Research ArticleCell Migration

ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X7 receptors

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Science Signaling  21 Nov 2017:
Vol. 10, Issue 506, eaah7107
DOI: 10.1126/scisignal.aah7107
  • Fig. 1 Compared to WT DCs, Panx1−/− DCs exhibit reduced ATP-induced uptake of DAPI.

    (A and B) Sequence of fluorescence images of WT (A) and Panx1−/− (B) DCs in a representative DAPI uptake experiment. Times, treatments, and some representative cells are indicated. Scale bar, 100 μm. Under each panel, the DAPI fluorescence plot profile is shown. (C) Analysis of the DAPI uptake experiments shown in (A) and (B) for WT DCs (open circles) and Panx1−/− DCs (green circles). After 5 min of recording, 500 μM ATP was added. Each point corresponds to the mean ± SEM of 30 cells. (D) Analysis of DAPI uptake by WT and Panx1−/− DCs under resting conditions and after treatment with ATP in control cells and in cells pretreated with 50 μM Cbx. Only the first component is depicted. Data are means ± SEM of nine experiments for control WT DCs, four experiments for Cbx-treated WT DCs, nine experiments for Panx1−/− DCs, and four experiments for Cbx-treated Panx1−/− DCs, with at least 30 cells analyzed per experiment. Data were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001 compared to basal uptake; #P < 0.05 when comparing the indicated treatments to ATP-treated WT DCs. (E) Percentages of cells (black) that exhibited ATP-induced uptake of DAPI or Etd. Data are from nine experiments for DAPI uptake by WT and Panx1−/− DCs and six experiments for Etd uptake by WT and Panx1−/− DCs. Fluo., fluorescence; A.U., arbitrary units.

  • Fig. 2 Functional Panx1 channels and ATP release are required during the ATP-stimulated migration of DCs.

    (A) Protocol to evaluate the effect of ATP [pulse or continuous (Cont.)] on cell migration. (B) Left: Confocal fluorescence images of DAPI in migrating WT and Panx1−/− DCs under control conditions or treated with ATP at 1 and 15 min of recording. The average (AVG) DAPI fluorescence after 15 min in >20 cells is shown in pseudocolor from a representative experiment. Scale bar, 5 μm. Right: DAPI uptake rate in untreated and ATP-treated WT and Panx1−/− DCs. Data are means ± SEM of three experiments. Data were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001 compared to WT. (C) Migration of WT DCs was evaluated after a 30-min pulse (Pulse) or continuous exposure (Cont.) to 500 μM ATP. ns, not significant. (D) Effect of apyrase (5 U/ml) on the instantaneous speed of WT DCs. (E) Migration of Panx1−/− DCs under the same conditions described in (C). In (C) to (E), the bars show 90% of the points and the median from one experiment that is representative of three experiments, with at least 100 cells analyzed per condition. The horizontal dashed line denotes the median in untreated DCs. Data were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001 and **P < 0.01 when compared to resting conditions. (F) ATP measurement in the culture medium of WT and Panx1−/− DCs under resting conditions or after an ATP pulse. Data are means ± SEM of three experiments. Data were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. *P < 0.05 when compared to WT cells.

  • Fig. 3 P2X7 receptor activation is required for the ATP-induced migration of DCs and membrane permeabilization.

    (A) Left: Western blotting analysis of the relative abundance of P2X7 receptor in total homogenates of WT and Panx1−/− DCs under resting conditions or 12 hours after an ATP pulse. Vinculin was used as loading control. Right: Normalized P2X7 receptor abundance expressed as a percentage of the abundance of the corresponding control cells (Ctrl). Data are means ± SEM of four experiments and were analyzed by unpaired t test. *P < 0.05. (B) Effect of 10 μM A-740003 (A74) on the instantaneous speed of untreated and ATP-treated WT and Panx1−/− DCs. (C) Effects of a pulse of 200 μM BzATP and treatment with apyrase (5 U/ml) on the instantaneous speed of WT DCs. For (B) and (C), the bars show 90% of the points and the median from one experiment that is representative of three experiments, with at least 100 cells analyzed per condition. The horizontal dashed line denotes the median in untreated WT DCs. Data were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001 when compared to the untreated condition. #P < 0.05 between the indicated treatments. (D) Left: Effect of preincubation with 10 μM A74 on DAPI uptake by WT and Panx1−/− DCs. Data are means ± SEM of 30 different cells. Right: Representative DAPI fluorescence plot profiles are shown for the indicated treatments. (E) Effect of 10 μM A74 on DAPI uptake by WT and Panx1−/− DCs. Only the first component is depicted. Data are means ± SEM of three experiments. Data were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001 when compared to the corresponding basal uptake. #P < 0.05 between the indicated treatments.

