Research ArticleCELLULAR NEUROSCIENCE

Building sensory axons: Delivery and distribution of NaV1.7 channels and effects of inflammatory mediators

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Science Advances  23 Oct 2019:
Vol. 5, no. 10, eaax4755
DOI: 10.1126/sciadv.aax4755
  • Fig. 1 Venus-NaV1.7-BAD localizes to nanoclusters on distal axons.

    (A) Diagram of the Venus-NaV1.7-BAD construct showing the fusion of Venus to the N-terminus and the insertion of the BAD into the S1-S2 loop of domain IV. (B to H) DRG neurons were cultured for 7 to 10 days in microfluidic chambers (MFCs). (B) Immunolabeling of DRG neurons in MFCs express both peripherin (cyan) and PGP9.5 (yellow) in both the somatic (left) and axonal (right) chambers. (C) Compressed confocal z-stack of a DRG neuron within the MFC somatic chamber expressing Venus-NaV1.7-BAD, showing the distribution of the Venus signal (top), surface labeling with SA-CF640 (middle), and merge (bottom). The surface labeling reveals a region of the proximal axon with increased density of NaV1.7 (white arrow). (D) A substack of the z-slices from (C) chosen from the center of the soma to demonstrate the surface labeling (magenta) surrounding the somatic cytoplasm containing the total NaV1.7 protein (green). Bottom panel shows line scans of the normalized fluorescence intensity of the Venus (green) and SA-CF640 (magenta) signals along the line scan (dashed white line; middle). The black arrowheads indicate the increased fluorescence intensity of the SA-CF640 surface labeling at the plasma membrane. (E) Compressed confocal z-stack of a distal axon from the MFC axonal chamber. Both the Venus signal (top) and surface labeling (middle) demonstrate localization of channels to nanoclusters along the axonal membrane (white arrows). (F) Line profile of the fluorescence intensity of the distal axon in (E), normalized to the maximum fluorescence intensity. Peaks in the line profile corresponding to the clusters of channels along the axon. This example shows two clusters that are noticeably brighter than the others [yellow arrows in (E) and black arrows in (F)]. (G) Estimation of the number of channels per axonal nanocluster. The average fluorescence intensity of single molecules was determined by creating a histogram of individual channel intensities. Fluorescence intensities of nanoclusters were divided by this value to approximate the number of channels in each nanocluster. (H) Single-molecule tracks of individual surface labeled Venus-NaV1.7-BAD channels on a distal axon. A compressed z-stack was used to demonstrate the overall morphology of a distal axon (outlined in white) expressing Venus-NaV1.7-BAD where the overlay between the Venus signal (green) and surface labeling (magenta) appears as white. Tracks are representative molecule behaviors from the regions indicated by the white boxes (boxes a to e). Molecules near the distal end and between nanoclusters show relatively high mobility (blue trajectories), while channels within the nanoclusters display low mobility (black trajectories).

  • Fig. 2 Anterograde Halo-NaV1.7 vesicles accumulate at the distal axon.

    (A) Diagram of the Halo-NaV1.7 construct showing addition of the β4 transmembrane and extracellular location of the HaloTag enzyme. (B) Schematic of the OPAL imaging technique used to visualize anterograde vesicles containing NaV1.7. DRG neurons expressing Halo-NaV1.7 were cultured in MFCs for 7 to 10 days. Immediately prior to imaging, JF549-Halo was added to the somatic chamber for 15 min. After removal of excess dye, axons within the axonal chamber were imaged using spinning disk microscopy to visualize anterograde vesicles traveling from the somatic chamber into the axonal chamber. (C) Representative axon in the axonal chamber labeled as described in (B). Colored arrowheads show locations of the identified vesicles over time. The corresponding kymograph of the Halo-NaV1.7 vesicles was created from a time-lapse image sequence taken at 3.3 frames/s. It shows vesicle position (x axis) as a function of time (y axis). Negative slopes on the kymograph represent movement in the anterograde direction, while vertical lines represent stationary vesicles. (D) Kymographs generated from an axon before and 60 min after either dimethyl sulfoxide (DMSO) or Noco treatment. (E) Average vesicle velocities before and after DMSO or Noco treatment. (F) The number of vesicles with a velocity >0.1 μm/s within a 30-μm segment of axon per 60 s. ****P < 0.0001, ***P < 0.001, two-way ANOVA with Tukey’s multiple comparisons test. (G) Compressed z-stack of an axon terminal in the axonal chamber of the MFC at 1 hour after somatic labeling, demonstrating the accumulation of vesicles near the distal end of the axon (white arrowhead). (H) Image sequence of Halo-NaV1.7 compressed over time to allow visualization of the distal axon. The kymograph of a line scan through this axon shows the behavior of vesicles within this region over time. The vertical lines demonstrate that the vesicles that have accumulated within the distal axon are generally stationary. A small number of channels leave the distal axon as retrograde vesicles (blue arrowheads). Anterograde vesicles move along the axon until reaching the distal axon where they become immobilized (red arrowhead in the black box that corresponds to the region delineated by the white box in the top panel). (I) Time series of a vesicle destined for the distal axon corresponding to the region indicated by the white box in (H), as well as the kymograph region outlined by the back box (H). The red arrowheads follow the position of the vesicle over time as it travels along the axon and then stops at the neck of the distal axon. The vesicle behavior over time is illustrated by the corresponding kymograph with the red arrowhead, denoting where it transitions from mobile to stationary.

