Research ArticleNotch Signaling

Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing

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Science Signaling  13 Jun 2017:
Vol. 10, Issue 483, eaag2989
DOI: 10.1126/scisignal.aag2989
  • Fig. 1 DTX4 is a membrane-associated positive regulator of Notch signaling.

    (A) Effect of knockdown of DTX isoforms on induction of the Notch target gene Hey1. RNA was extracted from U2OS-N1 cells transfected with the indicated siRNAs (NT, nontargeting) before coculture with OP9-Dll1 (with DAPT when indicated) or control OP9 cells, and transcripts were quantified by qRT-PCR and normalized to UBCH5B. Left: The amount of Hey1 transcript in the cells cultured with OP9-Dll1 cells was set to 100%. Right: Abundance of DTX transcripts. Data in both graphs are means ± SD of three independent experiments. (B) Effect of knockdown of DTX4 on Notch reporter gene induction. U2OS-N1 cells were transfected with both CSL-LUC (Notch reporter) and TK-renilla (internal control) 24 hours before coculture with OP9-Dll1 or OP9 cells. Data are means ± SEM of five experiments, analyzed by one-way analysis of variance (ANOVA) and Bonferroni post hoc test (****P < 0.0001). Reporter activity in cells cocultured with OP9-Dll1 with nontargeting siRNA was set to 100%. The siRNA used in (B) to target DTX4 was different from the siRNA used in (A). (C) Effect of knockout (KO) of DTX4 on Notch reporter gene induction. U2OS-N1 cells or genome-edited DTX4 KO cells (N1/DTX4-KO) were transfected with both CSL-LUC and TK-renilla 24 hours before coculture with or without OP9-Dll1. Data in the graph are means ± SEM of three independent experiments, analyzed by one-way ANOVA and Bonferroni post hoc test (****P < 0.0001). (D) Western blotting (WB) analysis of lysates from U2OS-N1 and N1/DTX4-KO cell, using a Notch antibody that recognizes the intracellular domain of N1 or α-tubulin antibody. p300 is the proform of N1 (not furin-processed); TM-IC is the nonactivated mature form of N1. Molecular weights are indicated on the right. Data are representative of three independent experiments. (E) Localization of N1 and DTX4 in permeabilized (P) and nonpermeabilized (NP) N1/DTX4-GFP cells alone or transiently transfected with DTX4-Cherry or mCherry. For the N1/DTX4-GFP cells, N1 was detected with the antibody recognizing HA, followed by fluorophore-conjugated secondary antibody (red) and DTX4 by GFP fluorescence (green). For the transiently transfected cells, DTX4-GFP is green, and DTX4-Cherry and mCherry are red. Hoechst labeling is in blue in the merged pictures. Scale bar, 10 μm. Insets correspond to threefold enlargements of the boxed regions. Arrows indicate colocalized vesicles. Data are representative of 60 cells analyzed from three experiments. (F) Live-cell imaging (movie S1) of DTX-GFP, mCherry, and DTX4-Cherry. Confocal images are from movies of N1/DTX4-GFP, N1/Cherry, and N1/DTX4-Ch cells. Data are representative of 10 cells analyzed from three experiments. (G) Analysis of the interaction between N1 and DTX4 by coimmunoprecipitation. Whole-cell extracts (WCE) or proteins that immunoprecipitated with the antibody recognizing GFP (IP GFP) from the indicated cell lines were analyzed by Western blotting for the indicated proteins. TM-IC is the mature furin-cleaved form of N1. The lower left blot has residual N1 signal (indicated with gray Notch1). The apparent molecular weights are indicated. Data are representative of three experiments.

  • Fig. 2 Transendocytosis is affected by events occurring in Notch-expressing cells.

