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Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells

A Corrigendum to this article was published on 28 January 2016

This article has been updated

Abstract

Dendritic cell (DC) migration in peripheral tissues serves two main functions: antigen sampling by immature DCs, and chemokine-guided migration towards lymphatic vessels (LVs) on maturation. These migratory events determine the efficiency of the adaptive immune response. Their regulation by the core cell locomotion machinery has not been determined. Here, we show that the migration of immature DCs depends on two main actin pools: a RhoA–mDia1-dependent actin pool located at their rear, which facilitates forward locomotion; and a Cdc42–Arp2/3-dependent actin pool present at their front, which limits migration but promotes antigen capture. Following TLR4–MyD88-induced maturation, Arp2/3-dependent actin enrichment at the cell front is markedly reduced. Consequently, mature DCs switch to a faster and more persistent mDia1-dependent locomotion mode that facilitates chemotactic migration to LVs and lymph nodes. Thus, the differential use of actin-nucleating machineries optimizes the migration of immature and mature DCs according to their specific function.

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Figure 1: LPS activation induces fast and persistent DC migration.
Figure 2: LPS activation of DCs modifies the dynamics of their actin cytoskeleton.
Figure 3: Arp2/3-dependent actin at the front of iDCs limits migration but promotes antigen uptake.
Figure 4: mDia1 is required for fast DC migration.
Figure 5: Cdc42 and RhoA respectively control the migration of iDCs and LPS-DCs.
Figure 6: mDia1 is required for the chemotactic response of mature DCs.
Figure 7: mDia1 is required for migration of mature DCs to LVs and LNs in vivo.

Change history

  • 23 December 2015

    In the version of this Article originally published, references 42 and 43 were incorrect. They have been replaced with a new ref. 42, and references 44–51 have been renumbered accordingly. These changes have been made in all online versions of the Article.

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Acknowledgements

The authors thank the PICT IBiSA platform at Institut Curie (CNRS UMR144, especially V. Fraisier) and the Institut Curie animal facility. P.V. thanks B. Goic for support, patience and critical reading of the manuscript. P.V. was supported by fellowships from Region Ile-de-France, Fondation pour la Recherche Médicale (FRM) and Institut Curie. M.B. and M.C. benefited from fellowships from Association pour la Recherche contre le Cancer and FRM respectively. S.M.K.-G. and A.S.A. were supported by the Grand Rapids Community and the Lunn Hope Foundations, the Van Andel Endowment, and the Purple Community. T.B. was supported by DFG SFB 1054-B03 and SFB 914-A06. This work was financially supported by grants from: the City of Paris, the European Research Council and the DCBIOL Labex (ANR-10-IDEX-0001-02-PSL and ANR-11-LABX-0043) to A.-M.L.-D. (Strapacemi 243103) and the Association Nationale pour la Recherche (ANR-09-PIRI-0027-PCVI) and the InnaBiosanté foundation (Micemico) to A.-M.L.-D., M.P. and R.V.

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Affiliations

Authors

Contributions

P.V. designed, performed and analysed most experiments, prepared all manuscript figures and strongly participated in article writing and project conception. P.M. conceived and performed trajectory analyses and modelling. M.B. set up and assisted with siRNA-mediated silencing, qPCR and macropinocytosis experiments. P.J.S. and H.-R.T. assisted in optimizing micro-channel experiments. P.J.S. further helped P.V. with article revision. P.P. built the program for actin distribution analysis in migrating DCs. M.M. created all codes for quantification of macropinocytosis and arrival of DCs to LVs. M.C. carried out the initial observation of increased migration speed and decreased antigen uptake in CK666-treated iDCs. E.T. helped with micro-fabrication. D.L. assisted P.V. with immunofluorescence experiments. D.O. performed immunoblots. M.R. set up collagen gel experiments. T.B. generated and provided Cdc42 and RhoA conditional KO mice. S.M.K.-G. and A.S.A. generated and provided mDia1KO mice. S.X., P.S. and R.L. generated and provided Arpc2 conditional KO mice. R.V. conceived and performed migration modelling. M.P. and A.-M.L.-D. designed the overall research and wrote the manuscript.

Corresponding authors

Correspondence to Pablo Vargas or Matthieu Piel or Ana-Maria Lennon-Duménil.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 DC migration becomes faster and more persistent upon LPS treatment.

