Research ArticleDENDRITIC CELLS

Lysosome signaling controls the migration of dendritic cells

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Science Immunology  27 Oct 2017:
Vol. 2, Issue 16, eaak9573
DOI: 10.1126/sciimmunol.aak9573
  • Fig. 1 TFEB translocates to the nucleus and is required for fast DC migration.

    (A) Immunofluorescence images of TFEB-GFP knock-in immature DCs (iDCs) and LPS-DCs. Red dotted circles show nuclei (×60 magnification; scale bar, 10 μm; one plane is shown). (B) The normalized TFEB nucleus-to-cytosol ratio from cells shown in (A) (n = 60 cells per condition, pooled from N = 4 independent experiments). (C) The normalized TFEB nucleus-to-cytosol ratio from immature DCs treated with Torin 2 (2.5 nM; n = 78 cells per condition, pooled from N = 2 independent experiments). (D to F) Analysis of BMDC migration in 4 μm–by–5 μm fibronectin-coated microchannels. Cells were imaged between 6 and 16 hours after LPS treatment (100 ng ml−1 for 30 min). (D) Sequential images of wild-type (WT) and TFEB-inducible KO LPS-DCs migrating in microchannels (×10 magnification; one image every 2 min; scale bar, 50 μm). (E) Mean instantaneous velocities of cells shown in (D) (from left to right: n = 57, 68, 77, and 54 cells; representative results from one of two experiments are shown). ns, not significant. (F) Mean instantaneous velocities of BMDCs treated with DMSO or Torin 2 (2.5 nM; n = 200 cells per condition; representative results from one of four experiments are shown). (B and C) For each experiment, data were normalized against the mean TFEB nucleus-to-cytosol ratio observed in immature DCs (A) or DMSO-treated immature DCs (B). Data are quoted as means, and error bars correspond to SEM. (E and F) Data are represented as boxes and whiskers; bars include 90% of the points, the line represents the median, and the box contains 75% of the data. Mann-Whitney test was applied.

  • Fig. 2 TRPML1 is required for fast DC migration, chemotaxis, and arrival at the LNs.

    (A) Localization of TRPML1-GFP (green), Lamp1 (red), and the nucleus (N) (DAPI; blue) in transfected, migrating LPS-DCs (8 μm–by–5 μm fibronectin-coated microchannels; spinning disk microscope, ×100 magnification; scale bar, 5 μm; one plane is shown). (B) Mean instantaneous velocities of BMDCs migrating in 4 μm–by–5 μm fibronectin-coated microchannels (n = 240 cells per condition; representative results from one of three experiments are shown). (C to F) Chemotaxis of LPS-DCs migrating in collagen gels (representative results from one of three experiments are shown). (C) Directionality of LPS-DCs migrating in the presence or absence of CCL21 (n = 455 and 532 cells for Trpml1+/+ and Trpml1−/− LPS-DCs with CCL21, respectively; n = 533 and 394 cells for Trpml1+/+ and Trpml1−/− LPS-DCs without CCL21, respectively). (D) Trajectories of LPS-DCs undergoing chemotaxis. The starting point of each trajectory was translated to the origin of the plot (n = 50 random trajectories per condition). (E) The mean instantaneous speed measured from the trajectories obtained in (C). (F) The mean square displacement (MSD) of LPS-DCs, measured from the trajectories obtained in (C). (G) In vivo migration of LPS-DC to LNs. DCs were stained with carboxyfluorescein diacetate succinimidyl ester or orange [5-(and-6)-(4-chloromethyl(benzoyl)amino]tetramethylrhodamine and co-injected into the footpad of wild-type recipients; 16 hours after the injection, flow cytometry was used to assess the proportion of DCs that had reached the popliteal LNs. An example of a fluorescence-activated cell sorting (FACS) dot plot is shown. (H) LN homing index for LPS-DCs (n = 6 mice per condition, pooled from two independent experiments). (I and J) Analysis of mDC populations in mouse inguinal LNs at steady state. (I) Frequency of mDCs as a proportion of live cells (n = 3 mice per condition; representative results from one of three experiments are shown). (J) Frequency of mDC2 cells as a proportion of live cells (n = 7 mice in total, from three independent experiments). (B and E) Data are represented as boxes and whiskers; bars include 90% of the points, the line represents the median, and the box contains 75% of the data. (H) Data are quoted as means, and error bars correspond to SEM. (B, H, and I) Mann-Whitney test was applied. (E) Welch two-sample t test was applied (P = 1.358 × 10−7). (J) Paired t test was applied.

  • Fig. 3 Fast DC migration relies on calcium release by TRPML1.

