Diffusion coefficient and mobile fraction under various pharmacological treatments.

*<p>P-values were obtained by comparing diffusion coefficients from each experiment to the untreated PBL data using a two-sample T-test. Except for PBL+Iono no significant differences in the diffusion coefficient were observed (P-values>0.05) No significant differences in the mobile fraction were observed (all P-values>0.05).</p

TCR mobility is modulated by intracellular calcium via the actin cytoskeleton.

<p>A. Mean FRAP recovery curves are shown for Peripheral blood T Lymphocytes (PBL, blue curve n = 61), PBL treated with cytochalasin D (PBL+CytoD, green curve n = 20), PBL treated with ionomycin (PBL+iono, orange curve n = 37), and PBL treated with ionomycin and cytochalasin D (PBL+Iono+CytoD, light blue curve n = 25). B. Mean FRAP recovery curves are shown for PBL (blue line, n = 61) PBL treated with latrunculin B (PBL+LatB, pink curve n = 10), PBL treated with ionomycin (PBL+iono, orange curve n = 37), and PBL treated with ionomycin and latrunculin B (PBL+Iono+LatB, red curve n = 10).</p

TCR mobility does not depend on the activation state of T cells.

<p>Mean FRAP recovery curves are shown for peripheral blood T Lymphocytes (PBL, blue curve n = 61), Cord Blood T Lymphocytes either freshly isolated (CBTL, green curve n = 20) or activated in culture (activated CBTL, red curve n = 20). Data are from 2 (for CBTL) and 4 (for PBL) independent experiments.</p

Fitting the diffusion and binding model to FRAP recovery curves.

<p>We show the fit of the pure diffusion model (red) and the binding+diffusion model (black) to the PBL data and to the ionomycin-treated PBL. Both models provide a good fit to the recovery curves but a statistical test reveals that the binding model is significantly better at explaining the FRAP recovery when the PBL are treated with ionomycin (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003913#pone-0003913-t002" target="_blank">Table 2</a>).</p

[Ca²⁺]_i increase induces actin polymerization in PBL.

<p>PBL were treated for 15 minutes with latrunculin B (50 nM, 100 nM or 500 nM) or with ionomycin (0.5 µg/ml, 1 µg/ml, 2.5 µg/ml). F-actin cellular content was measured by FACS analysis. Data are from one representative experiment out of three. In parallel experiments the vehicle of the drugs (DMSO) did not affect actin polymerization.</p

Model fitting to FRAP recovery curves.

<p>SSR – Sum of squared residuals, AIC – Akaike's Information Criterion, Prob – Probability that the model is the better descriptor of the FRAP recovery curve based on AIC.</p

Predictions of model of clonal interference for changes in mucoid frequencies with time.

<p>Simulations of the adaptive dynamics over the period of the experiment (30 days). The frequencies of mucoid phenotypes are plotted and can be compared to those observed in the experiments (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003802#ppat-1003802-g001" target="_blank">Fig. 1B</a>). The values of parameters used and the dynamics of haplotypes that compete for fixation are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003802#ppat.1003802.s009" target="_blank">Figure S9</a>.</p

<i>In vitro</i> evolved <i>E. coli</i> show increased virulence <i>in vivo</i>.

<p><b>A</b>) Survival of mice infected with different doses of ancestral (ANC, in blue), mucoid bacteria evolved in the presence of MΦ (MUC, in red) or bacteria evolved in the absence of MΦ (CON, in green). The number of mice are shown inside the bars, <b>B</b>) Survival probability of mice infected with ANC, MUC and CON, represented as lines from the fit of a binomial General Linear Model used to infer LD₅₀, <b>C</b>) Kaplan-Meier curves and <b>D</b>) % maximum reduction in temperature or weight at the LD₅₀ dose for the MUC (n = 10), ANC (n = 11) and CON (n = 5) (Error bars correspond to 2SE, * indicates p<0.05).</p

Emergency of morphological diversity in the bacterial populations adapting to MΦ.

<p>(A) Examples of the variability for colony morphology that emerged in <i>E. coli</i> populations adapting to MΦ, from left to right – ANC stands for morphology of ancestral, SCV for the small colony variants morphology and MUC for the mucoid colony morphology. (B) Dynamics of frequency change of the evolved phenotypes in each replicate evolving populations (M1 to M6): white squares indicate ANC, black triangles SCV, black circles MUC phenotypes.</p

Genetic characterization of adaptive mutations and the dynamics of their appearance.

<p>(A) Mutations identified in MUC1 to MUC6 clones isolated from M1 to M6 populations (evolved for 450 generations), represented along the <i>E. coli</i> chromosome. For simplicity, the genomes are represented linearly and are horizontally drawn. The types of mutations are represented in the following way: SNPs are shown as crosses, IS insertions as inverted triangles and deletions as triangles. Filled symbols represent mutation in the coding region of the gene and empty symbols in the regulatory region. (B) Emergence and spread of adaptive mutations in M1 to M6 populations. Dynamics of haplotype frequencies in evolving populations at different days of evolution experiment are represented by circles. The color and symbol (IS insertions are represented as circles and other mutations as crosses) of each sector represents different haplotypes and the area of the circle their frequency in the population. Grey area represents the frequency of clones in the population that were typed for existing mutations in the population and did not differ from ancestral haplotype.</p