cell division. Through tracking the localization proteins and motor proteins, we will investigate the localization and building process of bacterial flagellar motor. We can learn the flagellar growth, energy consumption, growth mechanism and polar localization mechanism from our proposed project. Through the gained new knowledge, we can apply and extend our capability of nano- manufacturing. Furthermore, the knowledge of flagellar growth and sheath will be valuable for the potential application s of microbial detection. |