    (A) A typical calcium response by Fluo4-AM–loaded Trpml1+/+ (black line) or Trpml1−/− (gray line) immature DCs. Cells were challenged with MLSA1 (10 μM) and thapsigargin (1 μM). Dotted lines indicate the treatment time. To prevent overlap between the two responses, we added an artificial offset (Trpml1+/+ signal + 100). (B) Quantification of calcium responses (data were pooled from N = 2 independent experiments with n = 29 and 30 cells for Trpml1+/+ and Trpml1−/− immature DCs, respectively). (C and D) Analysis of BMDC migration in 4 μm–by–5 μm fibronectin-coated microchannels. (C) Mean instantaneous velocities of DMSO- or MLSA1 (10 μM)–treated immature DCs (data were pooled from N = 2 independent experiments, with n = 316, 291, 206, and 163 cells from left to right). (D) Mean instantaneous velocities of DMSO- or Torin 2 (2.5 nM)–treated immature DCs (data were pooled from N = 2 independent experiments, with n = 215, 202, 201, and 166 cells from left to right). (E) Immunofluorescence images of TFEB-GFP knock-in control (siCtrl) or TRPML1 knockdown (siTRPML1-A) LPS-DCs (×60 magnification; scale bar, 10 μm; one plane is shown). (F) The normalized TFEB nucleus-to-cytosol ratio from cells shown in (E) (n = 40 cells per condition, pooled from N = 2 independent experiments). (G) Normalized TFEB nucleus-to-cytosol ratio from DMSO- or MLSA1-treated immature DCs (n = 60 cells per condition, pooled from N = 3 independent experiments). (B, F, and G) Data are quoted as means, and error bars correspond to SEM. (B) MLSA1 values were normalized according to the thapsigargin values, which were set to 100% in immature DCs. (F and G) For each experiment, data were normalized with respect to the mean TFEB nucleus-to-cytosol ratio obtained in siCtrl immature DCs (F) or DMSO-treated immature DCs (G). (C and D) Drugs (MLSA1 or Torin 2) were present during the recording. Data are represented as boxes and whiskers; bars include 90% of the points, the line represents the median, and the box contains 75% of the data. Mann-Whitney test was applied.

  • Fig. 4 TFEB controls actin cytoskeleton reorganization in response to microbial sensing.

    (A) LifeAct-GFP immature DCs and LPS-DCs migrating in 8 μm–by–5 μm fibronectin-coated microchannels. The middle (M) and cortical (C) planes were imaged. The inset (rectangle with a dashed boundary) highlights the proximity of the lysosomes (in red, labeled with WGA AF-647) to the F-actin patch observed in LPS-DCs. The red arrowhead indicates an actin cable that originates in the actin patch and extends toward the cell rear (spinning disk microscope, ×100 magnification; scale bars, 5 μm). (B) Sequential images of LifeAct-GFP siCtrl or TFEB-silenced (siTFEB) LPS-DCs (8 μm–by–5 μm fibronectin-coated channels; epifluorescence microscope, ×20 magnification; one image every 2 min; scale bars, 10 μm). (C) Mean LifeAct-GFP distribution in siCtrl or siTFEB DCs (from top to bottom, n = 40, 38, 29, and 35 cells; representative results from one of two experiments are shown). (D) Dynamic analysis of the fraction of time spent by the cells depicted in (C) with LifeAct-GFP at their front. (E) The mean F-actin front-back ratio. (D) Data are quoted as means, and error bars correspond to SEM. (E) Data are represented as boxes and whiskers; bars include 90% of the points, the line represents the median, and the box contains 75% of the data. Mann-Whitney test was applied.

  • Fig. 5 TRPML1 controls the organization of the DC actin cytoskeleton.

    (A) Sequential images of LifeAct-GFP LPS-DCs (8 μm–by–5 μm fibronectin-coated channels; epifluorescence microscope, ×20 magnification; one image every 2 min; scale bars, 10 μm). (B) Mean LifeAct-GFP distribution of DCs (from top to bottom: n = 76, 97, 69, and 71 cells; representative results from one of three experiments are shown). (C) Fraction of time spent by the cells depicted in (B) with LifeAct-GFP at their front. (D) Cortical LifeAct-GFP signal obtained in DMSO- and MLSA1 (10 μM)–treated DCs (8 μm–by–5 μm fibronectin-coated channels; spinning disk microscope, ×100 magnification; scale bar, 5 μm). (E) Mean LifeAct-GFP distribution in MLSA1-treated cells (from top to bottom: n = 41, 42, 45, 63, 78, and 57 cells; representative results from one of two experiments are shown). (F) The fraction of time spent by the cells depicted in (D) with LifeAct-GFP at their front (n = 58 and 60 cells for Trpml1+/+ and Trpml1−/− MLSA1-treated LPS-DCs, respectively). (C and F) Data are quoted as means, and error bars correspond to SEM. Mann-Whitney test was applied.

  • Fig. 6 Calcium release through TRPML1 controls myosin IIA retrograde flow at the rear of LPS-DCs.

