MEK1/2 inhibition promotes invasive-opposing leading edge lamellipodial structures in 3-D by increasing localised Rac1 activity and decreasing localised RhoA activity.

<p><b>A</b> Whole A2780 cells seeded on cell derived matrix and stained with Alexa Fluor 488 Phalloidin, imaged by confocal microscopy to reveal actin structures of cells arrested in position during migration subject to different treatment conditions: (left) a cell without cRGDfV stimulation and treated with DMSO ~2 hours prior to fixing exhibiting a more 3-D lamellipodial actin structure; (centre) a cell treated with cRGDfV and DMSO for vehicle ~2 hours prior to fixing exhibiting more pseudopodia and filopodia at the leading edge; (right) a cell treated with cRGDfV and PD184352 ~2 hours prior to imaging exhibiting a reversion back to a more lamellipodial leading edge. <b>B.</b> High resolution maximum Z projection images of the leading edge actin structures of cells fixed and stained as in A. A 0.7 Airy pinhole was used to delineate individual filopodia, with a zoom factor of 4.0 on a 100x objective for cells treated: (left) without cRGDfV, with DMSO which exhibits fewer, shorter filopodial protrusions and abundant actin veiling; (centre) with cRGDfV and DMSO for vehicle, which exhibits more, longer filopodia and little actin veiling; (right) with cRGDfV and PD184352 which exhibits a reversion to the basal phenotype with fewer, shorter filopodia with increased actin veiling. <b>C-D.</b> Average <b>C.</b> number and <b>D</b>. normalised length of filopodia per leading edge of fixed, polarised cells in maximum projection high resolution confocal Z-stack images under treatment conditions as indicated. N > 13 cells per condition across 3 experimental repeats, one way ANOVA with Holm-Sidak post hoc test, * indicates p<0.05, *** p<0.001. <b>E-H</b> Representative Ratiometric FRET images of whole A2780 cells on CDMs at single timepoints (main images) and leading edge quantified areas (panel of images, right). <b>E.</b> Cell transfected with Raichu-Rac1 probe, stimulated with cRGDfV and treated with DMSO for vehicle; <b>F.</b> Cell transfected with Raichu-Rac1 probe, stimulated with cRGDfV and treated with PD184352; <b>G.</b> Cell transfected with Raichu-RhoA probe, stimulated with cRGDfV and treated with DMSO for vehicle; <b>H.</b> Cell transfected with Raichu-RhoA probe, stimulated with cRGDfV and treated with PD184352. All images have the same custom look-up table (LUT) applied and set between 1.0 and 2.0 (shown, right of images), where red pixels denote high GTPase activity. <b>I.</b> Quantification of average FRET ratio in the leading edge of all analysed cells across all 20 timepoints in each 5 minute movie. N > 15 cells across 3 experimental repeats. Tukey boxplot used with mean indicated as +. Student t-tests used, * indicates p <0.05.</p

Logical simulation of integrin-driven cell migration.

