Jak3 Enables Chemokine-Dependent Actin Cytoskeleton Reorganization by Regulating Cofilin and Rac/Rhoa GTPases Activation

<div><p>We have previously shown that Jak3 is involved in the signaling pathways of CCR7, CCR9 and CXCR4 in murine T lymphocytes and that Jak3^−/− lymphocytes display an intrinsic defect in homing to peripheral lymph nodes. However, the molecular mechanism underlying the defective migration observed in Jak3^−/− lymphocytes remains elusive. Here, it is demonstrated for the first time, that Jak3 is required for the actin cytoskeleton reorganization in T lymphocytes responding to chemokines. It was found that Jak3 regulates actin polymerization by controlling cofilin inactivation in response to CCL21 and CXCL12. Interestingly, cofilin inactivation was not precluded in PTX- treated cells despite their impaired actin polymerization. Additionally, Jak3 was required for small GTPases Rac1 and RhoA activation, which are indispensable for acquisition of the migratory cell phenotype and the generation of a functional leading edge and uropod, respectively. This defect correlates with data obtained by time-lapse video-microscopy showing an incompetent uropod formation and impaired motility in Jak3-pharmacologically inhibited T lymphocytes. Our data support a new model in which Jak3 and heterotrimeric G proteins can use independent, but complementary, signaling pathways to regulate actin cytoskeleton dynamics during cell migration in response to chemokines.</p></div

Actin polymerization is decreased in human PBMCs stimulated with CXCL12.

<p>Primary human PBMCs pre-incubated with DMSO, WHI-P131 or PTX were stimulated with 300 ng/mL of CXCL12 at different time points. F-actin was detected as described above. Representative histograms (n = 4) (<i>top</i>) and graphs (<i>bottom</i>) of the F-actin increment are shown. The graph represents the average values of 4 independent experiments ± SEM. Significance was calculated using a paired Student's t test (one tailed). Asterisks indicate statistical significant values, *<i>p</i><0.05, **<i>p</i><0.01.</p

Summarized data describing Jak3 and G_αi dependent signaling pathways activated in response to chemokines.

<p>In control cells (<i>left</i>), actin polymerization (red), cofilin dephosphorylation (purple) and Rac1 activation (green) are early events (30 seconds) responsible for lamellipodia formation and initiation of leading edge organization. Subsequently, activation of RhoA (blue) takes place, leading to the uropod formation as well as the establishment of the migratory cell phenotype. Jak3 deficiency or Jak3-inhibition (<i>middle</i>) results in a reduction of F-actin, which is sufficient to allow cell polarization at the lamellipodia, and unstable leading edge formation. Increased levels of F-actin are also observed in the absence of Jak3. Cofilin increases its activation, but is not dephosphorylated after 30 seconds, leading to accumulation of active cofilin, up to 300 seconds. Rac1-GTP does not significantly increase in response to the chemokine stimulus, although basal active Rac1 levels are significantly increased. RhoA activation is absent within the time course of stimulation, preventing the migratory phenotype acquisition. PTX treatment (<i>right</i>) prevents actin polymerization, whereas p-cofilin kinetics is not affected. Rac1 activation does not significantly increase in response to the chemokine, while RhoA-GTP is absent after the chemokine stimulation. As a result, neither leading edge nor migratory cell phenotype occur in the absence of G protein activation. Color intensity increment in the depicted cells represents accumulation of protein in that condition or during activation.</p

Rac1 activation is diminished in Jak3-inhibited or G_αi-inactivated T lymphocytes after CCL21 stimulation.

<p><i>A</i>, Representative images are shown from DMSO-, WHI-P131- or PTX-treated cells stimulated for 0, 30 and 300 seconds with CCL21. One representative cell stained with Rhodamine-phalloidin (F-actin) and Rac1-GTP Alexa-Fluor 488 (activated Rac1) is shown for each condition. <i>B</i>, The graph represents the average of mean fluorescence intensity measurements of single cells (between 5-26 cells per coverslip). An average of 36 cells per condition were individually analyzed for GTPase activation from each experiment. Data are expressed as relative increment (RI) of the fluorescence in each sample compared to unstimulated control cells. Mean values ± SEM from 3 independent experiments are shown. Asterisks indicate statistical significance (*<i>p</i><0.05).</p

RhoA activation is prevented in the absence of Jak3 or G protein activity in T lymphocytes stimulated with CCL21.

<p><i>A</i>, Representative images are shown from DMSO-, WHI-P131- or PTX-treated cells stimulated for 0, 30 and 300 seconds with CCL21. Images of single cells are shown from each condition. Cells were stained with Rhodamine-phalloidin to detect F-actin and RhoA-GTP Alexa-Fluor 488 (activated RhoA), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#s2" target="_blank">materials and methods</a>. Data are expressed as RI, as described above. <i>B</i>, The graph values represent the average of mean fluorescence intensity measurements of single cells (between 3–19 cells per coverslip). An average of 36 cells were individually analyzed for GTPase activation per condition from each experiment. Mean values ± SEM from 4 independent experiments are shown. Asterisks indicate statistical significance (*<i>p</i><0.05).</p

Jak3 deficiency results in increased basal levels of F-actin in murine T lymphocytes.