    (A) Lysosomes are clustered in the vicinity of the myosin IIA–GFP pool located at the cell rear. The cortical plane of a migrating Trpml1+/+ LPS-DC (scale bar, 5 μm; lysosomes stained with WGA AF-647) is shown. (B) The normalized myosin IIA–GFP signal (myosin IIA–GFP concentration) measured in the region where lysosomes are located (data were pooled from N = 2 independent experiments, with n = 95, 81, 93, and 76 cells from left to right). (C) The mean myosin IIA–GFP distribution of Trpml1+/+ and Trpml1−/− BMDCs (from top to bottom: n = 74, 68, 58, and 44 cells; representative results from one of three experiments are shown). (D) The myosin IIA–GFP front-back ratio measured in the cells depicted in (C). (E) Cortical LifeAct-GFP signal obtained in DMSO- or para-nitroblebbistatin (p-blebb; 50 μM)–treated LPS-DCs (scale bars, 5 μm). (F) Left: Mean LifeAct-GFP distribution in LPS-DCs, DMSO- or para-nitroblebbistatin–treated cells (from top to bottom: n = 51 and 67 cells; representative results from one of three experiments are shown). Right: The fraction of time spent by these cells with LifeAct-GFP at their front. (G) Myosin IIA localizes on actin cables. Left: The overlay shows myosin IIA–GFP (green) and utrophin–red fluorescent protein (RFP; red) in an LPS-DC (scale bar, 5 μm; the cortical plane shown, and the nucleus indicated by white dashed lines). Right: The insets show sequential images of utrophin-RFP and myosin IIA–GFP (light blue rectangle). To highlight the myosin IIA retrograde flow without any contribution of cell velocity, all time points were aligned with respect to the cell rear (retrograde flow is indicated by white arrowheads; scale bars, 1 μm). Images were corrected for background and bleaching, and a median filter (2-pixel radius) was applied. (H) Myosin IIA–GFP at the cell rear undergoes retrograde flow, as indicated by red rectangles. Left: The cortical myosin IIA–GFP signal (C) obtained in a Trpml1+/+ LPS-DC is depicted; all time points were aligned as in (G) (an image was recorded every 400 ms; an image every 8 s is depicted; scale bar, 5 μm.) Right: Kymographs showing myosin IIA–GFP retrograde flow in migrating LPS-DCs. All time points were aligned as in (G). The tangent of the angle between the red dotted line (myosin IIA–GFP) and the y axis (cell rear) enables one to calculate the myosin IIA–GFP retrograde flow velocity (one image per 400 ms). (I) Myosin IIA–GFP retrograde flow in LPS-DCs (data were pooled from N = 2 to 4 independent experiments, with n = 14, 22, 11, and 12 cells from left to right). (J) Myosin IIA–GFP retrograde flow in immature DCs (n = 12 cells per condition, pooled from N = 2 independent experiments). (A to J) Experiments were carried out in 8 μm–by–5 μm fibronectin-coated microchannels; for (A), (E), (G), and (H), images were acquired on a spinning disk microscope (×100 magnification). (B) Data were normalized for each experiment with respect to the median ratio obtained in Trpml1+/+ immature DCs. (B and D) Data are represented as boxes and whiskers; bars include 90% of the points, the line represents the median, and the box contains 75% of the data. (F, I, and J) Data are quoted as means, and error bars correspond to SEM. (I and J) For each experiment, data were normalized against the mean retrograde flow for myosin IIA–GFP in immature DCs. Mann-Whitney test was applied.

  • Fig. 7 Inhibition of macropinocytosis activates the TFEB-TRPML1 axis.

    (A) Normalized TFEB nucleus-to-cytosol ratio from immature DCs treated with rottlerin (3 μM) or ML141 (50 μM) (data were pooled from N = 2 independent experiments, with n = 40, 45, 43, and 45 cells from left to right). (B) Mean instantaneous velocities of immature DCs migrating in 4 μm–by–5 μm fibronectin-coated microchannels, treated with rottlerin (3 μM) or ML141 (50 μM) (n = 109, 136, 140, 79, 77, and 120 cells from left to right). (C to F) Analysis of the localization of myosin IIA–GFP in immature DCs migrating in 8 μm–by–5 μm fibronectin-coated microchannels. (C) Mean myosin IIA–GFP distribution in immature DCs treated with rottlerin (3 μM) (from top to bottom: n = 50, 40, 47, and 50 cells; representative results from one of two experiments are shown). (D) The myosin IIA–GFP front-back ratio measured in the cells depicted in (C). (E) The mean myosin IIA–GFP distribution in immature DCs treated with ML141 (50 μM) (from top to bottom: n = 66, 49, 50, and 50 cells; representative results from one of two experiments are shown). (F) The myosin IIA–GFP front-back ratio measured in the cells depicted in (E). (A to F) Drugs were present during the recording. (A) Data were normalized for each experiment with respect to the mean TFEB ratio obtained in DMSO-treated immature DCs. Data are quoted as means, and error bars correspond to SEM. (B, D, and F) Data are represented as boxes and whiskers; bars include 90% of the points, the line represents the median, and the box contains 75% of the data. Mann-Whitney test was applied.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/2/16/eaak9573/DC1

    Materials and Methods

    Fig. S1. TFEB translocates to the nucleus and is required for fast migration of LPS-DCs (related to Fig. 1).