(a,b) Analysis by flow cytometry of surface expression of the DC activation marker CD86 and the chemokine receptor CCR7. iDCs were incubated for different times with LPS (100 ng/ml), washed and cultured overnight. 30 min was found to be the minimal time to get the highest level of activation and CCR7 expression. 1 representative experiment out of 3 is shown. (c) Mean instantaneous speed of migrating DCs in the presence of Pertussis toxin (Ptx, 200 μg/ml) in fibronectin- (c) or fibronectin plus CCL21- (d) coated micro-channels (n = 80, 94 and 91 for iDC, LPS-DC and LPS-DC Ptx respectively). 1 representative experiment out of 2 is shown. (d) Ptx does not affect the LPS-induced increment in DC speed (c) but decreases the velocity of LPS-DCs migrating in the presence of CCL21 (d) (positive control for the activity of Ptx, n = 92 and 100 for CCL21 and CCL21 Ptx respectively). 1 representative experiment out of 2 is shown. (e) Mean instantaneous speed of WT and TLR4KO DCs migrating in micro-channels (n = 150, 150, 152 and 122 for iDC, LPS-DC, iTLR4KO and LPS-TLR4KO cells respectively). 1 representative experiment out of 2 is shown. (f) Mean instantaneous speed of control and Myd88KO DCs migrating in micro-channels (n = 81, 74, 67 and 102 for iDC, LPS-DC, iMyd88KO and LPS-Myd88KO respectively). 1 representative experiment out of 3 is shown. The Mann-Whitney test was applied for statistical analyses in cf. In the box plots of panels cf the bars include 90% of the points, the center corresponds to the median and the box contains 75% of the data.

Supplementary Figure 2 Quantification of LifeAct-GFP dynamics in migrating DCs.

(ab) Method used to quantify LifeAct-GFP localization in migrating DCs. Sequential images of LifeAct-GFP DCs were acquired on an epifluorescence microscope every 1 min at 20×. Scale bar: 10 μm. (a) The LifeAct-GFP signal recorded at each time-point was integrated into a single image for all migrating cells. Cell alignment and cell size normalization were applied to generate the LifeAct-GFP density maps. Scale bar: 5 μm. (b) Mean intensities obtained for each cell were averaged into a single LifeAct-GFP density map, assigning equivalent weight to each cell. Scale bar: 2.5 μm. (c,d) Analysis of the LifeAct-GFP front/back ratio in iDCs and LPS-DCs migrating in micro-channels. The front was defined as the first third of the cell. Scale bar: 2.5 μm. Results obtained by analyzing the data showed in Fig. 2a–c (n = 31 and 27 cells for iDC and LPS-DC respectively). 1 representative experiment out of 4 is shown. Graphic shows mean and error bars correspond to S.E.M. The Mann-Whitney test was applied for all statistical analysis.

Supplementary Figure 3 Arp2/3 decreases the migration of iDCs but enhances their antigen uptake capacity.

(a) Immunoblot blot analysis of Arpc4 expression in BMDCs nucleofected with Arpc4-specific siRNA. (b,c) LifeAct-GFP density maps obtained from iDC or LPS-DC silenced (b) or inhibited (c) for Arp2/3. LPS-DCs were treated with the Arp2/3 inhibitor CK666 (25 μM) or silenced for Arpc4. Scale bar: 2.5 μm. 1 representative experiment out of 2 is shown. (d) Analysis by flow cytometry of surface expression of the DC activation marker CD86. iDCs were incubated 30 min with LPS (100 ng/ml), washed and cultured overnight. 1 experiment out of 4 is shown. (e) Mean instantaneous speed of DCs migrating in micro-channels and treated or not with CK666 as in c (n = 244, 192, 231 and 230 for iDC, iDC-CK666, LPS-DC and LPS-DC CK666 respectively). 1 representative experiment out of 3 is shown. (f) Immunoblot analysis of Arpc2 and tubulin expression in tamoxifen-treated DCs. TomatoFP+ (Arpc2WT) and conditional GFP+ (Arpc2KO) iDCs were sorted from 10-days- DC cultures in which tamoxifen was added during the last 8 days. (g) Analysis of the migration of Arpc2WT and KO iDCs under an agarose gel. Cells were imaged during 200 min and their cell speed was quantified after tracking. The analysis was performed on cells that had migrated >100 μm (n = 80 and 163 for Arpc2 WT and Arpc2 KO respectively). 1 representative experiment out of 2 is shown. (h) Analysis by flow cytometry of surface expression of the DC activation marker CD86 performed as in d. (i) Quantification of the size (left) and number (right) of vesicles present at the front of Arpc2WT and KO DCs migrating in micro-channels filled with fluorescent ovalbumin (n = 36 and 37 for Arpc2WT and Arpc2 KO respectively). Graphics show mean and error bars correspond to S.E.M. (j) Macropinocytic capacity of migrating DCs treated with the Arp2/3 inhibitor CK666 (n = 62 and 41 for iDC and iDC CK666 respectively). 1 representative experiment out of 3 is shown. (k) Macropinocytic capacity of iDCs and LPS-DCs (n = 39 and 33 for iDC and LPS-DC respectively). 1 representative experiment out of 2 is shown. (l) Density maps obtained from LifeAct-GFP DCs fixed while migrating in micro-channels and stained for Arp2 and GFP. Scale bar: 2.5 μm. In the box plots of panels e,g,j and k the bars include 90% of the points, the center corresponds to the median and the box contains 75% of the data. The Mann-Whitney test was applied for all statistical analyses.

