Figure 3.

Effect of light and de novo protein synthesis on the relocation of LCIB-Clover. A, Schematic of the liquid culture conditions. MBC-3 cells were cultured in medium aerated with 0.04% CO₂ illuminated at 120 µmol photons m⁻² s⁻¹ (white box) for 24 h were shifted to the dark (black box) for 2 h (indicated as I to the left of the white box) or 5 h (II). After incubation in the dark for 2 h, the concentrations of CO₂ aeration were switched to 0.001% for 3 h without adding CHX (III) or with the addition of CHX (IV). Red and yellow boxes indicate the time of observation and the addition of CHX, respectively. For all culture conditions, the pH of the medium was 7.0. B, Representative LCIB-Clover fluorescence images of MBC-3 cells. Roman numerals correspond to the culture conditions indicated in A. Ci concentrations in the culture medium were measured using gas chromatography, and the calculated CO₂ and ${\text{HCO}}_3^{–}$ concentrations are shown below the images. CO₂ concentrations shown in red indicate VLC (<7 µM CO₂) conditions. DIC, differential interference contrast image. Scale bars: 2 μm. C, Quantification of localization patterns of LCIB-Clover. Roman numerals correspond to the culture conditions indicated in A. The CV value of the fluorescence intensity was calculated in each cell to quantify LCIB-Clover localization. The median values derived from the analysis of MBC-3 cells are represented with error bars depicting the interquartile range (n = 5–7). Dunn’s multiple comparisons test was used to assess the statistical significance of LCIB-Clover localization between the different conditions. ^*P-value < 0.05, Kruskal–Wallis test with Dunn’s multiple comparison.