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. 2017 Jan 25;45(2):926-937.
doi: 10.1093/nar/gkw787. Epub 2016 Sep 12.

In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

Anne Plochowietz  1 , Ian Farrell  2   3 , Zeev Smilansky  2 , Barry S Cooperman  3 , Achillefs N Kapanidis  4
Affiliations

In vivo single-RNA tracking shows that most tRNA diffuses freely in live bacteria

Anne Plochowietz et al. Nucleic Acids Res. .

Abstract

Transfer RNA (tRNA) links messenger RNA nucleotide sequence with amino acid sequence during protein synthesis. Despite the importance of tRNA for translation, its subcellular distribution and diffusion properties in live cells are poorly understood. Here, we provide the first direct report on tRNA diffusion localization in live bacteria. We internalized tRNA labeled with organic fluorophores into live bacteria, applied single-molecule fluorescence imaging with single-particle tracking and localized and tracked single tRNA molecules over seconds. We observed two diffusive species: fast (with a diffusion coefficient of ∼8 μm2/s, consistent with free tRNA) and slow (consistent with tRNA bound to larger complexes). Our data indicate that a large fraction of internalized fluorescent tRNA (>70%) appears to diffuse freely in the bacterial cell. We also obtained the subcellular distribution of fast and slow diffusing tRNA molecules in multiple cells by normalizing for cell morphology. While fast diffusing tRNA is not excluded from the bacterial nucleoid, slow diffusing tRNA is localized to the cell periphery (showing a 30% enrichment versus a uniform distribution), similar to non-uniform localizations previously observed for mRNA and ribosomes.

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Figures

Figure 1.
Figure 1.
Internalization of labeled tRNA molecules into live Escherichia coli. (A) Schematic of labeled tRNA molecule. (B) Schematic of the internalization method using electroporation (EP). Labeled tRNA molecules are added to cell suspension of electrocompetent cells and are exposed to the discharge of a high-voltage electric field. Cells are quickly recovered and non-internalized tRNA molecules are washed off. Cell imaging is performed on an inverted microscope using HiLO illumination. Scale bar: 1 μm. (C) Efficient loading of E. coli cells with tRNAs labeled with organic fluorophores of different spectral range. Cells show high inner-cell fluorescence upon electroporation with 1 μM of tRNA-Cy3, tRNA-Cy5, and ssRNA-Cy5 (top row, left to right) and basal fluorescence of non-electroporated cells corresponding to cellular autofluorescence in the respective spectral channel (‘−EP’ cells, bottom row). More than 85% of cells were loaded when internalizing tRNA-Cy3, tRNA-Cy5 and ssRNA-Cy5 (>500 cells per dataset). Scale bar: 3 μm.
Figure 2.
Figure 2.
Counting and controlling the number of internalized tRNA molecules per cell. (A) Single-cell photobleaching step analysis of tRNA-Cy5 in live bacteria. Scale bar: 1 μm. (B) Photobleaching time-trace samples (blue) and Hidden–Markov Model fit (red) of single photobleaching steps corresponding to the single-molecule unitary intensity (gray bars). (C) Step height histogram (unitary intensity distribution) of tRNA-Cy5 (158 photobleaching time-traces, 324 fitted single photobleaching steps = unitary intensity) and Gaussian fit centered at (8.7 ± 2.4) a.u. corresponding to Cy5 brightness of (7500 ± 2100) photons/s. (D) Counting the number of internalized tRNA-Cy5 molecules per cell at different concentrations of labeled tRNA-Cy5 in cell suspension before electroporation. Cells are loaded with 1–10 tRNA molecules when adding 50 nM tRNA-Cy5 (mean: 1.4 molecules per cell, median: 0.9) and up to 250 nM tRNA-Cy5 (mean: 1.5 molecules per cell, median: 1.0) in the cell suspension before electroporation, which is an ideal regime for single-molecule studies. Cells are loaded with about 100 tRNA molecules per cell (mean: ∼140, median: ∼100, Supplementary Figure S5 for tRNA-Cy3 and tRNA-Cy5.5) at 1 μM tRNA-Cy5 initial concentration before electroporation.
Figure 3.
Figure 3.
Single-molecule tracking of tRNA-Cy5 and measurement of diffusion coefficient (Dapp) of tRNA in vivo. (A) Tracking of a single tRNA-Cy5 molecule for ∼1.5 s and reconstruction of the single-molecule trajectory in the bacterial cell (90 FOV, 700 cells and ∼20 000 localizations for live-cell dataset and ∼200 localizations for negative controls −tRNA + EP and +tRNA − EP). Scale bar: 1 μm. (B) Measurement of the mean-squared displacement (MSD) for tRNA-Cy5 in live cells (blue) and fixed cells (black). The MSD plateaus due to cellular confinement and motion blurring in live cells and due to the localization precision. We calculated the localization precision σ from the apparent Dapp of the fixed cell control Dapp(fixed cells) = 0.32 μm2/s = σ2/(4Δt), with Δt = 5ms and obtained σ = 0.08 μm, Supplementary Figure S6b. (C) Apparent Dapp distribution of tRNA-Cy5 and fit to two diffusive species. The slow diffusive species was constrained to ribosomal complex diffusion of Dapp(ribosome) = 0.5 μm2/s (red, 5%, Ref (26)) and free fit of fast diffusing species resulted in Dapp(tRNA-Cy5) = 3.6 μm2/s (blue, 95%). Similar fitting results were obtained when constraining the slow diffusing species to immobile molecules of the fixed-cell control, Dapp(fixed cells) = 0.32 μm2/s (Supplementary Figure S6b, c and g) or when allowing a free fit to one, two and three diffusive species (Supplementary Figure S6d–f). Including another independent dataset (90 FOV, 582 cells), the apparent Dapp of tRNA in live cells was obtained to Dapp(tRNA-Cy5) = (3.5 ± 0.2) μm2/s (Supplementary Figure S6h). (D) Cumulative distribution function (CDF) of tRNA-Cy5 tracking data was freely fitted to two diffusive species and the Dapp of the fast diffusing species was obtained to D2 = 7.6 μm2/s (slow diffusive species: D1 = 0.12 μm2/s).
Figure 4.
Figure 4.
Spatial distribution of tRNA molecules in live bacterial cells. Localizations of tRNA-Cy5 molecules were normalized along cell-length and cell-width and were represented in unit cell (‘Materials and Methods’ section). (Ai) Localizations of tRNA molecules classified as fast diffusing (Dapp > 1 μm2/s, blue) and slow diffusing (red) and combined (top) were shown in unit cells, respectively. (Aii) Probability distribution of fast and slow diffusing molecules along cell width (y-axis, short cell axis). Bias of slow diffusing molecules to cell periphery and fast diffusing molecules to mid-cell relative to uniform-distribution (dotted line). (B) Spatial distribution of tRNA molecules upon Chloramphenicol treatment blocking translation. (Bi) Representation as in Ai. (Bii) Fast and slow diffusing tRNA molecules were evenly distributed along cell widths. Results in (A and B) indicate that slow diffusing tRNA molecules at the cell periphery were utilized during translation. The spatial distribution of control measurements such as unspecific RNA, and fixed cells are shown along cell within Supplementary Figure S9 and within a unit cell in Supplementary Figure S10 for different cell length.
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