Supplementary Figure 4 Actin distribution at the back of DCs relies on the Formin mDia1 and is required for fast and persistent migration.

(a) LifeAct-GFP density maps of iDCs migrating in the presence or absence of the formin inhibitor Smifh2 (25 μM). 1 representative experiment out of 3 is shown. Scale bar: 2.5 μm. (b) Immunofluorescence analysis of mDia1 expression in LPS-DCs migrating in micro-channels analyzed by spinning disk microscope (100×). Scale bar: 5 μm. (c) Density maps obtained from LifeAct-GFP DC fixed while migrating in micro-channels and stained for mDia1 and GFP (2 independent experiments). Scale bar: 2.5 μm. (d) Quantitative PCR analysis of mDia1 expression in DCs nucleofected with mDia1-specific siRNA. Graphic shows mean and error bars correspond to S.E.M. (e) Immunoblot analysis of mDia1 and tubulin expression in immature mDia1WT and KO DCs. 1 representative experiment out of 3 is shown. (f) Mean instantaneous speed of LPS-DCs migrating in micro-channels and treated with different doses of Smifh2 (n = 125, 154, 134 and 141 for iDC, LPS-DC, LPS-DC 12 μM and LPS-DC 25 μM respectively). 1 representative experiment out of 3 is shown. (g) 10% of the highest instantaneous speed values of DCs migrating in micro-channels (n = 210, 238, 233 and 179 for iWT, LPS-WT, imDia1KO and LPS-mDia1KO respectively). 1 representative experiment out of 5 is shown. (h) Analysis by flow cytometry of surface expression of the DC activation marker CD86. iDCs were incubated 30 min with LPS (100 ng/ml), washed and cultured overnight. 1 experiment out of 4 is shown. (i) Mean instantaneous speed of mDia1WT and KO migrating in micro-channels and treated with CK666 (25 μM) (n = 119, 107, 52 and 111 for iWT, iWT CK, imDia1KO and imDia1KO CK respectively). 1 representative experiment out of 2 is shown. In the box plots of panels f, g and i the bars include 90% of the points, the center corresponds to the median and the box contains 75% of the data. The Mann-Whitney test was applied for all statistical analyses.

Supplementary Figure 5 Regulation of DC Arp2/3- and mDia1-dependent actin nucleation by Cdc42 and RhoA.

(a, b) iDCs were treated with LPS for 30 min, washed and further cultured at 37 °C during 16 h. a. Immunoblot analysis was performed as described in the supplementary experimental procedures. (b) Normalized intensity obtained from data showed in panel a. Graphics show mean individual values. (c) Mean instantaneous speed of iDCs migrating in micro-channels and treated with different doses of ML141 (n = 164, 163, 142, 143 and 39 for iDC, iDC 12 μM, iDC 25 μM, iDC 50 μM and iDC 100 μM respectively). 1 experiment out of 3 is shown. In the box plot the bars include 90% of the points, the center corresponds to the median and the box contains 75% of the data. The Mann-Whitney test was applied for statistical analysis. (d) Analysis by flow cytometry of surface expression of the DC activation marker CD86. iDCs were incubated 30 min with LPS (100 ng/ml), washed and cultured overnight.

Supplementary Figure 6 Analysis of chemokine gradient sensing by iDCs and LPS-DCs.

(a) Collagen gels were bathed with a solution containing fluorescent ovalbumin (OVA), to evaluate protein penetration in the gel. Gradient steepness was evaluated by measuring the changes in fluorescence intensity according to the protein source using an epifluorescence microscope. Areas closer to the protein source (red) reached a plateau faster than distal areas (blue). After 200 min the gradient was considered as stable at any distance from the source. All the images showed in the paper correspond to analysis between 200 min and 600 min after addition of the chemokine. (b) CCR7 and Clec-2 surface staining using flow cytometry was performed in DCs treated with LPS (100 ng/ml) for 30 min and further cultured overnight. Geometric mean of fluorescence in the CCR7 positive population is depicted. (c) Trajectories of iDCs (upper panel) and LPS-DCs (lower panel) along the CCL21 gradient formed in collagen gels. Directionality of segments of cell trajectories is shown in different colors: from red (toward the source) to blue (against it).

