Figure 1.

Dynamic RNase E-mKate2 foci are associated with single-cell homeostasis

(A) Representative time-lapse images of exponential-phase M. smegmatis RNase E-mKate2 fluorescent reporter. Phase-contrast (blue) and fluorescence (magenta) are merged. Scale bar, 10 μm. Numbers represent hours. Boxes zoom in on two first-generation (G1) cells, without (white arrow) or with (magenta arrow) RNase E fluorescent focus, which disappears in the second generation (G2). Scale bar, 5 μm. See also Video S1.

(B) Representative heatmaps of single-cell RNase E-mKate2 fluorescence as a function of generation time and percent of cell length.

(C and D) RNase E-mKate2 fluorescence frequency distribution (C) or averaged over the cell lifetime (D) in single cells segregated by the absence (purple, n = 180) or presence (magenta, n = 330) of RNase E foci. Black lines indicate mean ± SD. Significance by unpaired t-test. Data are from two independent experiments.

(E and F) Single-cell size (E) and growth rate (F) averaged over the cell lifetime in M. smegmatis wild type (CT) and in RNase E-mKate2 subpopulations identified in (C). Black lines indicate mean ± SD (100 ≤ n ≤ 330). Significance by one-way ANOVA followed by Tukey’s multiple comparison test: ∗p = 0.012 and 0.023; ∗∗∗p = 0.0003; ∗∗∗∗p < 0.0001.

(G) Distribution of RNase E foci as a function of cell length (n = 734). Black line is the fitting with a sum of two Gaussians. Old (O) and new (N) cell-pole are indicated. Data are from two independent experiments.

(H) Pearson correlation of RNase E-mKate2 fluorescence averaged over the cell lifetime between mother cells and their old- (black) and new-pole (white) daughters (left panel), and between siblings (right panel). Data are from two independent experiments (n = 170, per category).

(I) Representative snapshot images (left) of RNase E-mKate2 reporter stained with RNAselect dye. Phase-contrast (gray), RNase E-mKate2 (magenta), and RNAselect (green) fluorescence. Scale bar, 5 μm. Single-cell Pearson correlation of fluorescence (right panel) in exponential-phase cells without (gray) or with (magenta) RNase E foci. Data are from two independent experiments (n = 145).

(J) Representative time-lapse image series of exponentially growing RNase E-mKate2 reporter treated with 2.5 mM of the putative M5 RNase E inhibitor (Kime et al., 2015). Phase-contrast (blue) and fluorescence (magenta) are merged. Numbers represent hours. Scale bar, 10 μm. See also Video S2.

(K and L) Single-cell RNase E-mKate2 fluorescence (K) and size (L) from time-lapse microscopy (J). M5 inhibitor (gray shading). Black lines indicate mean ± SD (n = 142). Data are from two independent experiments. Significance by one-way ANOVA followed by Tukey’s multiple comparison test: ns, not significant; ∗∗∗∗p < 0.0001.