    (A) Effect of knockdown of DTX4 on transendocytosis. U2OS-N1 cells transfected with the indicated siRNAs were cocultured with OP9-Dll1 cells for 4 hours before immunofluorescence analysis using fluorescent anti-HA (green) and anti-VSV (red). The images are representative of five independent experiments. (B) Effect of knockdown of DTX4 or Dyn2 on transendocytosis. As in (A), except that cells were permeabilized or not before immunofluorescence. The images are representative of five independent experiments. Scale bars, 10 μm. (C) Quantification of transendocytosis events in (A). The percentage of colocalizations of the extracellular portions of Notch and Dll1 was determined, using the Colocalizer protocol under Icy software, as the ratio between HA- and VSV-positive spots over the total VSV spots. Equivalent acquisition settings were used within each experiment. The graphs represent mean ± SEM of five independent experiments (100 to 160 cells from 30 to 60 fields). Statistically significant differences are compared to the NT conditions (****P < 0.0001, **P < 0.01, *P < 0.05).

  • Fig. 3 ADAM10 acts downstream of DTX4 and transendocytosis in the Notch pathway.

    (A) Effect of knockdown of ADAM10 on transendocytosis, which was measured as in Fig. 2. The picture is representative of three independent experiments used for quantification (more than 60 cells from 30 fields) in the middle graph (mean ± SEM, statistical analysis by an unpaired t test; n.s., P > 0.05). The right graph shows ADAM10 expression normalized to HPRT expression, as quantified by qRT-PCR (unpaired t test, ****P < 0.0001). (B) Effect of knockdown of DTX4 and ADAM10 (A10) on Notch reporter gene induction. CSL-LUC activity was obtained as in Fig. 1, either after coculture with OP9-Dll1 cells or after culture on Dll-Fc–coated plates. DAPT or GI (at a suboptimal concentration) was applied all along the coculture. No stimulation was in the absence of Dll1. Data are means ± SEM of four experiments for Dll1-expressing cells (dark gray bars), analyzed by one-way ANOVA and Bonferroni post hoc test (****P < 0.0001, **P < 0.01). Data are means ± SEM of two experiments for coated Dll-Fc (light gray bars), each point in triplicate. (C) Effect of increasing doses of GI on Notch reporter gene induction. Curves giving the variation in luciferase activity (means ± SD of three independent experiments) as a function of the GI concentration, depending on the transfected siRNAs. 100% is control condition (siNT) without GI.

  • Fig. 4 N1 undergoes clustering and ubiquitylation upon activation by Dll-Fc.

    (A) Formation of clusters. N1/DTX4-Ch or U2OS-N1 cells were incubated with clustered Dll-Fc for 0 or 30 min at 37°C, fixed, permeabilized, and labeled with antibodies recognizing Fc (green), HA (on U2OS-N1 cells; red), and appropriate secondary antibodies. In N1/DTX4-Ch cells, red fluorescence is the DTX4-Cherry protein. Hoechst is in blue in merged images (first column). Insets are threefold enlargements of the boxed regions; arrows indicate colocalized vesicles. Scale bar, 10 μm. Data are representative of four independent experiments (>200 cells). (B) Ubiquitylation of N1. GFP/DTX4-Ch or N1-GFP/DTX4-Ch cells were transfected with the indicated siRNAs before activation with clustered Dll-Fc and GI for 30 min at 37°C. Whole-cell extracts or proteins that immunoprecipitated with the antibody recognizing GFP (IP GFP) were analyzed by Western blotting for the indicated proteins. White lane indicates that intervening lanes have been spliced out from the same blot. On the left are the molecular weights. The bracket indicates N1-derived ubiquitylated products; TM-IC is the mature form of N1. The table below gives the quantification of the ubiquitylated/total immunoprecipitated N1 reported to control conditions and the quantity of exogenous DTX4 (means from two experiments).

  • Fig. 5 Live imaging of Notch-GFP activation.