Supplementary Figure 7 Analysis of chemokine gradient sensing by mDia1KO LPS-DCs.

(a) Trajectories of WT (upper panel) and mDia1KO LPS-DCs (lower panel) along the CCL21 gradient formed in collagen gels. Speed of segments of cell trajectories is shown in different colors: from red (fast) to blue (slow). (b) Frequency of movement in the direction of the gradient in the 500 μm closer to the CCL21 source compared to random migration in absence of chemokines. Values are compared to equivalent position in the absence of chemokines. Analysis performed in 500 random tracks due to oversampling. (c) Mean speed of DCs in the 500 μm closer to the CCL21 source. Analysis performed in 500 random tracks due to oversampling. Error bars correspond to S.D. Student’s t-Test was applied for statistical analysis in b. Chi-squared Test was applied for statistical analysis in c. (d) Ear explant stained with the anti-Lyve-1 marker to highlight LVs. The area enclosed in the yellow square corresponds to the zone analyzed when monitoring DC migration to LVs. Scale bar 250 μm. (e) Scheme of different types of trajectories representing the behavior of mDia1WT and KO LPS-DCs in the ear explants. (f) mDia1WT and KO LPS-DCs were stained with CMTRM or CFSE and co-injected in the footpad of WT recipient mice. Their presence in the popliteal lymph node 16 h later was analyzed by flow cytometry. Example representative of 3 experiments is shown. (g) pMLC levels on DCs activated 30 min with LPS and cultured during the indicated time. The pMLC signal was normalized to tubulin levels.

Supplementary Figure 8 Unprocessed full scans of key blots.

(a) Full scans of immunoblots showed in Supplementary Fig. 5a. The whole membrane was divided in two based on the 100 kDa molecular weight marker (left panel). Each blot was sequentially incubated with the indicated primary antibody and the corresponding HRP-conjugated secondary antibody (central panel). For loading controls, the membrane containing the 50 kDa band was incubated sequentially with an anti-tubulin antibody and revealed with the corresponding HRP-conjugated secondary antibody (right panel). (b) Full scans of immunoblots showed in Supplementary Fig. 7g. Blots were obtained as described in a, except that the gel was cut based on the 37 kDa molecular weight marker, allowing detection of phospho-MLC and tubulin in the same run.

Supplementary information

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Distinct LifeAct-GFP dynamics in iDCs and LPS-DCs.

LifeAct-GFP-expressing DCs migrating in 8 μm × 5 μm fibronectin-coated micro-channels and time-lapsed on a spinning disk microscope (20×) at a frequency of 1 image/2 min. Scale bar 5 μm. (AVI 83 kb)

F-actin is enriched in ruffles at the front of iDCs.

LifeAct-GFP-expressing iDCs migrating in 8 μm × 5 μm fibronectin-coated micro-channels and time-lapsed on a spinning disk microscope (100×) at a frequency of 1 image/400 ms. The cortex of a representative cell is shown. Scale bar 5 μm. (AVI 1988 kb)

F-actin is enriched at the rear of LPS-DCs.

LifeAct-GFP-expressing LPS-DCs migrating in 8 μm × 5 μm fibronectin-coated micro-channels and time-lapsed on a spinning disk microscope (100×) at a frequency of 1 image/400 ms. The cortex of a representative cell is shown. Scale bar 5 μm. (AVI 948 kb)

LPS-DCs but not iDCs respond to CCL21 gradients.

iDCs and LPS-DC migrating along a CCL21 gradient in a collagen gel. The frequency of acquisition was 1 image/2 min (10×) on a video-microscope. The source of CCL21 is at the right of the movie. Scale bar 50 μm. (AVI 5470 kb)

Chemotaxis of wild-type and mDia1KO LPS-DCs in vitro.

mDia1 wild-type and KO LPS-DCs migrating along a CCL21 gradient in a collagen gel. 1 image/2 min (10×) was acquired on a video-microscope. The source of CCL21 is at the right of the movie. Scale bar 50 μm. (AVI 7071 kb)

Chemotaxis of wild-type and mDia1KO LPS-DCs in vivo.

mDia1 wild-type (red) and KO (green) LPS-DCs migrating in an ear explant. 1 image/2 min (20×) was acquired on a on a video-microscope. Scale bar 50 μm. (AVI 3012 kb)

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Vargas, P., Maiuri, P., Bretou, M. et al. Innate control of actin nucleation determines two distinct migration behaviours in dendritic cells. Nat Cell Biol 18, 43–53 (2016). https://doi.org/10.1038/ncb3284

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