Figure 3. Single-cell calcium flux predicts cellular aminoglycoside response.

(A) Strip chart of cells expressing GCaMP6f. Cells were imaged in PMM alone for 5 hr, then exposed to 10 µg/mL kanamycin for 4 hr. After 4 hr, PMM alone was flowed in for an additional 26 hr. The blue arrow indicates a cell that was able to divide after treatment with kanamycin, 5 µm scale bar. Time is shown in (HH:MM) format. (B) Individual GCaMP6 time traces from cells that regrow after treatment compared to a random selection of cells that do not regrow within 24 hr. (C) The average (line) and standard deviation (shaded region) of the moving SD from all cells that regrow (blue) vs those that do not regrow (red). (D) Strip chart showing GCaMP6 fluorescence (top), propidium iodide fluorescence (middle), and the merge. Cells were treated with 100 µg/mL kanamycin at time t = 0. Time is shown in (HH:MM) format. (E) The mean GCaMP6 standard deviation for the population is shown in blue. Yellow shows the population average of the PI fluorescence. (F) Time traces of individual cells showing the GCaMP6 fluorescence (blue) and the PI fluorescence (yellow) on the same cells. The PI fluorescence was not correlated with the onset of transients, although many cells did uptake PI during the course of the experiment.

Figure 3—figure supplement 1. Cells that did not experience calcium transients are not genetically resistant.

(A–C) Cells that regrow after 1 treatment of kanamycin are not genetically resistant. (A) Strip chart of E. coli with 0 µg/mL kanamycin (t = 0–2 hr), 10 µg/mL kanamycin (t = 2–6 hr), 0 µg/mL kanamycin (t = 6–23 hr), and 10 µg/mL kanamycin (t = 23–30 hr). Time is shown at each frame in (HH:MM) format. The red arrow indicates a cell that regrows after the first kanamycin exposure. (B) Image from A at t = 27.5 hr. Individual cells were manually highlighted and shown with three different colors. Each cell has it’s GCaMP6 time trace shown in (C) for just the time period of the second kanamycin addition. (C) GCaM6f fluorescence over time of individual cells in B. (D–F) Untreated cells that exhibit transients do not divide. (D) Strip chart of E. coli imaged via the GCaMP6 fluorescence under PMM without aminoglycoside treatment. The blue and red arrow mark cells that do not divide. The time is shown in (HH:MM) format. (E) Image taken after 10 min of imaging with individual cells segmented manually. Each color corresponds to the GCaMP6 fluorescent trace shown in (F). Any cell that exhibited transients in the untreated conditions did not divide within the movie, which ended with a fully overgrown field of view.