  • Fig. 3 Anterograde NaV1.7 vesicles are Rab6A positive.

    DRG neurons in MFCs were transfected with Halo-NaV1.7 and either EGFP-tagged Rab6A or Rab3A constructs. OPAL imaging was used to visualize anterograde Halo-NaV1.7 labeled with JF646-Halo, and two-color time-lapse imaging was performed. (A) Rab6A shows cotransport with Halo-NaV1.7. Selected frames from a time-lapse movie show the position of an anterograde vesicle containing Halo-NaV1.7 (magenta arrowheads) and EGFP-Rab6A (green arrowheads) over time. The overlay (white arrowheads) demonstrates comovement. The corresponding kymographs show a second vesicle containing both NaV1.7 and Rab6A as demonstrated by the overlapping lines on the kymograph. (B) Rab3A is an example of a Rab that does not cotransport with NaV1.7. The image sequence shows distinct NaV1.7 (magenta arrowheads) and Rab3A (green arrowheads), demonstrating independent movement. This can be visualized by distinct magenta and green lines on the kymograph. (C) DRG neurons in MFCs were transfected with NaV1.7 and either EGFP-tagged Rab1A, Rab2, Rab3A, Rab5A, Rab6A, Rab7, Rab8A, or Rab10. Anterograde NaV1.7 was labeled with JF646-Halo only in the somatic chamber, and two-color time-lapse imaging was performed. Kymographs were created for each Rab-NaV1.7 pair, and vesicle tracks were visually inspected and classified as Rab only, NaV1.7 only, or both. Rab6A shows a much higher association with NaV1.7 than any of the other Rabs investigated: Rab1A: 4%, 85 vesicles, 12 axons, 2 cultures; Rab2: 6%, 103 vesicles, 10 axons, 2 cultures; Rab3A: 7%, 115 vesicles, 8 axons, 2 cultures; Rab5A: 8%, 108 vesicles, 14 axons, 2 cultures; Rab6A: 62%, 104 vesicles, 10 axons, 4 cultures; Rab7A: 1%, 92 vesicles, 10 axons, 2 cultures; Rab8A: 13%, 83 vesicles, 10 axons, 2 cultures; Rab10: 12%, 99 vesicles, 12 axons, 2 cultures.

  • Fig. 4 NaV1.7 current density increases following treatment with IM.

    (A and B) Family of current traces evoked by 100-ms depolarizing voltage steps from −80 to +40 mV in 5-mV increments from a holding potential of −100 mV. Representative traces from NaV1.8-null DRG neurons expressing NaV1.7 channels in (A) control (black) and (B) IM-treated (IM; red) neurons. (C) Comparison of peak inward current between control (black) and IM-treated conditions (red). (D) Scatter plot showing enhanced current density in DRG neurons treated with IM. Current density was measured by normalizing maximal peak currents with cell capacitance. The bars indicate the means; *P < 0.05, two-sample t test.

  • Fig. 5 Anterograde NaV1.7 vesicular trafficking is enhanced by IM.

    DRG neurons expressing Halo-NaV1.7 were cultured in MFCs for 7 to 10 days. Treatment dishes were exposed to IM in both the somatic and axonal chambers for 4 to 5 hours before imaging. Anterograde vesicles labeled with JF549-Halo were visualized via OPAL imaging. (A) Representative frames from time-lapse movies showing Halo-NaV1.7 vesicles traveling along axons from control dishes (left) or dishes incubated with IM (right). Below are the corresponding kymographs showing vesicle movement over time. The images were processed and contrasted identically. (B) The number of vesicles moving along each axon per minute was significantly enhanced after incubation with IM (control: 12.7 ± 1.3 vesicles per axon per 60 s, n = 21 axons; +IM: 21.5 ± 1.5 vesicles per axon per 60 s, n = 20 axons; ***P ≤ 0.0001, two-sample t test). (C) The fluorescence intensity was measured for individual vesicles under either control or +IM conditions. The median fluorescence intensity of vesicles was significantly enhanced after incubation with IM (control: 1840 A.U., n = 312 vesicles; +IM: 6220 A.U., n = 285 vesicles; P ≤ 0.0001, Mann-Whitney U test). (D) Vesicle tracks from kymographs were analyzed to measure the average velocity, including stops and pauses. The average vesicle velocity was significantly faster after incubation with IM (control: 1.01 ± 0.03 μm/s, n = 330 vesicles; +IM: 1.58 ± 0.04 μm/s, n = 532 vesicles; ***P ≤ 0.0001, Mann-Whitney U test). (E) Vesicle tracks were broken into segments that could be fit with a straight line, representing movement of a consistent velocity or instantaneous velocity. Stops or pauses were excluded. The histogram shows the counts for the number of line segments of each velocity under control (gray) and +IM (red) conditions. (F) Compressed z-stacks (inverted signal) showing accumulation of Halo-NaV1.7 vesicles at the distal axon under control (top) and +IM (bottom) conditions. (G) Quantitation of fluorescence intensity from the distal 60 μm of axons. The fluorescence intensity was significantly greater in axons from the +IM condition versus control (control: 1825 ± 223 A.U., n = 28 axons; +IM: 4535 ± 770 A.U., n = 27 axons; *P ≤ 0.0014, Mann-Whitney U test). (H) Kymographs generated from an axon from the +IM condition before and 60 min after either DMSO or Noco treatment. (I) Average vesicle velocities before and after DMSO or Noco treatment. ****P < 0.0001, two-way ANOVA with Tukey’s multiple comparisons test. (J) The number of vesicles with a velocity >0.1 μm/s within a 30-μm segment of axon per 60 s. ****P < 0.0001, **P < 0.003, two-way ANOVA with Tukey’s multiple comparisons test.

