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.

Figure 1

(a) Schematic depiction of the segregation assay. An upstream process (Clp protease localization) affects a downstream process (substrate degradation) that can be measured in daughter cells originating from cells with and without an FP tag on the upstream component. If the tag is non-intrusive, heterogeneity of the downstream process in the daughter cells should be independent of the tag. (b) Schematic of the mCherry-ssrA degradation reporter. (c) The plot shows single-cell degradation rates as measured by time-lapse fluorescence microscopy in daughter cells after cell division in the indicated bacterial strains. The daughter with the faster degradation rate is plotted on the x-axis. The spread along the diagonal is due to pulse-induction of the mCherry-ssrA reporter. Diagonal lines represent no cell-to-cell variability (gray line), 2× variability (dashed black line) and 5× variability (solid black line). Degradation rates are in arbitrary units (a.u.).

Figure 2

(a) Immunofluorescence microscopy of ClpX in wildtype (left), ClpX-Venus YFP (middle) and ΔclpX (right) strains. Insets are phase images and a close-up is shown for the wildtype. (b) Fluorescence images show bacteria expressing the ClpP-SNAP tag labeled with TMR (tetramethylrhodamine), compared to wildtype (right). Insets show phase images. (c) Cartoons of a fluorescent protein (yellow) forming a weak anti-parallel dimer and of avidity effects potentially clustering tagged ClpX hexamers (blue). (d) Fluorescence images of bacteria expressing the indicated constructs. The cell outline (red) is shown for cells with weak cytoplasmic signal. (e,f) HILO microscopy of gently fixed cells with ClpX-mGFPmut3 (e) and ClpP-mGFPmut3 (f). (g) Live-cell HILO microscopy of cells expressing ClpP-mGFPmut3. Scale bars,1 μm.

Figure 3

The plots show single-cell segregation assays for bacteria expressing the indicated proteins. Post-division single-cell degradation rates were measured by time-lapse fluorescence microscopy at 37 °C (upper row) and 30 °C (lower row) for both daughter cells. The ClpP-mGFPmut3 strain, the wildtype and a foci-forming control (Supplementary Fig. 11) were imaged at 30 °C because ClpP-mGFPmut3 levels were reduced at 37 °C but similar to the wildtype at 30 °C (Supplementary Fig. 3). Diagonal lines represent no cell-to-cell variability (gray line), 2× variability (dashed black line) and 5× variability (solid black line). Degradation rates are in arbitrary units (a.u.).