Aggregate distribution and associated fluorescence levels along the cell axis. Shown is IbpA-YFP foci localization along the cells' normalized longitude internal coordinate, oriented from the new pole (0) to the old pole (1) of each cell. Binned histograms show foci localization (A–D, 100 bins; E, 200 bins) at first appearance (A), at the last movie frame before first division (B), at the first movie frame after first division (C), at the first movie frame after two consecutive divisions (D), and cumulative over all movies' frames (E). (F) Foci maximal fluorescence intensity (arbitrary gray-level units) as a function of their localization.

Growth rate and aggregation accumulation in bacterial lineages. Bacterial lineage representation of average growth rates (A) and fluorescence intensity (B) of new-pole cells (blue) and old-pole cells (red) of the MGAY strain. Summits represent the measured value, and lines follow the lineage propagation. Values of the ordinates are normalized by the generation mean value and averaged over all 12 films. (A) A clear pattern of gradual loss of growth rate of old-pole cells and fast recovery of new-pole cells is discerned. In 90.5% of offspring cell pairs the old-pole cell grows more slowly than the new-pole cell and its mother cell (79.4%) (aging), whereas in 74.6% the new-pole cell grows faster than its mother cell (rejuvenation). (B) Net fluorescence intensity of foci at first time point after division. (C) The above patterns result in a negative correlation between the cells' growth rate and aggregate load (measured as foci fluorescence) whereby the two subpopulations [old-pole cells (red) and new-pole cells (blue)] can be discerned.

IbpA-YFP in vivo localization. IbpA-YFP overlaps with inclusion bodies and is independent of division machinery or nucleoid occlusion. (A) Representative phase contrast image of MGAY cells after treatment with streptomycin. (B) Overlay of A with the fluorescence image (IbpA-YFP depicted in green). (C) Representative phase contrast image of MGAY cell in the presence of an insoluble protein [antibody single-chain Fv fragment plasmidic expression (38)]. (D) Overlay of C with the fluorescence image. Similar inclusion body and IbpA-YFP colocalization was observed upon heat shock (data not shown). (E) Representative phase contrast image of MG1655(ibp::cat) cell after treatment with streptomycin. (F and G) IbpA-YFP foci are localized in cellular poles or near formed septums, visualized by expression of the ftsZ-CFP fusion (depicted in red) in the MGAY strain. (H) IbpA-YFP foci in the MGAY(minCD) strain lacking the ability to localize the division septum to mid-cell. (I) IbpA-YFP foci in the MGAY strain (blue, DAPI DNA stain; red, FM4-64 membrane stain). (J) IbpA-YFP foci in 2-aminopurine-treated dam⁻ strain where the chromosomes were degraded [blue, DAPI stain (none detected); red, FM4-64). Images were taken using 5% (B, D, and F–H) and 100% (I and J) of available excitation light. (Scale bars: 1 μm.)

Individual aggregates are located to the old pole through cycles of cell divisions. Because movement of foci is rarely observed, the location of an aggregate within the cell after division is determined by its location in the mother cell. Those that were at the one- or three-quarter positions are found concentrated around the mid-cell point after division (Step I). Aggregates at the mid-cell are subsequently located in the new pole, with equal probability to be in either of the two cells (Step II). Those that are found in the new-pole end of the cell immediately before division remain in the same pole; however, that pole, having been formed in the previous division event, is now an old pole in the offspring cell (Step III). Note that aggregates can be initially detected at polar, mid-cell, or quarter-cell positions but are eventually located to an old pole. Once there, they are consistently inherited by the old-pole cell after division. Aggregates are indicated by green dots. Red cell ends are old poles, and blue cell ends are new poles.

Aging correlation with presence of protein aggregation. The aging effect calculated from the relative growth rate (GR) difference between old-pole and new-pole offspring [e.g., (GR_old − GR_new)/GR_offspring] of newborn mother cells where inclusion body is inherited by the old-pole cell (population 1) or the new-pole cell (population 2). See Table 1 for actual values. IB, inclusion bodies.