<p><b>A.</b> Reconstructed network describing signalling events leading to GTPase activity. The model consists of one input node, EGF, two nominated output nodes, Rac1 and RhoA, and 38 intermediate nodes. Reactions included in the model are activation or inhibition, where some reactions need cooperation of two or more upstream nodes via AND gates. See <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004909#pcbi.1004909.s012" target="_blank">S1 Table</a> for references to all reactions included in the model. <b>B, C.</b> Time-course simulation outputs for the first 50 time increments of the model for different Rac1 activator/inhibitor hierarchies, where the outputs of interest Rac1 and RhoA (yellow box) and the dominant Rac1 activator/inhibitor (green box) are highlighted: <b>B.</b> pRacGAP1 dominates Rac1 activity above all Rac1 activator. As pRacGAP1 activity is ‘switched’ ON, Rac1 activity is ‘switched’ OFF after one time increment, RhoA activity is ‘switched’ ON a further time increment later and Rac1/RhoA remain OFF/ON respectively as t → ∞; <b>C.</b> Sos1E dominates Rac1 activity over pRacGAP1 which in turn dominates Rac1 activity over Vav2 and RalbP1. Initially pRacGAP1 activation switches OFF Rac1 which switches on RhoA later as before, however when Sos1E is ‘switched’ ON (green box), Rac1 is ‘switched’ ON after one time increment and then RhoA is ‘switched’ OFF after one further time increment. When Sos1E is later ‘switched’ OFF, Rac1 and subsequently RhoA are switched OFF/ON respectively, leading to cyclic activity of Rac1 nd RhoA as t → ∞. <b>D-F.</b> Steady-state outputs of the model for simulations with example different Rac1 activator/inhibitor hierarchies (full list of hierarchies in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004909#pcbi.1004909.s014" target="_blank">S3 Table</a>), where activator/inhibitor dominance is visualised by reaction arrow thickness: <b>D.</b> All Rac1 activators Vav2, RalbP1 and Sos1E (Sos1-Eps8-Abi1 complex) (thick black arrows) dominate Rac1 activity over the Rac1 inhibitor pRacGAP1 (thin red arrow); <b>E.</b> pRacGAP1 (thick red arrow) dominates Rac1 activity over Vav2, RalbP1 and Sos1E (thin black arrows); <b>F.</b> Sos1E (thick black arrow) dominates Rac1 activity over pRacGAP1 (medium red arrow) which in turn dominates Rac1 activity over Vav2 and RalbP1 (thin black arrows). Note steady-state outputs in the Boolean simulations can only be stable activity where the node is ON for all time as t → ∞ (green), stable inactivity where the node is OFF for all time as t → ∞ (red) and cyclic activity where the node is encapsulated in a stable limit cycle and cycles regularly between ON and OFF activity as t → ∞ (yellow). All simulations performed in CellNetAnalyzer.</p

<i>In silico</i> node knockouts predict a Sos1/Ras/Raf/MEK1/2/ERK1/2/p90RSK negative feedback loop affects Rac1 and RhoA dynamics via the Rac1 activation Sos1-Eps8-Abi1 complex.

<p><b>A.</b> Effect of individual <i>in silico</i> node knockout of every non-output node in the model on RhoA/Rac1 output dynamics. Node knockouts were performed by removing all inward edges for each node individually such that the knocked out node remained OFF for all time, except for inhibitory nodes with no input edges PTEN, Ship2 and PP2a which were set to ON. Note ‘Rac1 to RhoA switch’ output corresponds to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004909#pcbi.1004909.g001" target="_blank">Fig 1B and 1E</a> and cyclic output corresponds to <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004909#pcbi.1004909.g001" target="_blank">Fig 1C and 1F</a>. <b>B-C.</b> Selected steady-state node activity for the Sos1E > pRacGAP1 > RalbP1, Vav2 cyclic activity inducing hierarchy for proteins affecting Rac1 and RhoA dynamics given knockout of <b>B</b> MEK1/2 and <b>C</b> Eps8. MEK1/2 is a critical node in the Sos1/Ras/Raf/MEK1/2/ERK1/2/p90RSK negative feedback loop, whereby removal of MEK1/2 keeps ERK1/2 and p90RSK inactive, which removes the inhibition of Sos1 keeping the Sos1-Eps8-Abi1 complex active which keeps Rac1 active and RhoA inactive and abrogates the pro-invasive Rac1 to RhoA switch. Eps8 removal prevents formation of the Sos1-Eps8-Abi1complex which prevents activation of Rac1 regardless of activity state of Sos1/Ras/Raf/MEK1/2/ERK1/2/p90RSK negative feedback loop nodes. <b>D-E.</b> Western blots showing steady state endogenous levels of phosphorylated p44 and p42 MAP Kinase (Erk1 and Erk2) and total levels of ERK2 (for loading) for cells treated with a single MEK1/2 inhibitor, PD184352 or AZD6244, or DMSO for vehicle for different cell types: <b>D.</b> H1299s, a non-small lung cell carcinoma, either stably expressing mutant p53 (right) or with an empty for p53 (left); <b>E.</b> A2780s, an epithelial ovarian cancer cell line, with (right) and without (left) stimulation with cRGDfV. Numbers below each band denote the intensity level normalised to the corresponding DMSO control. <b>F-J.</b> Effects of MEK1/2 inhibition on 2-D cell migration in >10 hour scratch wound experiments. <b>F.</b> Average speed of tracked H1299 cells over the time taken for wounds to close or 16 hours (whichever comes first). Cells expressing mutant p53 or with an empty vector for p53 were treated with DMSO or MEK1/2 inhibitors ~1 hour prior to imaging. <b>G.</b> Average persistence of the same tracked cells as in F. persistence is the measure of the distance between the cells first time-point position and last time-point position divided by the total distance travelled by cell between every time point position. <b>H.</b> Average speed of tracked A2780s cells for the same conditions as in F, cells were treated with cRGDfV at the same time as DMSO/MEK1/2 inhibitors ~1 hour prior to imaging. <b>I.</b> Average persistence for same tracked A2780 cells. Data are representative of 3 experiments, and 90 cells were tracked per condition. Graphs are Tukey boxplots where + represents the mean of each condition. **** indicates one way ANOVA with post-hoc Tukey HSD with p-value < 0.0001, * indicates p-value < 0.05 <b>J.</b> Representative images of H1299 cells in a sub-domain of an image at t = 0 (the initial frame) and the same sub-domain at t = 5 hours (30 10 minute frames later), for cells without/with mutant p53 expression and treated with DMSO or MEK1/2 inhibitor AZD6244. Yellow arrow heads indicate lamellipodial leading edge actin, while red arrow heads indicate more spike-like protrusions. The same cell is highlighted with arrow heads at t = 0 and t = 5 hours for each different condition.</p