    (A) Activation in N1-GFP/DTX4-Ch cells. Sequential confocal images from movie S2 showing N1-GFP upon activation by clustered Dll-Fc over a 40-min period. Insets are threefold enlargements of the squared boxes at each time point, with Notch-GFP in green and DTX4-Cherry in red. Blue arrows indicate immobile clusters, and yellow arrows indicate mobile vesicles. The image is representative of five independent experiments, with more than 10 cells in randomly selected visual fields recorded in each experiment. (B) Tracking of mobile vesicles. Sequential confocal images (4 s apart) of N1-GFP/DTX4-Ch cells, 60 min after the addition of clustered Dll-Fc, extracted from movie S3. Scale bars, 10 μm. The tracking of four rapidly moving vesicles (four colors) is indicated in green, as well as their position on each image by arrowheads. The image is representative of five independent experiments, with more than 10 cells in randomly selected visual fields recorded in each experiment. Nucl, nucleus.

  • Fig. 6 ADAM10 is recruited to Notch after internalization.

    (A) Colocalization of N1-GFP, DTX4-Cherry, and ADAM10. N1-GFP/DTX4-Ch cells were incubated at 37°C with GI, clustered Dll-Fc, and an antibody recognizing ADAM10 extracellular domain for 15, 30, or 60 min, and then fixed, permeabilized, and incubated with fluorescent secondary antibody against ADAM10 antibody (blue in the merged pictures). Insets correspond to threefold enlargements of the boxed regions; yellow arrows indicate N1-GFP (green) and DTX4-Cherry (red) colocalized vesicles, devoid of ADAM10 staining, and white arrows point to vesicles with the three proteins. The data are representative of five independent experiments, with more than 10 cells in randomly selected visual fields observed in each. Scale bar, 10 μm. (B) Effect of Dynasore treatment. N1-GFP/DTX4-Ch cells were incubated as in (A) for 60 min, in the presence or absence of clustered Dll-Fc and Dynasore, and then fixed and analyzed as in (A). Insets are threefold enlargements of the boxed regions; white arrows point to vesicles with the three proteins. Images are representative of three independent experiments. (C) Quantification of N1-ADAM10 colocalization in activated N1-GFP/DTX4-Ch cells from (B). The percentage of N1-ADAM10 colocalization was determined as the ratio between GFP- and ADAM10-positive spots over the total GFP spots. Equal acquisition settings were used within each experiment used for the graphs (mean ± SEM from two independent experiments; 342, 245, and 143 cells for the three conditions). The results of the third experiment in (B) were not amalgamated with the other two because of different acquisition settings; however, the quantification results were similar. Statistically significant differences are compared to the NT conditions (one-way ANOVA and Bonferroni post hoc test, ****P < 0.0001). (D) Effect of knockdown of DTX4, Dyn2, or AP2 on N1-ADAM10 colocalization. U2OS-N1 cells were transfected with the indicated siRNAs and then incubated at 37°C with GI, clustered Dll-Fc, and a fluorescent antibody recognizing ADAM10 extracellular domain for 60 min before fixation and subsequent staining with fluorescent antibody recognizing HA. The percentage of Notch-ADAM10 colocalization was the ratio between HA- and ADAM10-positive spots over the total HA spots. Equivalent acquisition settings were used within each experiment. The graph represents mean with SEM from two independent experiments (>200 cells from 30 fields), and statistically significant differences are compared to the NT conditions (one-way ANOVA and Bonferroni post hoc test, ****P < 0.0001). Representative images are shown in fig. S7B. (E) Representative blotting of N1-GFP processing products. N1-GFP/DTX4-Ch cells were cultured for 7 hours on coated Dll-Fc in the presence or not of DAPT and Dynasore, as indicated. Cell extracts were analyzed by Western blot with antibodies recognizing GFP, S3 product (V1744), and tubulin. TM-IC (S1) is the nonactivated, mature form of Notch; S2 and S3 result from ADAM and γ-secretase processing, respectively. The apparent molecular masses are indicated. Below is the quantification of S2 abundance, as the ratio of S2 over (S1 + S2) in the presence of DAPT. The graph represents means with SEM from six independent experiments, with statistical analysis by unpaired t test (****P < 0.0001).

