Research ArticleCELL BIOLOGY

Rapid, directed transport of DC-SIGN clusters in the plasma membrane

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Science Advances  08 Nov 2017:
Vol. 3, no. 11, eaao1616
DOI: 10.1126/sciadv.aao1616
  • Fig. 1 Trajectories of DC-SIGN clusters and labeled MTs.

    (A) Fluorescence micrograph of ensconsin microtubule-binding domain (EMBD)–3XGFP–labeled MTs (green) in an MX DC-SIGN cell. Note that, to adequately visualize the internal MTs proximate to the ventral surface, we used “dirty TIRF”. GFP, green fluorescent protein. (B) Highly linear trajectories of DC-SIGN clusters exhibiting directed motion collected over the course of 400 s (red). (C) Some of the Brownian (light blue) and subdiffusive (dark blue) trajectories extracted with u-track from this video record. (D) Most directed trajectories are colinear with observable MTs. Trajectories shown were produced from the u-track analysis of a fluorescence video with DCN46-Fab labeling of DC-SIGN, as described in Materials and Methods.

  • Fig. 2 Instantaneous velocities of representative DC-SIGN trajectories.

    MX DC-SIGN cells (A) and dendritic cells (B). Top panels show the actual trajectories from which the instantaneous velocities were extracted using TrackMate analysis. Bottom panels show the instantaneous velocities measured along each trajectory. Because of the high instantaneous velocities in MDDC, portions of the track velocities plots were double-checked manually.

  • Fig. 3 Evidence that labeled DC-SIGN is on the surface.

    (A) KI quenches DC-SIGN fluorescence on MX DC-SIGN cells (left, before KI; right, after KI). MX DC-SIGN cells were fixed with 4% paraformaldehyde and labeled with DCN46–Alexa Fluor 647 monoclonal antibody (mAb). The after-KI image was taken 2 min after adding KI at a final concentration of 300 mM. (B) KI quenches DC-SIGN fluorescence on primary dendritic cells (left, before KI; right, after KI). Dendritic cells were fixed and labeled with DCN46–Alexa Fluor 647 mAb. The after-KI image was taken 1 min after adding KI at a final concentration of 300 mM. Images for (A) and (B) were taken on a confocal microscope. (C) KI quenches DC-SIGN fluorescence on live MX DC-SIGN cells, as visualized by TIRF microscopy [top row: left, before KI; middle, after KI; right, epifluorescence (epi) image]; by contrast, when we deliberately permitted entry of anti–DC-SIGN Fab fragments into the cell by incubating at 37°C for more than 30 min and by not adding chlorpromazine (bottom row), internal fluorescence was not quenched either in TIRF (middle) or epifluorescence (right) microscopy. Video S3 shows another example of the total quenching process.

  • Fig. 4 Box and whisker plots of the areal density of longer (>2 μm), directed DC-SIGN tracks in MX DC-SIGN cells with and without cytoskeletal inhibitors and in dendritic cells.

    Chlorpromazine (CPZ)–treated cells, representing the DC-SIGN tracks on control MX DC-SIGN cells without additional drug treatments. Nocodazole, latrunculin A (Lat A), and ciliobrevin data sets were collected on MX DC-SIGN cells treated with CPZ and additional drugs, correspondingly, as indicated. The MDDC data set was collected on primary dendritic cells without drug treatment and is shown for reference. P values were obtained from the standard Student’s t test. *P = 0.11 (between CPZ and ciliobrevin), **P = 0.016 (between CPZ and Lat A), ***P = 0.0016 (between CPZ and nocodazole).

  • Fig. 5 DC-SIGN motion along projections of MX DC-SIGN cells and movement of DENV along projection in MDDC.

    (A) Fluorescence micrograph of silicon rhodamine (SiR)–tubulin–labeled MTs (two left panels, red), which extend into dendritic projections (white arrows) of MDDCs (leftmost), as imaged in dirty TIRF (that is, the incident angle was reduced until MT labels were excited); the three right panels show an anti-tubulin confocal image of an MDDC [left, immunofluorescence; middle, differential interference contrast (DIC) image; right, merged image]. (B and C) Both DC-SIGN and DENV bound to projections undergo rapid, predominantly retrograde transport. (B) Kymographs showing significantly greater flux of DC-SIGN clusters in the retrograde direction in projections in MX DC-SIGN cells. Selected retrograde and anterograde motions and stalls are highlighted in magenta, cyan, and green, respectively. (C) DENV bound to projections in an MDDC also undergoes rapid, primarily retrograde directed transport. One selected DENV trajectory along a projection of more than ~12.5 s is highlighted in red. (D) Confocal microscopy (see the Supplementary Materials) demonstrates that DENV colocalized with DC-SIGN on the dendritic projections of an MDDC (white arrows); right panels show inset in the left panel magnified to show colocalization of DC-SIGN and DENV on filopodial-like dendritic tips. Magenta indicates DC-SIGN staining, and DC-SIGN was labeled with DCN46–Alexa Fluor 647 mAb. Green represents labeled DENV, and DENV was directly conjugated with Alexa Fluor 488.