  • Fig. 4 Panx1-independent Ca2+ influx is required for the ATP-induced migration of DCs.

    (A) Effect of extracellular Ca2+ chelation with 2 mM BAPTA and CaMKII inhibition with 10 μM KN-62 (KN) on the migration of untreated and ATP-treated WT and Panx1−/− DCs. The bars show 90% of the points, and the line corresponds to the median from one experiment that is representative of three independent experiments, with at least 100 cells analyzed per condition. The dashed line denotes the median instantaneous speed of WT DCs under resting conditions. Data were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001 compared to the control condition. ###P < 0.001, ##P < 0.01, and #P < 0.05 when compared to ATP-treated WT cells. (B) Top: Sequence of fluorescence images of WT and Panx1−/− DCs loaded with Fura-2 at indicated times after treatment with 500 μM ATP. Scale bar, 20 μm. Bottom: Ca2+ signal traces in WT and Panx1−/− DCs before and after ATP treatment. Each point corresponds to the means ± SEM of 40 different cells in one experiment that is representative of three experiments. (C) Maximal Ca2+ signal at baseline (1 min; blue circle), at the peak (3 min; red circle), and at the plateau (9 min; green circle). Data are means ± SEM of 40 different cells from one experiment that is representative of three independent experiments. Data were analyzed by Mann-Whitney test. (D) Confocal images showing the intracellular distribution of Panx1 as evaluated by immunofluorescence in WT and Panx1−/− DCs under control conditions or after ATP treatment, as indicated. Scale bar, 5 μm. Images are representative of three independent experiments.

  • Fig. 5 ATP stimulates reorganization of the actin cytoskeleton to enable the fast migration of DCs.

    (A) Different confocal images of LifeAct-GFP showing the indicated planes of F-actin in untreated (Control) and ATP-pulsed (500 μM) DCs. Scale bars, 5 μm. (B) Left: Density maps of LifeAct-GFP at the cortical plane showing the average distribution in control and ATP-treated DCs. Right: Analysis of the fraction of the time that LifeAct-GFP spent at the first third of the cell (front) in LifeAct-GFP DCs. Data are means ± SEM of three experiments, with at least 30 cells analyzed per condition. Data were analyzed by Mann-Whitney test. ***P < 0.001. (C) Actin density maps (left) and graph (right) showing the effects of apyrase (Apy; 5 U/ml), 10Panx1 (200 μM), A74 (10 μM), and KN (10 μM) on the mean fraction of time that LifeAct-GFP spent at the front in control (Ctrl) and ATP-treated DCs. Data are means ± SEM of three experiments and were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001 compared to Ctrl.

  • Fig. 6 Panx1 contributes to the 3D migration of DCs and their homing to lymph nodes but not to their ATP-induced maturation.

    (A) Scheme showing the 3D migration of DCs confined in collagen gels. Cell tracks (>150 cells each condition from one experiment that is representative of three experiments) of WT and Panx1−/− DCs migrating in collagen gels under resting conditions or after ATP treatment. Cells were imaged for 6 hours. Scale bars, 50 μm. (B) Cell displacements under the conditions shown in (A). Data are means ± SEM of three experiments and were analyzed by Kruskal-Wallis test, followed by Dunn’s multiple comparison test. ***P < 0.001. (C) Left: Flow cytometric analysis showing the presence of WT [5-(and-6)-{[(4-chloromethyl)benzoyl]amino}tetramethylrhodamine (CMTMR), red] and Panx1−/− [5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), green] DCs in the popliteal lymph nodes of mice 16 hours after these cells were injected in the footpad. Positive populations are depicted. Data are from one experiment and are representative of eight experiments. Right: Homing index (see Materials and Methods) indicating the ratio of Panx1−/− DCs compared to WT DCs that migrated to the lymph nodes. Data are means ± SEM of eight experiments. (D) Flow cytometric analysis of the cell surface abundance of CD86 in WT (left) and Panx1−/− (right) DCs under the indicated conditions. The dotted lines indicate the gate for positive cells. Data are from one experiment and are representative of three independent experiments.