  • Fig. 6 Enhanced surface expression of NaV1.7 channels after incubation with IM.

    DRG neurons expressing Venus-NaV1.7-BAD were cultured in MFCs for 7 to 10 days. Treatment dishes were exposed to IM in both the somatic and axonal chambers for 4 to 5 hours before imaging. (A) Compressed z-stacks of distal axons under control conditions (left) and after exposure to IM (right). Under both conditions, nanoclusters can be seen along the distal axon with both the Venus signal (green) and CF640 surface labeling (magenta). (B) Line profile of the axons in (A) showing fluorescence intensity normalized to the maximum fluorescence intensity, with peaks representing nanoclusters of NaV1.7. (C and D) Quantification of the fluorescence intensity of the Venus signal (C) and CF640 surface labeling (D) shows significantly more surface labeling at distal axons after incubation with IM versus control conditions. *P< 0.01, two-sample t test (C), Mann-Whitney U test (D). (E) Single-molecule tracks of individual surface labeled Venus-NaV1.7-BAD channels on a distal axon. A compressed z-stack is used to demonstrate the overall morphology of a distal axon expressing Venus-NaV1.7-BAD where the overlay between the Venus signal (green) and surface labeling (magenta) appears as white. The white dots along the axon represent the location of axonal NaV1.7 nanoclusters. The lines below the picture represent the trajectories of single molecules over time, tracked from time-lapse movies. Black lines represent molecule tracks with relatively low mobility, while blue lines represent molecule tracks with higher mobility. Tracks are representative of single-molecule behaviors from the regions indicated by the white boxes (boxes a to d).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaax4755/DC1

    Fig. S1. Venus-NaV1.7-BAD and Halo-NaV1.7 display WT currents and express in cultured DRG neurons (related to Figs. 1A and 2A).

    Fig. S2. Venus-NaV1.7-BAD surface labeling does not display bleed-through, and DRG neurons display autofluorescence (related to Fig. 1).

    Fig. S3. Venus-NaV1.7-BAD and Halo-NaV1.7 nanoclusters are visible in DRG axons (related to Fig. 1).

    Fig. S4. Methods for estimating number of channels per nanocluster (related to Fig. 1G).

    Fig. S5. Retrograde, but not anterograde, vesicles can be visualized using the fluorescent protein signal (related to Fig. 2).

    Fig. S6. Rab6 expression in cultured DRG neurons (related to Fig. 3).

    Fig. S7. Fluorescence intensity for individual JF549-Halo molecules (related to Fig. 5).

    Fig. S8. Modification of MFCs (related to Materials and Methods).

    Movie S1. Anterograde trafficking of Halo-NaV1.7.

    Movie S2. Anterograde trafficking of Halo-NaV1.7 under control conditions and after exposure to IM.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Venus-NaV1.7-BAD and Halo-NaV1.7 display WT currents and express in cultured DRG neurons (related to Figs. 1A and 2A).
    • Fig. S2. Venus-NaV1.7-BAD surface labeling does not display bleed-through, and DRG neurons display autofluorescence (related to Fig. 1).
    • Fig. S3. Venus-NaV1.7-BAD and Halo-NaV1.7 nanoclusters are visible in DRG axons (related to Fig. 1).
    • Fig. S4. Methods for estimating number of channels per nanocluster (related to Fig. 1G).
    • Fig. S5. Retrograde, but not anterograde, vesicles can be visualized using the fluorescent protein signal (related to Fig. 2).
    • Fig. S6. Rab6 expression in cultured DRG neurons (related to Fig. 3).
    • Fig. S7. Fluorescence intensity for individual JF549-Halo molecules (related to Fig. 5).
    • Fig. S8. Modification of MFCs (related to Materials and Methods).
    • Legends for movies S1 and S2

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Anterograde trafficking of Halo-NaV1.7.
    • Movie S2 (.avi format). Anterograde trafficking of Halo-NaV1.7 under control conditions and after exposure to IM.

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