Investigating stator dynamics and distributions of Escherichia coli flagellar motors by PALM
林再順1*, 羅健榮2
1Physics, National Central University, Jhongli, Taiwan
2Department of Physics and Graduate Institute of Biophysics, National Central University, Jhongli, Taiwan
* presenting author:再順林, email:tsaishun.lin@gmail.com
Introduction
Escherichia coli (E. coli) swims in liquid by flagellar motors, striving for nutrient-rich environment. The diameter of an E. coli flagellar motor complex is about 50 nm assembled with stator-units, which are the torque generating protein complex. Stator-units drive flagella motor to rotate by deriving energy from transporting ions into cytoplasm and ion-motive force. However, the real position of stators in the motor is not clear because of the small size of motor (50 nm) and the high dynamics of motor components in cell membrane.
Method
The conventional fluorescent microscope possess a resolution limit about 250 nm that is impossible to resolve the nanostructure of bacteria flagella motor. In this study, we set up a newly developed Super Resolution Microscope, photoactivated localization microscopy (PALM), to investigate spatial distributions and temporal dynamics of stator-units. We constructed photoactivated green fluorescent proteins, PAGFPs, to fuse to the subunit (PomA) of stators. By collecting the positions of each PAGFP along the time, we were able to reconstruct high-resolution PALM images (20-nm resolution) in a cell. Also, we studied the stator-unit dynamics by activating low number of PAGFP at one time and tracking their positions on the membrane.
Results
Results showed the nonhomogeneous distributions of the stators in the cell. The polar part of the cell had higher density of stators than other positions. We also counted about 10 stators at position of rotor. From single molecular tracking, we measured the mean squared displacement (MSD) of stator to explain the dynamics in membrane.
Conclusions
We proved the distribution of stators in E. coli with high-resolution images under optical microscope. Moreover, the MSD of stator showed the temporal local motion dynamics in high resolution. This is the first time that we cans study the dynamical stator-units in functional motors.


Keywords: Flagellar motor, E. coli, stator, PALM