  • Fig. 7 Proposed model for the contribution of Panx1 channels and P2X7 receptors to the migration of DCs.

    Under resting conditions (top), the Panx1 channels and P2X7 receptors exhibit low levels of activity, but these are increased after the concentration of extracellular ATP is increased (bottom). The opening of both Panx1 channels and P2X7 receptors leads to the activation of the DCs, which then release ATP through Panx1 channels to further activate P2X7 receptors. P2X7 receptor–mediated Ca2+ signaling modifies the actin cytoskeleton, thus increasing the speed of migration of the DCs.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/506/eaah7107/DC1

    Fig. S1. Extracellular ATP increases the permeability of the plasma membrane in DCs to DAPI.

    Fig. S2. The abundances of Panx2, Panx3, Cx43, and Cx45 are similar between WT and Panx1−/− DCs.

    Fig. S3. ATP-induced dye uptake by DCs is selective.

    Fig. S4. Extracellular ATP contributes to migration of DCs.

    Fig. S5. WT and Panx1−/− DCs have functional P2Y receptors that do not contribute to ATP-induced migration or membrane permeabilization.

    Fig. S6. ATP stimulates the migration, but not the death, of DCs.

    Fig. S7. Role of extracellular Ca2+ and Cx hemichannels in DC migration.

    Fig. S8. ATP-induced reorganization of the actin cytoskeleton.

    Fig. S9. The ATP-induced cell surface expression of CD40 and CD86 is Panx-independent.

    Movie S1. WT and Panx1−/− DCs in a representative DAPI uptake experiment.

    Movie S2. WT and Panx1−/− DCs migrating inside microchannels under resting conditions or after exposure to ATP.

    Movie S3. WT LifeAct-GFP DCs migrating inside microchannels.

    Movie S4. ATP-pulsed WT LifeAct-GFP DCs migrating inside microchannels.

    Movie S5. ATP-pulsed WT and Panx1−/− DCs migrating randomly in collagen gels.

  • Supplementary Materials for:

    ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X7 receptors

    Pablo J. Sáez,* Pablo Vargas, Kenji F. Shoji, Paloma A. Harcha, Ana-María Lennon-Duménil,* Juan C. Sáez*

    *Corresponding author. Email: pablo.saez{at}curie.fr (P.J.S.); ana-maria.lennon{at}curie.fr (A.-M.L.-D.); jsaez{at}bio.puc.cl (J.C.S.)

    This PDF file includes:

    • Fig. S1. Extracellular ATP increases the permeability of the plasma membrane in DCs to DAPI.
    • Fig. S2. The abundances of Panx2, Panx3, Cx43, and Cx45 are similar between WT and Panx1−/− DCs.
    • Fig. S3. ATP-induced dye uptake by DCs is selective.
    • Fig. S4. Extracellular ATP contributes to migration of DCs.
    • Fig. S5. WT and Panx1−/− DCs have functional P2Y receptors that do not contribute to ATP-induced migration or membrane permeabilization.
    • Fig. S6. ATP stimulates the migration, but not the death, of DCs.
    • Fig. S7. Role of extracellular Ca2+ and Cx hemichannels in DC migration.
    • Fig. S8. ATP-induced reorganization of the actin cytoskeleton.
    • Fig. S9. The ATP-induced cell surface expression of CD40 and CD86 is Panx-independent.
    • Legend for movies S1 to S5

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 2.47 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). WT and Panx1−/− DCs in a representative DAPI uptake experiment.
    • Movie S2 (.avi format). WT and Panx1−/− DCs migrating inside microchannels under resting conditions or after exposure to ATP.
    • Movie S3 (.avi format). WT LifeAct-GFP DCs migrating inside microchannels.
    • Movie S4 (.avi format). ATP-pulsed WT LifeAct-GFP DCs migrating inside microchannels.
    • Movie S5 (.mp4 format). ATP-pulsed WT and Panx1−/− DCs migrating randomly in collagen gels.

    Citation: P. J. Sáez, P. Vargas, K. F. Shoji, P. A. Harcha, A.-M. Lennon-Duménil, J. C. Sáez, ATP promotes the fast migration of dendritic cells through the activity of pannexin 1 channels and P2X7 receptors. Sci. Signal. 10, eaah7107 (2017).

    © 2017 American Association for the Advancement of Science

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