Dynamics of the Serine Chemoreceptor in the Escherichia coli Inner Membrane: A High- Speed Single-Molecule Tracking Study  Dongmyung Oh, Yang Yu, Hochan Lee, Barry L. Wanner, Ken Ritchie  Biophysical Journal  Volume 106, Issue 1, Pages 145-153 (January 2014) DOI: 10.1016/j.bpj.2013.09.059 Copyright © 2014 Biophysical Society Terms and Conditions

Figure 1 Single-molecule imaging of Tsr-Venus in live E. coli cell at 30 Hz. (a) Bright-field and (b) corresponding fluorescence image showing Tsr molecules localized at the poles in steady state. (c) Selected images from a video and (e) the corresponding trajectory of a confined Tsr-Venus molecule. (d) Selected images from a video and (f) the corresponding trajectory of a mobile Tsr-Venus molecule. Scale bars are 2 μm. (g) Typical fluorescence intensity of an individual Tsr-Venus molecule showing single-step activation and photobleach. The sharp dips in fluorescence intensity during the trace are due to fluorophore blinking. (h) Fluorescence lifetime of the Tsr-Venus molecules under our illumination conditions. This corresponds to a distribution of observed trajectory lengths. To see this figure in color, go online. Biophysical Journal 2014 106, 145-153DOI: (10.1016/j.bpj.2013.09.059) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 2 Trajectory analysis of individual Tsr-Venus molecules on the inner membrane of live E. coli. (a) Distribution of diffusion coefficients for Tsr-Venus molecules classified as confined in the polar and central regions, and for those molecules classified as mobile. Also shown is the measured diffusion coefficient for Venus molecules immobilized on glass. (b and c) The MSD, averaged over all trajectories, for (b) confined polar and (c) mobile fractions of Tsr-Venus. To see this figure in color, go online. Biophysical Journal 2014 106, 145-153DOI: (10.1016/j.bpj.2013.09.059) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 3 Inner membrane structure of E. coli visualized by single-molecule imaging. (a) Example trajectory of a Tsr-Venus molecule imaged at 1000 Hz. (b) Complete MSD-Δt plot observed over the full range of video rates (30 – 1000 Hz). Data are fitted with linear regressions in the three distinct linear regions. (c) Same MSD-Δt plot fitted with analytical solutions given by Eq. 3. (d) Fitting of a general fractional power law for anomalous subdiffusion fails to fit the curve at long times. (Data subsets: green, 1000 fps; blue, 400 fps; red, 260 fps; and black, 30 fps.) To see this figure in color, go online. Biophysical Journal 2014 106, 145-153DOI: (10.1016/j.bpj.2013.09.059) Copyright © 2014 Biophysical Society Terms and Conditions

Figure 4 Effect of A22 treatment on Tsr clusters and individual Tsr molecular mobility. Image of the Tsr-Venus localization after (a) ∼3 h and (b) ∼4 h incubation in 1.6% A22 in media. Note the rounding of the cells and the dispersion of the large polar cluster over time. (c) Distribution of diffusion coefficients determined for the restricted and mobile fractions of Tsr-Venus in A22-treated cells after ∼4 h incubation. (d and e) The MSD, averaged over all trajectories, for (d) restricted and (e) mobile fractions of Tsr-Venus in panel c. To see this figure in color, go online. Biophysical Journal 2014 106, 145-153DOI: (10.1016/j.bpj.2013.09.059) Copyright © 2014 Biophysical Society Terms and Conditions