Asymmetric segregation of native PAs raised by exposure to sublethal heat shock.

(A and C) Representative phase contrast, GFP epifluorescence (reporting IbpA concentration and localization), and superimposed images of a TLFM image sequence of two MG1655 ibpA-msfgfp cells growing into microcolonies after exposure to a sublethal heat shock (47 °C, 15 min). Scale bars correspond to 5 μm. (B and D) Corresponding lineage trees in which the length of lines connecting cells to their progeny is proportional to the relative growth rate of that cell, and the color of these lines is proportional to the relative average GFP fluorescence of that cell. 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 left side of the division pair. (E) Correlation between average cellular GFP fluorescence and growth rate of individual fourth- and fifth-generation MG1655 ibpA-msfgfp cells (nmicrolonies = 10, ncells = 478) after exposure to a sublethal heat shock (47 °C, 15 min). Color indicates whether the respective cell was PA-bearing or PA-free. No strong correlation could be observed between both variables (Pearson’s r = 0.1269, p-value = 8.31 × 10−2), not even within PA-bearing cells (Pearson’s r = 0.0927, p-value = 3.43 × 10−1, n = 34). (F) Violin plots comparing the distribution of growth rates of the same PA-free and PA-bearing cells (n = 444 and n = 34, respectively). Although a significant difference could be detected between the average growth rate of both classes (3.97%, Student t test, p-value = 1.67 × 10−3), a permutation test revealed that this could be attributed to the underlying age structure of PA-bearing cells (p-value = 0.56). (G) Distribution of fitness defects observed for permutations (100,000) comparing randomly selected PA-free cells (of similar age as the observed PA-bearing cells) to all other PA-free cells. Green line indicates the empirically observed fitness defect for PA-bearing cells (3.97%). The numerical data underlying this figure can be found in S1 Data. A.U., arbitrary unit; GFP, green fluorescent protein; IbpA, inclusion body binding protein A; msfGFP, monomeric superfolder GFP; PA, protein aggregate; TLFM, time-lapse fluorescence microscopy.