Research ArticleBiochemistry

The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane

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Science Signaling  08 May 2018:
Vol. 11, Issue 529, eaao4006
DOI: 10.1126/scisignal.aao4006
  • Fig. 1 TrkB receptors are predominantly monomeric at the plasma membrane.

    (A) Trk immunolabeling in SCNs after 5 min of BDNF compared to Neurobasal media only. dSTORM focused at the basal cell membrane plane. Top: Representative Trk localizations after dSTORM detection, depicted as red dots; scale bar, 2 μm. Bottom: Zoomed-in views of Trk aggregates; scale bar, 100 nm. Histograms are the radii of Trk clusters based on dSTORM localization in representative BDNF- and media-treated SCN, grouped in 10-nm bins; n = 337 and 290 clusters, respectively. Experiment was repeated three times. Ab, antibody. (B) Single-molecule counting of TrkB-ACP-488 by bleaching step analysis in TrkB-ACP–transfected HEK cells and SCNs labeled with coenzyme A (CoA)–488, treated with BDNF or media for 5 min, fixed, and imaged by TIRFM. Representative time series of spot bleaching in a single step (monomer) and in two steps (dimer) from two HEK cells is presented in the upper part. Lower traces: Representative intensity over frame of bleaching particles from two spots in a single neuron. Right graph: Data are means ± SEM ratio of TrkB-ACP dimers or oligomers to the total number of spots analyzed per cell; n = 22, 15, 18, and 20 cells for HEK (media), HEK (BDNF), SCN (media), and SCN (BDNF), respectively, pooled from three experiments. a.u. arbitrary units. (C) Representative colocalization images and a quantitative graph (upper left) of dual-labeled TrkB-ACP in HEK cells imaged by TIRF after fixation and analysis of colocalization between the detected spots in the two channels. Scale bar, 2 μm. Data are means ± SEM ratio (per cell) of colocalized spots in media- and in BDNF-treated cells (at 5 min) compared with the ratio after the simulated localization of randomized spot positions. n = 33 cells (media) and 22 cells (BDNF), pooled from three experiments. (D) HEK and SCN cells transfected with EGFR or TrkB after media or BDNF treatment then treated with bis(sulfosuccinimidyl)suberate (BS3) cross-linker or phosphate-buffered saline (PBS) only and subjected to Western blot analysis for EGFR or TrkB. Representative images are based on four and three experiments in HEK and SCN, respectively. Vertical lines indicate blot image cropping and concatenation; all concatenated images are from the same blotted membrane. IB, immunoblot. (E) TIRF-FRET (Förester resonance energy transfer) analysis of HEK cells after 5-min incubation in control media or BDNF treatment. The FRET ratio was normalized to donor (TrkB- or GB1a-Cerulean) signal. (F) Representative TIRF-FRET ratio images are presented on the left; scale bar, 5 μm. Right graph: FRET ratio means ± SEM (per cell) from n = 11 (TrkB-media), 18 (TrkB-BDNF), and 24 (GB1a + GB2) cells pooled from three experiments. ***P < 0.001, Student’s two-tailed t test. Difference between media and BDNF conditions was not significant (P > 0.1).

  • Fig. 2 TrkB binds to BDNF and is activated at the plasma membrane as a monomer.

    (A) TIRF images of TrkB-ACP and Qdot-605 (QD-605)–labeled BDNF puncta comovement in HEK cells. TrkB-ACP was labeled with CoA-488, then BDNF-QD was added, and cells were subsequently imaged up to 60 min after addition. Arrowheads in green and magenta mark individual puncta. White arrowheads mark colocalized TrkB and BDNF. Scale bar, 1 μm. (B) Traces of tracked TrkB and BDNF spots shown in (A) were plotted in a three-dimensional trace. Image at the lower base of the plot depict first frame of analysis. Images and tracking in (A) and (B) are representative of two experiments. (C) TIRF image of colocalized TrkB-ACP-488 and streptavidin-647–labeled biotin-BDNF in fixed HEK cells. Arrowheads mark colocalized spots. Quantification of colocalization is shown in (G). Scale bar, 2 μm. Image is representative of three experiments. (D) Representative TIRF images of phospho-tyrosine immunofluorescence and TrkB-ACP in HEK cells treated with media or BDNF for 5 min. Scale bar, 5 μm. (E) TIRF imaging analysis of the density of phospho-tyrosine (pTyr) spots in HEK cells and SCNs treated as in (D). Data are means ± SEM density per cell from n = 50 (HEK + media), 56 (HEK + BDNF), 27 (SCN + media), and 26 (SCN + BDNF) cells pooled from three experiments; ***P < 0.001, Student’s two-tailed t test. (F) Depiction of the analysis presented in (D) and (F). TrkB and BDNF or phospho-tyrosine spots were detected, and the ratio of TrkB-ACP monomers and dimers was assessed by photobleaching step analysis. (G) The ratio of monomers (Mono.) and dimers or oligomers (Di/oligo.) from TrkB-ACP spots detected in colocalization with streptavidin-647–labeled biotin-BDNF and immunolabeled pTyr in HEK and SCN cultures. Data are from n = 28 (HEK and BDNF), 16 (SCN and BDNF), 37 (HEK and pTyr), and 25 (SCN and pTyr) spots are pooled from three experiments.