MEK1/2 inhibition significantly reduces α5β1/RCP driven 3-D invasion, Eps8 reverses the ME1/2 inhibition effect.

<p><b>A-B.</b> Images of cells stained with calcein-AM following 48 hours of invasion into a fibronectin supplemented collagen matrix. Images shown are individual Z positions 14.97 μm apart, with the direction of invasion left to right for the images (the far left image for each condition is the bottom of the transwell). Cells were transfected with control siRNA or Eps8 siRNA 24 hours prior to seeding onto inverted transwells. MEK1/2 inhibitors or DMSO were used 24 hours after invasion was initiated to allow for proliferation. <b>A.</b> A2780 cells stimulated with cRGDfV throughout invasion assay with control/Eps8 siRNA and DMSO/PD184352 as indicated. 45 μm line has been taken as threshold for invasion, beyond which cells are thought of as invasive. <b>B.</b> As in A for H1299 cells stably expressing mutant p53. H1299 cells are less invasive so the 30 μm line has been taken as threshold for invasion. <b>C-D.</b> Quantification of at least 3-independent invasion assay experiments. Invasive proportion is calculated as the total GFP fluorescence for all the images above the invasive threshold (45 μm for A2780s, 30 μm for H1299s) divided by the total GFP fluorescence in all images. All data has been normalised to the Eps8 siRNA, DMSO treated condition to give relative invasion levels for all conditions for: <b>C.</b> A2780 cells stimulated with cRGDfV; <b>D.</b> H1299s cells stably expressing mutant p53. Graphs are Tukey boxplots with mean represented as +, individual student t-tests used as indicated, * indicates p <0.05, ** indicates p <0.01, *** indicates p < 0.001.</p

Perivascular T cells form long-lasting interactions with CX₃CR1^+/GFP in the brains of mice infected with <i>Pb</i> ANKA.

<p>hCD2-DsRed X CX₃CR1^GFP/GFP dual reporter mice were infected with 10⁴<i>Pb</i> GFP ANKA (n = 5 from three experiments). <b>(A)</b> Maximum intensity projections from intravital two-photon microscopy movies (283x283x30 μm) showing interaction of perivascular T cells (red) with CX₃CR1^+/GFP cells (green) in brains of mice on day 7 p.i. Blood vessels (cyan) were visualized by i.v. injection of Qtracker non-targeted quantum dots. GFP⁺ pRBCs (green) can also be seen within the lumen of the vessels. White arrows highlight selected perivascular T cells forming stable interactions with CX₃CR1^+/GFP cells. <b>(B)</b> Selected cropped frames from a time-lapse movie showing a perivascular T cell [number one in <b>(A)</b> in contact with a CX₃CR1^+/GFP cell over a 17-minute period. <b>(C)</b> Three dimensional section showing the same T cell and CX₃CR1^+/GFP cell interacting in the XY, XZ and YZ planes. Scale bars: (A) 30 μm, (B) 5 μm, (C) 10 μm. (D) Mean duration ± SD of individual hCD2-DsRed T cell contacts with CX₃CR1^+/GFP cells in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s021" target="_blank">S8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s022" target="_blank">S9</a> Videos</b>. (E) Mean number ± SD of CX₃CR1^+/GFP cells contacted by individual perivascular hCD2-DsRed T cells in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s021" target="_blank">S8</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.s022" target="_blank">S9</a> Videos</b>, over a 17-minute period.</p

