Segregation of molecules at cell division reveals native protein localization

Abstract

We introduce a nonintrusive method exploiting single-cell variability after cell division to validate protein localization. We found that Clp proteases, widely reported to form biologically relevant foci, were uniformly distributed in Escherichia coli cells, and that many commonly used fluorescent proteins caused severe mislocalization when fused to homo-oligomers. Retagging five other reportedly foci-forming proteins with the most monomeric fluorescent protein tested suggests that the foci were caused by the fluorescent tags.

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Figure 1: Reporter tag validation by segregation analysis.
Figure 2: Localization of Clp proteins in single cells.
Figure 3: Single-cell segregation assays.

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Acknowledgements

We thank D. Rudner, T. Mitchison and M. El Karoui for valuable comments on the manuscript, E. Toprak for help with instrumentation, M. Elowitz (Caltech) for image analysis tools, P. Malkus (Harvard Medical School) for pPM1, pPM14, pPM16 and pPM88, N. Lord for assistance with Tn7 integration, A. Hilfinger and D. Huh for discussions, members of the Harvard Medical School Nikon Imaging Center, and the developers of Micro-manager. This work was supported by US National Institutes of Health grants GM081563 and GM095784.

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Authors

Contributions

D.L. and J.P. designed the study, devised the experimental strategies, interpreted the results and wrote the paper. D.L. constructed the plasmids and strains, performed all experiments and analyzed the data. B.O. built the HILO imaging setup, performed the HILO microscopy with D.L. and participated in writing the manuscript. P.C. and T.A.B. provided crucial reagents, and participated in the discussions and in writing the manuscript.

Corresponding author

Correspondence to Johan Paulsson.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1–5, Supplementary Note 1, Supplementary Methods 1 (PDF 17132 kb)

Supplementary Video 1

Micro-colony growth of E. coli cells with ClpP-Venus foci. ClpP-Venus formed bright fluorescent foci in live E. coli cells. The ClpP-Venus foci were not present in all cells, the foci localized preferentially to cell poles and mid-cell region and showed binary segregation at cell division. Cells without a focus had a cytoplasmic yellow fluorescence signal (not visible in this movie) and usually formed a yellow fluorescence focus in the next cell cycles. A few ClpP-Venus foci appeared blurred because they were in different focal planes (z-stacks were required to detect all foci in a microcolony; data not shown). Some cells had small foci, which are very faint and barely visible in the movie. The yellow fluorescence images of this movie were acquired with 2 × 2 binning (effective pixel size was 129 nm). The cell boundary (red) was determined by segmenting the respective phase-contrast images. Scale bar (white), 1 μm. The movie is part of a dual-color experiment; see Supplementary Video 2 for the corresponding red fluorescence movie. (MOV 8233 kb)

Supplementary Video 2

Degradation of mCherry-ssrA in the ClpP-Venus strain showed that the ClpP-Venus foci generate cell-to-cell variability after division. Synthesis of mCherry-ssrA was pulse-induced before the cells were monitored. The red fluorescence images were subjected to a 'per-frame auto-scaling' to better display the variability between daughter cells after division. The foci in the red fluorescence image were not due to spectral bleedthrough from ClpP-Venus foci but probably represent immortal mCherry molecules bound to the ClpP-fluorescent protein foci (Supplementary Fig. 1). Red fluorescence images of this movie were acquired with 2 × 2 binning (effective pixel size was 129 nm). The cell boundary (red) was determined by segmenting the respective phase-contrast images. Scale bar (white), 1 μm. (MOV 8233 kb)

Supplementary Video 3

Degradation of mCherry-ssrA in the wild-type strain (protease was not tagged) displayed very low cell-to-cell variability after cell division. The mCherry-ssrA reporter was pulse-induced before imaging. mCherry-ssrA is specifically degraded by ClpXP and ClpAP. The red fluorescence images were per-frame auto-scaled to better illustrate the low variability after cell division. The yellow fluorescence images of this movie were acquired with 2 × 2 binning (129 nm effective pixel size). The cell boundary (red) was determined by segmenting the respective phase images. Scale bar (white), 1 μm. (MOV 13938 kb)

Supplementary Video 4

Live-cell HILO microscopy of cells expressing ClpA-mGFPmut3. Cells have ~50 particles, which are not localized in foci but move rapidly and seem to sample the entire cell. HILO imaging was performed as described in Online Methods. Scale bar (white), 1 μm. The images of the movie sequences were subjected to a quantitative grayscale scaling (left) and to a 'per-frame auto-scaling' (right) to better display the particle movement despite fast photo-bleaching (see Online Methods). (MOV 2345 kb)

Supplementary Video 5

Live-cell HILO microscopy of cells expressing ClpP-mGFPmut3. Cells have ~50 particles, which are not localized in foci but move rapidly and seem to sample the entire cell. HILO imaging was performed as described in Online Methods. Scale bar (white), 1 μm. The images of the movie sequences were subjected to a quantitative grayscale scaling (left) and to a 'per-frame auto-scaling' (right) to better display the particle movement despite fast photo-bleaching (see Online Methods). (MOV 3136 kb)

Supplementary Video 6

Live-cell HILO microscopy of cells expressing ClpX-mGFPmut3. Cells have ~50 particles, which are not localized in foci but move rapidly and seem to sample the entire cell. HILO imaging was performed as described in Online Methods. Scale bar (white), 1 μm. The images of the movie sequences were subjected to a quantitative grayscale scaling (left) and to a 'per-frame auto-scaling' (right) to better display the particle movement despite fast photo-bleaching (see Online Methods). (MOV 3123 kb)

Supplementary Video 7

Live-cell HILO microscopy of cells expressing mGFPmut3 alone (that is, not fused to another protein) expressed from the clpX promoter at the endogenous locus. The fluorescence signal displays a uniform cytoplasmic distribution. HILO imaging was performed as described in Online Methods. Scale bar (white), 1 μm. The images of the movie sequences were subjected to a quantitative grayscale scaling (left) and to a 'per-frame auto-scaling' (right) to better display the particle movement despite fast photo-bleaching (see Online Methods). (MOV 2726 kb)

Supplementary Video 8

Micro-colony growth of E. coli cells with Hfq-mGFPmut3, PepP-mGFPmut3, IbpA-mGFPmut3, MviM-mGFPmut3 and FruK-mGFPmut3 fusions. The fusions are constructed at the endogenous gene loci. Cells were grown to exponential phase in imaging medium and micro-colony growth was filmed on an agar pad with exposures every 10 min. Cell growth and imaging was performed at 30 °C. Green fluorescence images with 1-s exposure time were acquired every 10 min. Green fluorescence images of the movies were acquired with no binning (effective pixel size is 64.5 nm) and were subjected to a quantitative grayscale scaling. Scale bar (white), 1 μm. (MOV 3227 kb)

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Landgraf, D., Okumus, B., Chien, P. et al. Segregation of molecules at cell division reveals native protein localization. Nat Methods 9, 480–482 (2012). https://doi.org/10.1038/nmeth.1955

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