Rapid manifestation of PA-induced heterogeneity in terms of survival probability and resuscitation time.

(A) Representative phase contrast images superimposed with CFP epifluorescence images (reporting mCer-cI78EP8 concentration and localization) of a TLFM image sequence of MG1655 ΔlacY pTrc99A-mCer-cI78EP8 cells before and after application of a semilethal HS (52 °C, 10 min). Scale bar corresponds to 2 μm. (B) Survival probability of PA-free and PA-bearing cells within sibling pairs (n = 258 pairs) containing at least 1 PA upon application of the HS (52 °C, 10 min). Asterisk indicates a significant difference could be detected between the survival probability of both classes (Student t test, p-value = 1.77 × 10−13). Numbers in black indicate the number of cells included in each group. (C) Cumulative resuscitation time distributions of PA-bearing and PA-free MG1655 ΔlacY pTrc99A-mCer-cI78EP8 cells (n = 133 and n = 96, respectively) surviving the heat treatment (52 °C, 10 min). The time individual surviving cells needed to resume growth was determined and binned to create the resuscitation time distributions. Surviving PA-bearing cells displayed significantly shorter resuscitation times than their surviving PA-free counterparts (K-S test, p-value = 1.17 × 10−31). (D) PAs were detected as non-diffraction-limited spots in the CFP epifluorescence image, their size was determined and binned to create the PA size distribution. Representative phase contrast, CFP epifluorescence, and CFP epifluorescence images with detected regions are shown. Scale bar corresponds to 2 μm. (E) Distribution of PA sizes. (F) Survival probability of cells (stemming from the PA-bearing cells shown in (B)) harboring PAs of different sizes (n = 352 cells). No significant difference in survival probability could be detected when compared to the average cellular survival (indicated by the dotted green line; Fisher’s exact test). Numbers in white indicate the number of cells included in each bin. The numerical data underlying this figure can be found in S1 Data. CFP, cyan fluorescent protein; HS, heat shock; K-S, Kolmogorov-Smirnov; mCer, monomeric cerulean; PA, protein aggregate; TLFM, time-lapse fluorescent protein.