Parasite specific CD8⁺ T cells are comparably activated within the brains of mice infected with <i>Pb</i> ANKA and <i>Pb</i> NK65 parasites.

<p>C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA or <i>Pb</i> NK65 pRBCs. <b>(A)</b> Representative flow cytometric plots and calculated percentages showing frequencies of CD8⁺ T cells (gated on live leukocytes) within the brains of uninfected and infected (day 7 p.i.) mice (n = 5). <b>(B)</b> Absolute numbers of CD8⁺ T cells within the brains of uninfected and infected (day 7 p.i.) mice (n = 19–22). <b>(C)</b> Percentage of CD8⁺ T cells expressing high levels of the surrogate antigen specificity marker, CD11a (n = 5). <b>(D)</b> Activation phenotype of CD8⁺CD11a^high T cells from uninfected and infected (day 7 p.i.) mice (n = 5). Results are representative of two independent experiments <b>(A, C and D)</b> or four combined experiments <b>(B)</b>. Bars represent mean number ± SD. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).</p

T cells exhibit equivalent perivascular compartmentalisation but distinct behaviours during <i>Pb</i> ANKA and <i>Pb</i> NK65 infections.

<p>hCD2-DsRed C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA or <i>Pb</i> NK65 pRBCs or left uninfected. Transcranial two-photon microscopy of the meninges was performed on days 5 p.i. (<i>Pb</i> ANKA), and 7 p.i. (<i>Pb</i> ANKA and <i>Pb</i> NK65). <b>(A)</b> Maximum intensity projections from intravital two-photon microscopy movies (283x283x30 μm) showing Qtracker non-targeted quantum dot-labeled blood vessels (cyan) and DsRed T cells (red) within the meninges of infected mice on day 7 p.i. with <i>Pb</i> ANKA and <i>Pb</i> NK65 and in uninfected mice. <b>(B)</b> Mean number ± SD of hCD2-DsRed T cells (including luminal and perivascular) in imaged tissue sites in uninfected control mice and on day 5 (<i>Pb</i> ANKA, n = 2) and 7 p.i. (<i>Pb</i> ANKA, n = 7; and <i>Pb</i> NK65, n = 5). <b>(C)</b> Mean proportion ± SD of hCD2-DsRed T cells located perivascularly in the meninges of <i>Pb</i> ANKA and <i>Pb</i> NK65 infected (day 7 p.i.) mice. <b>(D)</b> Mean distance ± SD (μm) of perivascularly located T cells from abluminal vessel wall in mice infected with <i>Pb</i> ANKA and <i>Pb</i> NK65 (day 7 p.i.). <b>(E)</b> Representative cropped tile scanned images showing heterogeneity of T cell clusters around pial vessels within the brains of mice infected with <i>Pb</i> ANKA and <i>Pb</i> NK65 (day 7 p.i.). <b>(F)</b> Quantification of average perivascular T cell speeds, arrest coefficient (proportion of time points when instantaneous velocity is <2 μm/min) and confinement ratio (track displacement/track length) from individual three-dimensional T cell tracks. Each point represents an individual DsRed T cell. Results are pooled two experiments, n = 4 for both groups. <b>(G)</b> Graphical illustrations summarizing the XY movement of individual perivascularly located T cells from normalized starting positions in the brains of mice on day 7 p.i. with <i>Pb</i> ANKA and <i>Pb</i> NK65 parasites. Scale bars: (A) 30 μm, (C) 150 μm. *p ≤ 0.05, ****p<0.0001 (Student’s unpaired t test or one-way ANOVA with Tukey's multiple comparisons test).</p

Parasite specific OT-I CD8⁺ T cells that directly cause ECM are perivascular and are highly arrested in the brain.