  • Fig. 7 ADAM10 recruitment to N1 in coculture.

    (A) N1 and DTX4 colocalization upon activation by OP9-Dll1 cells. N1-GFP/DTX4-Ch cells were cocultured without or with OP9-Dll1 (second panel) for 4 hours, fixed but not permeabilized (NP), and incubated with antibody recognizing VSV and secondary antibody coupled to Alexa Fluor 647 (blue). A star indicates the position of the closest murine Dll1-expressing cell of the N1-expressing cell (N). Insets on the right correspond to the boxed region; green arrows point to a probable endocytic vesicle. The second panel is representative of the small proportion of cells in which N1 has not been activated, where N1-GFP was still cell surface–localized together with Dll1-VSV. Images are representative of two independent experiments (>20 cells observed). (B) Colocalization of N1 and ADAM10. N1-GFP/DTX4-Ch cells were cocultured with OP9-Dll1 for 4 hours, incubated with the antibody recognizing ADAM10 for an additional 30 min, fixed, permeabilized (P) or not (NP), and labeled with secondary antibody coupled to Alexa Fluor 647 (red). Insets correspond to threefold enlargements of the boxed regions; white arrows indicate N1- and ADAM10-positive vesicles. Images are representative of two independent experiments (>40 cells observed). Scale bars, 10 μm. (C) Model depicting the early steps of Notch activation. Upon Dll1 binding (left), Notch (green) and DTX4 (blue) are clustered. DTX4-dependent ubiquitylation (orange) of Notch leads to the recruitment of the endocytic machinery, including Dyn2 and AP2 complex (middle), enabling bilateral endocytosis and eventually Notch heterodimer dissociation (right). ADAM10 is internalized separately and cleaves Notch in endocytic vesicles (right).

  • Table 1 Cell lines used for analysis.

    In some experiments, additional proteins were transiently expressed as indicated by a “+” and the name of the transiently expressed protein. The HA-tagged form of N1 (N1-HA) had the HA tag after the epidermal growth factor (EGF) repeat 22, which was detectable without permeabilization, but did not interfere with activation. The fusion proteins between DTX4 and GFP or Cherry had the fluorescent protein fused to the C terminus of DTX4. N1 fused to GFP had the GFP fused to the amino acid 2097 of N1. Dll1 fused to the VSV tag had the tag on the amino acid 45, which was exposed extracellularly. See fig. S1 for diagrams of the proteins.

    Cell line nameParent cellsStable expressionTransient expressionGene deletion
    OP9-Dll1OP9Dll1-VSVNoneNone
    U2OS-N1U2OSN1-HA+DTX4-VSV
    +DTX4-GFP
    +DTX4-Cherry
    +DynK44A
    +siDTX4
    +siDTX1
    +siDTX3
    +siAP2
    +siDyn2
    +siADAM10
    None
    N1/DTX4-KOU2OSN1-HANoneDTX4
    N1/DTX4-GFPU2OSN1-HA
    DTX4-GFP
    +DTX4-Cherry
    +Cherry
    None
    DTX4-ChU2OSDTX4-CherryNone
    N1-GFP/DTX4-ChU2OSN1-GFP
    DTX4-Cherry
    None
    GFP/DTX4-ChU2OSGFP
    DTX4-Cherry
    None
    N1/DTX4-ChU2OSN1-HA
    DTX4-Cherry
    None
    N1/CherryU2OSN1-HA
    Cherry
    None
    N1ΔE-GFP/DTX4-ChU2OSN1ΔE-GFP
    DTX4-Cherry
    None
  • Table 2 Summary of the live-cell imaging analysis.

    Live-cell imaging in three independent experiments was analyzed under Icy software, sequentially using Spot Detector, Spot Tracking, and Motion Profiler plugins. Tracks over 10 s of duration were quantified; immobile vesicles were defined as having a net displacement under 1 μm. Because the speed is not constant over the whole tracking of a given vesicle, we monitored the average speed and the maximal speed of the vesicles. Speeds are expressed as means from the total mobile vesicles. Note that because the movies are acquired using a fixed z-stack, vesicle movements can be underestimated when they become out of focus. The number of analyzed cells for each condition is indicated in the table. Dll-Fc stimulation is with clustered Dll-Fc. n.a., not applicable.

