Presence of PAs increases survival probability of individual cells within microcolonies upon semilethal HS.

(A-B) Representative phase contrast and GFP epifluorescence (reporting IbpA expression/production and localization) images of a TLFM image sequence of growing MG1655 ibpA-msfgfp cells before and after application of an HS (51 °C, 7 min) killing approximately half of the cells. Shown are (A) MG1655 ibpA-msfgfp cells exposed to a prior sublethal HS (47 °C, 15 min) and (B) unstressed MG1655 ibpA-msfgfp cells. Scale bars correspond to 5 μm. (C) Average survival probability of sublethally heat-shocked (47 °C, 15 min) and unstressed control MG1655 ibpA-msfgfp cells (nmicrolonies = 38, ncells = 550 and nmicrolonies = 22, ncells = 315, respectively) upon exposure to a more lethal HS (51 °C, 7 min). No significant difference could be detected in survival frequency (Student t test, p-value = 0.98). Survival was defined as the ability of a cell to resume growth and subsequent division within 8 h after treatment. (D) Average survival probability of PA-free and PA-bearing MG1655 ibpA-msfgfp cells exposed to a prior sublethal HS (n = 498 and n = 52 cells, respectively) upon exposure to a second, more lethal HS (51 °C, 7 min). Asterisk indicates a significant difference in survival frequency between both cellular classes (Student t test, p-value = 1.33 × 10−3). (E) For PA-containing microcolonies, the fraction of cells surviving the second HS (51 °C, 7 min) is binned by the average cellular fluorescence before this HS. The dotted line indicates average survival of all cells; asterisks indicate bins that significantly differ (Fisher’s exact test, p-values = 1.50 × 10−2). Numbers in white indicate the number of cells included in each bin. Error bars for (C)–(E) indicate bootstrapped estimates of the standard error of the mean fraction of surviving cells. (F) Lineage tracing for the microcolony displayed in (A) illustrating growth, division, and survival of cells throughout the course of the entire experiment. Green lines indicate the PA-bearing cells. Red crosses indicate cells that did not survive the semilethal HS. At each division except the first—in which the identity of the poles is not known, and cells are positioned randomly—the cell inheriting the old pole is placed on the bottom side of the division pair. The numerical data underlying this figure can be found in S1 Data. GFP, green fluorescent protein; HS, heat shock; IbpA, inclusion body binding protein A; msfGFP, monomeric superfolder GFP; PA, protein aggregate; TLFM, time-lapse fluorescence microscopy.