從我們現在超高解析取得的結果顯示，固定子(PomA)在大腸桿菌細胞裡的分布不均勻，兩極的分布會高於細胞的中心。這樣的結果可能是由於固定子取自於Vibrio alginolyticus的鞭毛馬達系統，這種鞭毛馬達在Vibrio身上主要出現於兩極。同時我們亦紀錄了固定子的數量，固定子在大腸桿菌裡的數量經過90分鐘的紀錄，可以記錄到約2000個PomA在過表現細胞約100奈米的厚度裡。我們也做了雙色的螢光實驗，同時標記轉子與固定子的位置。透過PALM影像的重建，我們建立出在200奈米解析度下的固定子在細胞的影像，這樣的影像相似於螢光顯微鏡在螢光蛋白同時發光時會拍攝到的影像。在雙色螢光實驗的影像，固定子分布高的地方與轉子分布高的地方相吻合，確定了固定子分布在馬達轉子周圍，這是第一次藉由光學的方法看到固定子在鞭毛馬達轉子附近的分布情形，無論這些蛋白質在細胞裡是否固定我們都可以看到相似的結果。 ;This thesis aims to investigate stators assembly of flagella motor in Escherichia coli. E. coli can swim at a speed about 20 µm/s in the liquid at low Reynolds number environment, which is fast compared to the 2 µm cell length. The motor is driven by stators that are nonrotating part of motor. Two kinds of stators have been found, MotA/MotB couples proton flux and PomA/PomB couples sodium ion to the motor rotation. At least 11 stators units surround the motor basal part and work independently. However, the real position of stators around motor basal part is not clear. The diameter of a motor is only 45 nm that is below the resolution of conventional optical microscope. In order to study the stator-assembly and dynamics, we set up a super-resolution microscope to visualize the stator proteins in real motor.
Fluorescent microscopes such as confocal microscopes have resolution limit about 200 nm. It is impossible to resolve the nanostructure of bacteria flagella motor by conventional microscope. The resolution limit is confined at the condition that all the fluorescent probe emission at the same time. If there is only one fluorescent probe emission at one time, it would be possible to localize the central position accurately to only one nanometer. By using photoactivatable green fluorescent protein (PA-GFP) fused stator protein, we can visualize each stator position and monitor the dynamics of stators. By controlling the UV photoactivation laser power to switch on PA-GFP one by one, we can localize each PA-GFP separately at different time to reach 10 nm resolution. Our current results show the distributions of PomA in cells are not uniform. PomA have higher density in the polar region of cells. It is reasonable because the stator PomA/PomB come from the polar flagella of Vibrio alginolyticus. Total number of PomA is also counted. After 90 minutes recording, there are about 2000 PomA in one cell. Furthermore, in dual color experiment, rotor protein (FliN) and stator proteins (PomA) are colocalized in both fixed and living cells. The dynamical properties of stator proteins are important for the understanding of motor dynamics. Further investigations are needed using our newly developed PALM experimental system.