<p><i>A</i>, Jak3^+/− and Jak3^−/− (<i>top</i>) or, wild type cells pre-treated with DMSO, WHI-P131 or PTX (<i>bottom</i>) were analyzed as described above. The graphs represent mean values of F-actin from 4 independent experiments (Jak3^+/− vs Jak3^−/−, top graph) or 6 independent experiments (WT DMSO versus WHIP-131/PTX treated cells, bottom graph), respectively. Asterisks indicate statistical significance: **<i>p</i><0.01, ***<i>p</i><0.001. Significance was determined using a Student's unpaired (<i>top</i>) or paired t-test (<i>bottom</i>). <i>B</i>, human PBMCs with the same treatments are also shown. Histograms from a representative experiment from human PBMCs is shown (<i>left</i>). Graph represents the mean values obtained from 4 independent experiments <i>(right)</i>.</p

Motility and migration is impaired in the absence of Jak3 activity.

<p>Time-lapse video-microscopy analysis of primary lymphocytes from C57BL/6 mice pre-treated with DMSO, WHI-P131 or PTX, and stimulated for 25 min with CCL21 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#pone.0088014.s003" target="_blank">videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#pone.0088014.s004" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#pone.0088014.s005" target="_blank">S3</a>). <i>A</i>, schematic representation of the phenotype observed in time-lapse sequences and considered for cell quantifications (<i>top</i>). Images selected from the time-lapse sequences at the indicated time points (one representative of 3 independent experiments) (<i>bottom</i>). Arrows indicate responding cells (polarized cells). <i>B</i>, measurements of response observed during the recording of stimulated cells. Graphs show the percentage of cells with change of shape, represented as responding cells (<i>left</i>) or as percentage of cells displaying migratory structures (<i>right</i>), classified as “polarized” (showing lamellipodia) or “migratory phenotype” (with a leading edge and uropod) The bars represent the average values of three independent experiments ± SEM. Statistical significance was determined with a Student's paired t-test (one-tailed). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Jak3 is required for actin polymerization in murine T lymphocytes stimulated with CCL21.

<p>Cells were stimulated with 300/mL of the chemokine CCL21 for 0–300 seconds and F-actin was detected by staining with NBD-phallacidin-FITC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#s2" target="_blank">materials and methods</a>. Representative histograms (<i>top</i>) and graph (<i>bottom</i>) of F-actin increment are shown. <i>A</i>, Wild type (blue line) or Jak3-deficient (red line) lymphocytes were stimulated with the chemokine. Graph represents the average of 4 independent experiments ± SEM. Statistical significance was calculated using an unpaired Student's t-test (one-tailed). Asterisks indicate *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. <i>B</i>, Primary lymphocytes from C57BL/6 mice pre-treated with DMSO (blue line), WHI-P131 (red line) or PTX (green line), stimulated with the chemokine at the same time points. Representative histograms (<i>top</i>) and graph (<i>bottom</i>) of the F-actin increment are shown. The graph represents the average values of 9 independent experiments ± SEM. *<i>p</i><0.05, **<i>p</i><0.01. Statistical significance was determined with a paired Student's t-test (one-tailed).</p

Jak3 inhibition affects cofilin phosphorylation in response to chemokines.

<p><i>A</i>, PLN lymphocytes from C57BL/6 mice pre-treated with DMSO, WHI-P131 or PTX, were stimulated for 0 to 300 seconds with CCL21 and p-cofilin levels were analyzed at indicated time points. Cells were lysed, supernatants were prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088014#s2" target="_blank">materials and methods</a> and western blots were performed with anti phospho-cofilin antibody. Anti-actin antibody was used as loading control. Pooled PLN lymphocytes from 4 mice were used in each assay. One representative experiment (of a total of 3) is shown. <i>B</i>, Primary human PBMCs with the same pre-treatments were stimulated with CXCL12 for 0 to 300 seconds and p-cofilin analysis was performed at the indicated time points. Densitometric analysis of the blots was performed as described above for each group. One healthy donor was used for each experiment. A representative experiment is shown (n = 3).</p

Jak3 and G protein play complementary and independent roles in chemokine receptor mediated signaling.

<p>Chemokine receptor activates both signaling pathways G_αi (green lines) and Jak3 (yellow lines). Dotted lines indicate proposed pathways, while continuous lines indicate already reported pathways. First, G_αi, activates Cdc42 and Arp2/3 complex generating membrane protrusions through actin bundles leading to filopodia formation. Then, Jak3 is activated independently of G_αi and both lead to Rac1 activation and its association with the Arp2/3 complex, driving rearrangement of the actin network to for lamellipodia. This last step is accompanied of SSH1L activation which is required for dephosphorylation of both cofilin (activation) and LIMK1 (inactivation). Cofilin activation elicits free sites for Arp2/3 complex association with the actin filaments allowing branched actin polymerization which contributes to the assembly of the actin network. Next, both Jak3 and G_αi, are required for RhoA activation, which leads to activation of downstream effectors ROCK and MLC, resulting in the actomyosin complex assembly and function. At the same time, cofilin is phosphorylated by LIMK1 at the leading edge, which formation depends on Jak3 but no on G protein activity. Also, LIMK2 activation is a later event dependent on ROCK that in turn inactivates cofilin at the rear end of the cell and prevents actomyosin complex disassociation and uropod formation.</p