  • Fig. 3 TrkB clusters and dimerizes in BDNF-induced endosomes.

    (A and B) Confocal images of SCN expressing TrkB-ACP that was dual-labeled with 647- and 488-conjugated CoA. After labeling, SCNs were incubated with BDNF for 30 min, then fixed, and immunolabeled with Rab7 (A) or treated with streptavidin-568–tagged biotin-BDNF for 60 min (B). Arrowheads mark intracellular colocalization between dual-labeled TrkB-ACP and Rab7 or BDNF puncta. Zoomed insets show internalized particles. Images are representative of two experiments. Scale bars, 10 μm and 2 μm (inset). (C) dSTORM localization images of SCNs treated with biotin-BDNF for 30 min, then fixed, and stained with Trk-647 and Qdot-525–streptavidin to label TrkB (red) and BDNF (green). Zoomed insets show intracellular Trk-BDNF clusters. Images are representative of two experiments. Scale bars, 2 μm, 200 nm, and 100 nm (left to right). (D and E) Representative oblique illumination images of HEK cells coexpressing TrkB-Citrine and TrkB-Cerulean for FRET measurement, treated with BDNF–streptavidin-647 (D), or with streptavidin-647 alone (E) for 30 min. Arrowheads denote intracellular endosomal puncta containing TrkB donor and acceptor signals (E) and TrkB together with labeled BDNF (D). Zoomed insets show areas with internalized TrkB and TrkB with BDNF. Scale bars, 5 μm. (F) FRET ratio analysis of intracellular TrkB-Citrine and TrkB-Cerulean spots. Endosomes that were positive (BDNF-streptavidin-647) or negative for BDNF (in streptavidin-647–treated cells) were delineated, and the FRET ratio was calculated per spot. Data are means ± SEM FRET ratio in endosomes of cells 30 min after treatment with media with streptavidin-647 only or BDNF-647. ***P < 0.001, Student’s two-tailed t test; n = 160 and 77 endosomes for streptavidin-only and BDNF treatments, respectively. The left image shows exemplary FRET measurement of the BDNF-TrkB endosome. Scale bar, 2 μm. Results and images in (D) and (F) are representative of three experiments.

  • Fig. 4 Dynamin-mediated endocytosis facilitates BDNF-induced AKT but not ERK1/2 signaling in SCNs.

