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. 2021 Jun 11;118(24):e2104686118. doi: 10.1073/pnas.2104686118

Fig. 2.

Fig. 2.

Cytoplasmic shrinkage is sensitive to the extent and duration of nutrient removal and increases cell dry-mass density. (A) Representative phase-contrast, cytoplasmic GFP, and periplasmic mCherry images of KC1193 cells after transitioning from LB to M9 salts for 4 h. Shrinkage of the cytoplasm was evident by the bright periplasmic mCherry signal. (B) The relative cytoplasmic area of KC1193 cells (as measured by the GFP signal relative to the total cell area with GFP or mCherry signal) plateaued only after multiple washes. n > 100 cells for each condition, and P values were calculated from two-tailed Student’s t tests. N.S., not significant. (C) After shifting from LB to M9 salts in a microfluidic chamber, the cytoplasmic area of KC1193 cells continued to decrease for several hours. (D) The rate of shrinkage was faster in cells transitioned from LB to M9 salts compared to transitioned from M9 glucose to M9 salts. The data points are mean ± SEM for n > 100 cells in each condition. (E) Cytoplasmic shrinkage occurs predominantly at the new pole. After pulse-chase labeling with Alexa488-WGA to label the cell wall, the pole with separation between the cytoplasm and cell wall was almost always the unlabeled (new) cell pole. Inset: a typical cell with shrinkage at the new (dark) pole. (F) Quantitative phase imaging demonstrated that suddenly starved cells have higher dry-mass density than log-phase cells, yet the density is still lower compared to stationary-phase cells (n > 200 cells for each condition). (G) The total dry mass of suddenly starved cells was similar to that of log-phase cells. Inset: typical quantitative phase images for each condition, with the white arrow highlighting the pole with shrinkage (scale bar, 1 µm). (H) Proteomics measurements highlighted classes of proteins involved in translation and DNA replication with significantly lower abundance in suddenly starved cells (P < 0.01, false discovery rate < 0.05 with bootstrapping). The colored circles represent data from individual proteins. The white circles are median values across all proteins in the class, and error bars are 1.5 times the interquartile range.