    Fig. S2. TRPML1 is required for fast DC migration, chemotaxis, and arrival at LNs (related to Fig. 2).

    Fig. S3. Fast DC migration relies on calcium release by TRPML1 (related to Fig. 3).

    Fig. S4. TFEB controls actin cytoskeleton reorganization in response to microbial sensing (related to Fig. 4).

    Fig. S5. TRPML1 controls the organization of the DC actin cytoskeleton (related to Fig. 5).

    Fig. S6. Calcium released through TRPML1 controls myosin IIA retrograde flow at the rear of LPS-DCs (related to Fig. 6).

    Fig. S7. Inhibition of macropinocytosis activates the TRPML1-TFEB axis (related to Fig. 7).

    Fig. S8. Fascin expression, lysosomal area, and cholesterol homeostasis in migrating DCs.

    Table S1. Source data for all graphs with n < 25.

    Movie S1. F-actin is in proximity to the endolysosomes at the rear of the LPS-DCs.

    Movie S2. F-actin dynamics in control (siCtrl) and TFEB-silenced (siTFEB) LPS-DCs.

    Movie S3. F-actin dynamics in Trpml1+/+ and Trpml1−/− LPS-DCs.

    Movie S4. Myosin IIA–GFP dynamics in Trpml1+/+ and Trpml1−/− LPS-DCs.

    Movie S5. F-actin dynamics in LPS-DCs treated with DMSO or para-nitroblebbistatin.

    Movie S6. Myosin IIA moves along actin cables.

    References (4452)

  • Supplementary Materials

    Supplementary Material for:

    Lysosome signaling controls the migration of dendritic cells

    Marine Bretou,* Pablo J. Sáez, Doriane Sanséau, Mathieu Maurin, Danielle Lankar, Melanie Chabaud, Carmine Spampanato, Odile Malbec, Lucie Barbier, Shmuel Muallem, Paolo Maiuri, Andrea Ballabio, Julie Helft, Matthieu Piel, Pablo Vargas,* Ana-Maria Lennon-Duménil*

    *Corresponding author. Email: marine.bretou{at}curie.fr (M.B.); pablo.vargas{at}curie.fr (P.V.); amlennon{at}curie.fr (A.-M.L.-D.)

    Published 27 October 2017, Sci. Immunol. 2, eaak9573 (2017)
    DOI: 10.1126/sciimmunol.aak9573

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. TFEB translocates to the nucleus and is required for fast migration of LPS-DCs (related to Fig. 1).
    • Fig. S2. TRPML1 is required for fast DC migration, chemotaxis, and arrival at LNs (related to Fig. 2).
    • Fig. S3. Fast DC migration relies on calcium release by TRPML1 (related to Fig. 3).
    • Fig. S4. TFEB controls actin cytoskeleton reorganization in response to microbial sensing (related to Fig. 4).
    • Fig. S5. TRPML1 controls the organization of the DC actin cytoskeleton (related to Fig. 5).
    • Fig. S6. Calcium released through TRPML1 controls myosin IIA retrograde flow at the rear of LPS-DCs (related to Fig. 6).
    • Fig. S7. Inhibition of macropinocytosis activates the TRPML1-TFEB axis (related to Fig. 7).
    • Fig. S8. Fascin expression, lysosomal area, and cholesterol homeostasis in migrating DCs.
    • Legend for table S1
    • Legends for movies S1 to S6
    • References (44–52)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Source data for all graphs with n < 25.
    • Movie S1 (.mp4 format). F-actin is in proximity to the endolysosomes at the rear of the LPS-DCs.
    • Movie S2 (.mp4 format). F-actin dynamics in control (siCtrl) and TFEB-silenced (siTFEB) LPS-DCs.
    • Movie S3 (.mp4 format). F-actin dynamics in Trpml1+/+ and Trpml1−/− LPS-DCs.
    • Movie S4 (.mp4 format). Myosin IIA?GFP dynamics in Trpml1+/+ and Trpml1−/− LPS-DCs.
    • Movie S5 (.mp4 format). F-actin dynamics in LPS-DCs treated with DMSO or para-nitroblebbistatin.
    • Movie S6 (.mp4 format). Myosin IIA moves along actin cables.

    Files in this Data Supplement:

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