    (A and B) Western blot analysis of phospho-AKT (pAKT) (A) and phospho-ERK1/2 (pERK1/2) (B) in SCNs treated with BDNF or BDNF and Dynasore (Dyn) for the duration specified. Whole-cell lysates were gel electrophoresed and blotted to assess the phosphorylation of AKT and pERK1/2. Blots of pERK1/2 were reprobed for total ERK1/2; because both pAKT and AKT antibodies are rabbit-derived, detection of pAKT and total AKT was performed on separate blots. Image concatenation is denoted by vertical lines. Graph shows means ± SEM of a normalized signal from n = 5 and 3 experiments (for pERK1/2 and pAKT, respectively). *P < 0.05, **P < 0.01 by Student’s two-tailed t test. (C) Representative time-series images of SCNs expressing a PH-AKT-GFP probe by live TIRF imaging. Signal represents AKT recruitment to the plasma membrane in SCNs treated as described in (A) and measured before BDNF exposure or 5, 30, and 60 min after. Scale bar, 5 μm. (D) Graph shows means ± SEM of normalized PH-AKT-GFP TIRF signals per cell in each time point after treatments as in (A) and (C). *P < 0.05, Student’s two-tailed t test; n = 26 [dimethylsulfoxide (DMSO)], 51 (BDNF), and 44 (BDNF + Dynasore) cells. Data and images in (C) and (D) are from 4 (DMSO), 7 (BDNF), and 6 (BDNF + Dynasore) experiments, respectively. (E) Representative trace of a PH-AKT-GFP signal after addition of BDNF or BDNF with Dynasore. SCNs were imaged by TIRF before treatment and then every 5 s from 20 s to 5 min. (F) Means ± SEM of duration (left) and peak amplitude over baseline (right) of PH-AKT-GFP TIRF signal rise events after BDNF or BDNF + Dynasore treatments. **P < 0.01, Student’s two-tailed t test; n = 43 and 32 cells for BDNF and BDNF + Dynasore conditions, respectively, representative of four experiments. (G) Schematic model of TrkB endosome-specific dimerization and signaling role in SCN: TrkB receptor monomer binds to BDNF at the plasma membrane to activate ERK1/2 signaling. Dimerization of TrkB and robust downstream activation of the PI3K-AKT pathway occur in BDNF-induced endosomes.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/529/eaao4006/DC1

    Fig. S1. Cultured SCNs specifically express TrkB.

    Fig. S2. ACP tagging of TrkB receptor enables plasma membrane–specific labeling and maintains signaling capacity in response to BDNF.

    Fig. S3. Comparison of TrkB and EGFR dimer or oligomer ratio by step-bleaching analysis.

    Fig. S4. Cell-surface cross-linking of TrkA and TrkB.

    Fig. S5. Short BDNF treatment induces cell-surface TrkB-ACP pTyr.

    Fig. S6. Dynamin-dependent endocytosis of TrkB.

    Fig. S7. ERK1/2 and AKT signaling in response to BDNF is mediated by Trk.

    Fig. S8. Dynasore alone does not reduce PH-AKT-GFP signal at the plasma membrane.

    Fig. S9. Effects of DN dynamin on ERK1/2 signaling, TrkB TIRF-FRET, and BDNF internalization.

    Fig. S10. dSTORM and TIRF microscopy systems resolution calibration.

    Data file S1. Mass spectrometry analysis of the 100 to 130 kDa proteome in HEK cells.

    Data file S2. Mass spectrometry analysis of the 100 to 130 kDa proteome in rodent SCNs.

  • Supplementary Materials for:

    The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane

    Eitan Erez Zahavi, Noam Steinberg, Topaz Altman, Michael Chein, Yuvraj Joshi, Tal Gradus-Pery, Eran Perlson*

    *Corresponding author. Email: eranpe{at}post.tau.ac.il

    This PDF file includes:

    • Fig. S1. Cultured SCNs specifically express TrkB.
    • Fig. S2. ACP tagging of TrkB receptor enables plasma membrane–specific labeling and maintains signaling capacity in response to BDNF.
    • Fig. S3. Comparison of TrkB and EGFR dimer or oligomer ratio by step-bleaching analysis.
    • Fig. S4. Cell-surface cross-linking of TrkA and TrkB.
    • Fig. S5. Short BDNF treatment induces cell-surface TrkB-ACP pTyr.
    • Fig. S6. Dynamin-dependent endocytosis of TrkB.
    • Fig. S7. ERK1/2 and AKT signaling in response to BDNF is mediated by Trk.
    • Fig. S8. Dynasore alone does not reduce PH-AKT-GFP signal at the plasma membrane.
    • Fig. S9. Effects of DN dynamin on ERK1/2 signaling, TrkB TIRF-FRET, and BDNF internalization.
    • Fig. S10. dSTORM and TIRF microscopy systems resolution calibration.
    • Legends for data files S1 and S2

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Mass spectrometry analysis of the 100 to 130 kDa proteome in HEK cells.
    • Data file S2 (Microsoft Excel format). Mass spectrometry analysis of the 100 to 130 kDa proteome in rodent SCNs.

    [Download Data files S1 and S2]


    © 2018 American Association for the Advancement of Science

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