Single Cell Biophysics Experiments I(3/3)

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.