    CellsStimulationNumber of analyzed
    cells
    Percent immobile
    vesicles
    Average speed of
    mobile vesicles (μm/s)
    Maximum speed of mobile
    vesicles (μm/s)
    Movie
    N1/DTX4-GFP/3560.21.2Movie S1
    N1-GFP/DTX4-ChDll-Fcn.a.n.a.n.a.n.a.Movie S2
    N1-GFP/DTX4-ChDll-Fc23620.20.9Movie S3
    N1-GFP/DTX4-ChDll-Fcn.a.n.a.n.a.n.a.Movie S4
    N1-GFP/DTX4-ChDll-Fc + DAPT12610.21Movie S5
    N1-GFP/DTX4-ChDll-Fc + GI15670.21Movie S6
    N1/DTX4-GFPDll-Fc6370.31.3Movie S7

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/483/eaag2989/DC1

    Fig. S1. Diagram of the constructs used in this study.

    Fig. S2. Characterization of the cell lines used in this study.

    Fig. S3. Effect of DTX silencing and dynamin K44A overexpression on transendocytosis.

    Fig. S4. Specificity of GI on ADAM-mediated cleavage of N1.

    Fig. S5. Clustering of N1 with DTX4 at the cell surface upon Dll-Fc activation.

    Fig. S6. Ubiquitylation of N1 upon Dll-Fc–mediated activation.

    Fig. S7. Colocalization of N1 and ADAM10 upon Dll-Fc–mediated activation.

    Movie S1. DTX4-GFP in unstimulated cells.

    Movie S2. The first 40 min of N1 activation.

    Movie S3. N1 in moving vesicles after 60 min of activation.

    Movie S4. Tracking N1 from the cell surface into fast-moving vesicles.

    Movie S5. N1 in moving vesicles after 60 min of activation in the presence of DAPT.

    Movie S6. Notch in moving vesicles after 60 min of activation in the presence of GI.

    Movie S7. DTX4-GFP in stimulated cells.

  • Supplementary Materials for:

    Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing

    Patricia Chastagner, Eric Rubinstein, Christel Brou*

    *Corresponding author. Email: christel.brou{at}pasteur.fr

    This PDF file includes:

    • Fig. S1. Diagram of the constructs used in this study.
    • Fig. S2. Characterization of the cell lines used in this study.
    • Fig. S3. Effect of DTX silencing and dynamin K44A overexpression on transendocytosis.
    • Fig. S4. Specificity of GI on ADAM-mediated cleavage of N1.
    • Fig. S5. Clustering of N1 with DTX4 at the cell surface upon Dll-Fc activation.
    • Fig. S6. Ubiquitylation of N1 upon Dll-Fc–mediated activation.
    • Fig. S7. Colocalization of N1 and ADAM10 upon Dll-Fc–mediated activation.
    • Legends for movies S1 to S7

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.80 MB

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). DTX4-GFP in unstimulated cells.
    • Movie S2 (.mov format). The first 40 min of N1 activation.
    • Movie S3 (.mov format). N1 in moving vesicles after 60 min of activation.
    • Movie S4 (.mov format). Tracking N1 from the cell surface into fast-moving vesicles.
    • Movie S5 (.mov format). N1 in moving vesicles after 60 min of activation in the presence of DAPT.
    • Movie S6 (.mov format). Notch in moving vesicles after 60 min of activation in the presence of GI.
    • Movie S7 (.mov format). DTX4-GFP in stimulated cells.

    Citation: P. Chastagner, E. Rubinstein, C. Brou, Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing. Sci. Signal. 10, eaag2989 (2017).

    © 2017 American Association for the Advancement of Science

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