Distributions of mitochondrial motility parameters in WT- and <i>dotA^–</i>infected S2 cells during the first 4 hours post-infection.

<p>Mean velocity (A), duty cycle (B), and persistence (C) are shown, data are pooled from 24 (wild type) or 25 (dotA-) movies form 4 separate infection experiments. No differences in distribution were detected that would indicate specific effects of infection on mitochondrial motility. Similar comparisons were made separately for each of the four hourly time points and both bacterial strains (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062972#pone-0062972-g005" target="_blank">Figure 5</a>). The majority of the data proved not to follow normal distributions as determined by a one-sample Kolmogorov-Smirnov test. Only the combined active duty cycle distributions (B) were shown to be significantly different, with WT <i>L. pneumophila-</i> infected S2 cells showing a reduced mitochondrial duty cycle relative to <i>dotA⁻ L. pneumophila-</i> infected cells (P<0.001; Kolmogorov-Smirnov two-sample test).</p

Fluorescent images illustrating the angles tracked for MTR-labeled mitochondrial movement in GFP expressing WT and <i>dotA</i><i>⁻</i><i>L. pneumophila-</i> infected cells (A).

<p>“V” represents the vacuole containing the bacterium, “i” indicates the initial position of the mitochondrion, and “f” indicates the final position of the mitochondrion. The relative frequency of the angle of mitochondrial movement towards or away from the vacuole at 1 hour post infection of WT and <i>dotA⁻ L. pneumophila</i>-infected S2 cells are shown in (B, C). The data are represented as the relative frequencies (in 15° bins) as indicated by the length of the lines displayed over 180°. The origin of all radiating relative frequency lines corresponds to the initial position “i,” as shown in (A), for each mitochondrion. All angle measurements considered included only the smaller θ between the three points; thus allowing all angles to be plotted on a semi-circle. Non-moving mitochondria were excluded from the analysis. The distribution of directions of movement was significantly different between the WT and <i>dotA⁻</i> infected cells at 1 hr post-infection (p<0.01; Kolmogorov-Smirnov two-sample test). Scale bar, 20 µm.</p

Frequency distributions of mitochondrial motility parameters detailed for each time point.

<p>The mitochondrial velocity (A), duty cycle (B), and persistence (C) for WT- and <i>dotA^–</i>infected cells from the experiments summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062972#pone-0062972-g004" target="_blank">figure 4</a> are broken out for 1, 2, 3 and 4 hours point post-infection. Most of the data were found not to follow a normal distribution using the 1-sample Kolmogorov-Smirnov test. The only significant differences in mitochondrial motility found between WT- and <i>dotA^–</i>infected cells (indicated by (*)) were (i) the mitochondrial velocity distribution at 1 hr post-infection (P<0.01) and (ii) the duty cycle distribution at both 2 hr and 3 hr post-infection (P<0.025), using the Kolmogorov-Smirnov two-sample test.</p

Quantification of mitochondrial distribution near vacuoles formed by WT and <i>dotA</i><i>⁻</i><i>L. pneumophila</i>.

<p>(A) Representative fluorescent images of S2 cells infected with WT and <i>dotA⁻</i> showing placement of 50-pixel ROIs around vacuoles. Mitochondria are stained red with MTR, while <i>L. pneumophila</i> are visualized via their expressed GFP. Mitochondrial fluorescence intensities measured within a circular region with a 25-pixel (5.3 µm) diameter (B) or a 50-pixel (10.6 µm) diameter (C) showed no difference in mitochondrial recruitment between the WT and <i>dotA⁻</i> infections (N = 3; two-way ANOVA; p>0.05). Error bars = SEM. Scale bar, 20 µm.</p

Quantitative measures of mitochondrial behavior in <i>L. pneumophila</i>-infected S2 cells.

<p>(A) shows images of live S2 cells with mitochondria stained green with R123, and <i>L. pneumophila</i> apparent via mCherry expression. The white traces show typical paths for individual mitochondria followed for 140–320 sec in cells infected with WT (left) or <i>dotA⁻</i> (right) bacteria. Tracking of individual mitochondrial movements in these cells was used to calculate mitochondrial velocity (B), duty cycle (% of time spent moving, C) and persistence (net displacement/total path length, D) in cells at 1, 2, 3 and 4 hrs post-infection. Analysis of mean values for these measures revealed no significant differences in mitochondrial motility between WT- (black bars) and <i>dotA^–</i>infected (gray bars) cells at any of these time points (Friedman two-way analysis of variance; p>0.05). The white numbers within each histogram bar indicate the number of separate cells in which mitochondria were tracked. Error bars = SEM. Scale bar, 10 µm.</p