  • Table 1 Mean values of parameters characterizing longer (>2 μm), directed transport trajectories of DC-SIGN clusters in native MX DC-SIGN cells and dendritic cells and drug-treated MX DC-SIGN cells.
    CellLength of tracks
    (μm ± SEM)
    Contour velocity (μm/s ± SEM)End-to-end velocity (μm/s ± SEM)Number of tracks
    Dendritic cells3.1 ± 0.0603.9 ± 0.0502.4 ± 0.070367
    MX DC-SIGN2.8 ± 0.102.2 ± 0.0500.97 ± 0.040348
    Nocodazole2.2 ± 0.0702.9 ± 0.0900.91 ± 0.07045
    Latrunculin A2.7 ± 0.102.0 ± 0.0501.0 ± 0.050119
    Ciliobrevin2.3 ± 0.0403.5 ± 0.141.5 ± 0.1180

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/11/eaao1616/DC1

    video S1. Fluorescence video of DC-SIGN clusters exhibiting long, highly directed excursions.

    video S2. Fluorescence video of EMBD-labeled MTs (green, left) in an MX DC-SIGN cell (red, middle) with superimposed trajectories of DC-SIGN clusters exhibiting highly directed, superdiffusive motion (right).

    video S3. Quenching of DC-SIGN fluorescence by KI in a TIRF video of MX DC-SIGN cells (to accompany Fig. 3C).

    video S4. Effect of ciliobrevin on lysosmal transport.

    video S5. Effect of ciliobrevin on lysosmal transport.

    video S6. DC-SIGN–directed transport in MX DC-SIGN dendritic projections favors the retrograde direction (to accompany Fig. 5).

    video S7. When DENV binds to projections on MDDCs, it also undergoes rapid, directed transport.

    fig. S1. MSS analysis results for DC-SIGN clusters.

    fig. S2. KI (300 mM) does not quench Calcium Orange AM inside the cells.

    fig. S3. Effect of nocodazole on MT status in MX DC-SIGN cells.

    fig. S4. Effect of latrunculin A on actin filament status.

    fig. S5. Conceptualization of a ventral MT occupying a channel within the actin cortex with a putative MT motor driving the directed motion of DC-SIGN.

    table S1. Maximum instantaneous and average (including stall time) speeds of selected DENV trajectories in the MDDC projection shown in Fig. 5D and video S5.

  • Supplementary Materials

    This PDF file includes:

    • Legends for videos S1 to S7
    • fig. S1. MSS analysis results for DC-SIGN clusters.
    • fig. S2. KI (300 mM) does not quench Calcium Orange AM inside the cells.
    • fig. S3. Effect of nocodazole on MT status in MX DC-SIGN cells.
    • fig. S4. Effect of latrunculin A on actin filament status.
    • fig. S5. Conceptualization of a ventral MT occupying a channel within the actin cortex with a putative MT motor driving the directed motion of DC-SIGN.
    • table S1. Maximum instantaneous and average (including stall time) speeds of selected DENV trajectories in the MDDC projection shown in Fig. 5D and video S5.

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    Other Supplementary Material for this manuscript includes the following:

    • video S1 (.avi format). Fluorescence video of DC-SIGN clusters exhibiting long, highly directed excursions.
    • video S2 (.avi format). Fluorescence video of EMBD-labeled MTs (green, left) in an MX DC-SIGN cell (red, middle) with superimposed trajectories of DC-SIGN clusters exhibiting highly directed, superdiffusive motion (right).
    • video S3 (.avi format). Quenching of DC-SIGN fluorescence by KI in a TIRF video of MX DC-SIGN cells (to accompany Fig. 3C).
    • video S4 (.avi format). Effect of ciliobrevin on lysosmal transport.
    • video S5 (.avi format). Effect of ciliobrevin on lysosmal transport.
    • video S6 (.avi format). DC-SIGN–directed transport in MX DC-SIGN dendritic projections favors the retrograde direction (to accompany Fig. 5).
    • video S7 (.avi format). When DENV binds to projections on MDDCs, it also undergoes rapid, directed transport.

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