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Title: 利用單分子顯微術觀察大腸桿菌鞭毛蛋白的群聚現象
The Clustering Phenomenon of Flagella in E. coli revealed by Single-Molecule Microscopy
Authors: 張宜仁
Chang, Yi-Ren
Sung, Yun-Sing
Keywords: 鞭毛
Ripley’s K函數
Single Molecule Microscopy
Ripley’s K function
Issue Date: 2020
Abstract: 大腸桿菌的鞭毛在大腸桿菌中扮演相當重要的角色。在與趨向系統(Chemotaxis system)協同作用下,鞭毛能有順時鐘與逆時鐘旋轉的方向。逆時鐘方向旋轉時,鞭毛能讓大腸桿菌朝單一方向游泳前進;順時鐘旋轉時,鞭毛會讓大腸桿菌原地翻滾(tumbling)以改變方向。儘管對於鞭毛旋轉機制有透徹的了解,對於鞭毛生長的位置的認知卻相較貧乏。然而鞭毛的生長位置會影響細胞游泳與使用能量的效率,當鞭毛分佈集中在一端時,細胞能以較有效率的方式進行移動。對於大部分原核生物而言,其體內存在調控鞭毛生長位置與數量的蛋白,但在大腸桿菌中卻沒有相對應的蛋白與序列。故此,普遍對於大腸桿菌的鞭毛生長位置的認知為隨機分佈。 先前的研究中有發現大腸桿菌的鞭毛具有集中在舊端(old pole)所在的半側,然而此研究結果並沒有完全反駁鞭毛生長位置的隨機性。為了能驗證鞭毛生長位置是否屬於隨機,我們用螢光蛋白標定最先嵌入細胞膜的FlhA與FliF蛋白,在讓其基因過度表達後觀察其細胞分佈,得到非隨機的分佈。更進一步,我們利用光漂白後螢光恢復(Fluorescence Recovery After Photobleaching, FRAP) 發現在原先長有鞭毛結構的周圍會聚集FliF與FlhA的堆積。因此,我們猜測此群聚現象是一種細胞調控生長鞭毛結構位置的一種機制。 利用單分子顯微術來定位FlhA與FliF,並藉由Ripley’s K函數與基於密度的聚類演算法 (Density-Based Spatial Clustering of Applications with Noise, DBSCAN)得到FlhA與FliF的堆積半徑與分子參與堆積的比例。觀察在不同數量條件下的堆積半徑與分子參與堆積比例,我們發現當數量的增加,FlhA反而會分離原本的堆積並與FliF嘗試組織成新的結構。此結果可視為鞭毛蛋白從堆積組成結構的一種調控方式。
Flagella play important roles in Escherichia coli. Cooperate with chemotaxis system, flagella can rotate in clockwise and counterclockwise. When the flagella rotate with counterclockwise, an E. coli cell can swim toward a direction smoothly. In contrast, if the flagella rotate with clockwise, the cell will be tumbling to change direction of movement. Despite of how a flagellum rotates had been fruitfully researched, the detail mechanism of where and how a flagellum locate is still unrevealed, though the locations of the flagella is the key factor of the efficiency of the cellular energy regulation. For instance, when the flagella locate at its one pole concentrated, a cell can move more fluently. Since most of the Prokaryote depends on specific proteins to regulate the number and location of flagella, the flagella was assumed to randomly assemble on the cell surface in E. coli cells, which lack any analogs of the regulation proteins in other species of bacteria. In previous study, although more flagella were observed at the half side of the cell by the old pole, it still does not rule out the random assembly model since the old pole was synthesized anciently. To verify the random assembly model, we tagged the first two types of proteins embedded on membrane, i.e., FlhA and FliF, with fluorescence proteins. According to the images of the cells over-expressed FlhA and FliF, the non-uniform distributions of the proteins reject the random assembly model. In addition, we found out that FlhA and FliF clustered around the basal body via fluorescence recovery after photobleaching. Therefore, it implies that the clustering might be a regulation of how cell determined the location of basal body. Based on single molecule microscopy, the position of FlhA and FliF on the cell surface were super-resolved, and the radii of the clusters and the ratios of molecule in the clusters were further analyzed by Ripley’s K function and DBSCAN. The results suggest that when the number of protein increased, FlhA would separate the clusters and tried to assemble new cluster with FliF. It may provide a pathway of the regulation of flagellar structure assembly.
Other Identifiers: G060741004S
Appears in Collections:學位論文

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