<p>C57BL/6 (n = 9) and P14 (n = 10) mice were infected with 10⁶ SIINFEKL-expressing <i>Pb</i>-TG pRBCs. Prior to infection, 10⁶ naïve DsRed-expressing OT-I CD8⁺ T cells were adoptively transferred into CFP⁺ P14 host mice (n = 6). Development of ECM (grey area) was monitored by assessing <b>(A)</b> peripheral parasitaemia ± SD and <b>(B)</b> survival. <b>(C)</b> Maximum intensity projections from an intravital two-photon microscopy movie showing accumulation of DsRed⁺CD8⁺ OT-I T cells (orange) within the brain of a P14 recipient on day 6 p.i. infection. Endothelial cells (blue) were visualized by CFP expression. <b>(D)</b> Proportion ± SD of CD8⁺ OT-I T cells located perivascularly on day 6 p.i. (n = 8). <b>(E)</b> Quantification of average perivascular T cell speeds, arrest coefficient and confinement ratio from individual three-dimensional T cell tracks. Results are pooled from two experiments.</p

Hypothetical model for the CD8⁺ T cell-dependent development of ECM.

<p>(1) <i>Pb</i> ANKA infection leads to the upregulation of adhesion molecules and cross-presentation of parasite antigen by MHC-I on brain microvascular endothelial cells [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.ref085" target="_blank">85</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005210#ppat.1005210.ref087" target="_blank">87</a>]. (2) This promotes transient interaction of <i>Pb</i> ANKA-pRBCs and rolling of activated CD8⁺ T cells on the luminal aspect of the brain microvessel endothelial cells. Beginning one day prior to signs of ECM, parasite-specific CD8⁺ T cells are recruited to the perivascular space, either via direct diapedesis across the endothelium, or migration via the highly permissive choroid plexus. In the perivascular space, parasite-specific CD8⁺ T cells form immune synapses with (3) parasite Ag-expressing APCs and (4) the basolateral membrane of cross-presenting endothelial cells. Perivascular APCs may acquire parasite antigen as a result of either transport of material across the vessel wall preceding generalized breakdown of the barrier or subsequent to breach of cerebral vascular integrity. (5) The interaction between CD8⁺ T cells and the basolateral membrane of endothelial cells leads to release of cytotoxic perforin and granzyme molecules in the perivascular space that down regulate intercellular tight junction proteins, damaging the vascular integrity and causing vasogenic edema. (6) Through an undefined mechanism, perivascular CD8⁺ T cells also communicate across the glia limitans to induce astrocyte and microglial activation. Activation of these cells further amplifies cerebral inflammation and dysfunction and provides a source of VEGF that concomitantly induces vascular leakage and resistance of endothelial cells to apoptosis. In non-ECM malarial infections, parasites do not accumulate in the brain, brain endothelial cells do not phagocytose or cross-present malaria antigen and perivascular CD8⁺ T cells fail to recognize their cognate antigen, restricting their pathogenic activity and preventing ECM development.</p

Depletion of systemically and perivascularly located phagocytic cells from day 5 p.i. does not prevent ECM.

<p><b>(A)</b> C57BL/6 mice were infected with 10⁴<i>Pb</i> ANKA pRBCs. On day 5 p.i. mice were injected i.p. with 300 μL clodronate liposomes with (n = 22) or without (n = 6) 8 μL clodronate liposomes i.c.v., or left untreated (n = 9). Mice were monitored daily for development of ECM (grey area). Results are pooled from five experiments. <b>(B)</b> C57BL/6 (n = 11) and CX₃CR1-iDTR mice (n = 5) were infected with 10⁶<i>Pb</i>ANKA pRBCs and treated with tamoxifen and diphtheria toxin as described in the methods to systemically deplete CX₃CR1⁺ cells in the latter group. Mantel-Cox test showed no statistically significant differences in survival. <b>(C)</b> Representative coronal cryosection of brains showing depletion of Iba1⁺microglia (green), a CX₃CR1⁺ population, in CX₃CR1-iDTR mice (right), compared to parallel C57BL/6 control (left), with ECM. Scale bars: (C) 50 μm.</p