Analysis of single-molecule images for the subcellular localization and dynamics of proteins.

(A) Single-molecule image analysis. Spots were detected in each frame (highlighted with yellow circles), and tracks were created across frames (different colors were chosen for different tracks). (B) Cell detection. Cell outlines were determined from bright-field images. Only non-dividing cells were analyzed (indicated by white outlines). (C) Normalized position of spots of RNase E along short (x) and long (y) axes of an example cell. Red spots are inside the cell endcaps, and cyan spots are in the cylindrical region of the cell. (D) xNorm histogram of RNase E and LacY. Only spots in the cylindrical region of cells (like cyan spots in C), over n = 143,000 spots, were included. The standard error of the mean (SEM) calculated from bootstrapping is displayed as a shaded area but is smaller than the line width (see Fig. S1 for details). (E) The membrane binding percentage (MB%) of RNase E, LacY, and LacZ. Error bars are from the 95% confidence interval. (F) Histogram of absolute xNorm and model fitting of RNase E, LacY, and LacZ to determine MB%. Orange highlights indicate the range of xNorm expected based on the standard deviations in the parameter values estimated by MCMC. The white scale bars in panels A-B are 1 μm. See Table S6 for data statistics.

Effects of mRNA, MTS, and CTD on dynamics and localization of RNase E.

(A) MSD versus time delay (τ) of RNase E. Ensemble-averaged time-averaged (EATA) MSD was calculated by averaging the time-averaged MSD of individual tracks. (B-D) Change in the mean diffusion coefficient of RNase E (B), LacY (C), and ribosome L1 protein (D) when cellular RNAs were depleted by rifampicin treatment. (E) Linear representation of RNase E monomer. Note that in this study we define CTD as the region following MTS. The numbers indicate amino acid residues. (F) xNorm histograms of various RNase E mutants. The SEM from bootstrapping is displayed but smaller than the line width. (G) Mean diffusion coefficients of various RNase E mutants, lacking MTS and/or CTD. (H) Mean diffusion coefficients of RNase E upon removal of different RNA degradosome components. (I) Expected mass of fully occupied RNA degradosome upon removal of different degradosome components. Error bars in panels B-D and G-H are the SEM. At least,1,100 tracks for diffusion data or 90,000 spots for xNorm data were used in the analysis. See Table S6 for data statistics.

Localization and diffusion of membrane-binding motifs.

(A) Cartoon schematic of the membrane-binding motifs used in this study (not to scale). The orange circles indicate mEos3.2 used for imaging. (B) xNorm histograms of membrane-binding motifs. The SEM from bootstrapping is displayed but smaller than the line width. Data are from at least 107,000 spots. (C) Mean diffusion coefficients of membrane-binding motifs. Error bars are the SEM from at least 3,000 tracks. (D) Estimated mass of membrane-binding motifs based on the amino acid sequence including linkers and mEos3.2. (E) Snapshots from all-atom MD simulation of MTS and LacY2 in the E. coli membrane. The proteins are displayed in purple, and lipid tails are shown in cyan. Nitrogen and phosphorus atoms of the lipid head groups are represented in the van der Waals form in blue and grey, respectively. (F) Diffusion coefficients of MTS and LacY2 from the simulation. For panels B and C, see Table S6 for data statistics.

Localization and diffusion of chimeric RNase E with or without CTD.

(A-B) Cartoon schematic of RNase E chimeric variants with CTD (A) and without CTD (B). They are not to scale. (C-D) xNorm histograms of chimeric RNase E localization compared with that of LacY. The SEM from bootstrapping is displayed but smaller than the line width. (E-F) MB% of chimeric RNase E mutants without CTD (E) or with CTD (F) with various membrane-binding motifs. Error bars are from a 95% confidence interval. (G-H) Mean diffusion coefficients of chimeric RNase E without CTD (G) or with CTD (H). Error bars are the SEM. Each data set contains at least 70,000 tracks for diffusion or 72,000 spots for xNorm (Table S6).

lacZ mRNA degradation rates in chimeric RNase E strains.

(A-B) lacZ mRNA levels in RNase E with MTS and CTD or WT RNase E (A, strain SK595) and in RNase E-LacY2-CTD (B, strain SK505) when lacZ transcription was induced with 0.2 mM IPTG at t = 0 s and re-repressed with 500 mM glucose at t = 75 s. Blue and yellow regions indicate where kd1 and kd2 were measured by the exponential fitting of lacZ 5’ mRNA (Z5) in individual replicates. (C-D) Co-transcriptional and post-transcriptional lacZ mRNA degradation rates, kd1 (C) and kd2 (D), respectively, in various strains of chimeric RNase E with different membrane-binding motifs, either ΔCTD (light bars) or with CTD (solid bars). The dotted line indicates the kd1 value of cytoplasmic RNase E ΔMTS (strain SK339)15. RNase E ΔCTD based on LacY6 and LacY12 showed zero kd1. In all panels, error bars are the standard deviations from 3 biological replicates.