Research ArticleCell Biology

The depalmitoylase APT1 directs the asymmetric partitioning of Notch and Wnt signaling during cell division

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Science Signaling  02 Jan 2018:
Vol. 11, Issue 511, eaam8705
DOI: 10.1126/scisignal.aam8705
  • Fig. 1 Activity of the depalmitoylating enzyme APT1 is required for asymmetric localization of Numb and β-catenin.

    (A and B) Images of dividing MDA-MB-231 cells stained to show endogenous Numb (A) and β-catenin (B) in red, acetylated tubulin in green, and nuclei in blue. Arrowheads indicate asymmetric localization of Numb and β-catenin. Scale bars, 15 μm. (C and D) Distribution dot plots showing the difference in mean fluorescence pixel intensity of endogenous Numb (C) and β-catenin (D) across dividing MDA-MB-231 cells. The distribution of the percentage differences of all quantified cells was plotted, and cells with a difference of >20% (black dotted line) were scored as asymmetric. n = 508 to 582 cells scored for each experimental group. Each dot represents a single cell. Asterisks indicate statistically significant differences between the indicated groups. (E and F) Quantification of dividing MDA-MB-231 cells showing asymmetric Numb (E) and β-catenin (F) localization after treatment with Palmostatin B (PalmB) or dimethyl sulfoxide (DMSO). (G and H) Quantification of the number of dividing MDA-MB-231cells showing asymmetric Numb (G) and β-catenin (H) localization when APT1 was knocked down with shAPT1 and when wild-type APT1 (APT1WT) or the catalytically inactive APT1S119A mutant was coexpressed with shAPT1. Cells expressing a scrambled (Scr) short hairpin RNA (shRNA) sequence were used as a negative control for APT1 knockdown, and cells expressing the empty vector were used as a negative control for the APT1 rescue experiments. (I and J) Quantification of the number of dividing MDA-MB-231 cells showing asymmetric Numb (I) and β-catenin (J) localization when DHHC20 was knocked down with shDHHC20. (K to M) Immunoblots showing biotin-labeled Numb (K), β-catenin (L), and ERK (M) in MDA-MB-231 cell lysates after acyl-biotin exchange (ABE) assays and pulldown on streptavidin beads (PD). Cells were grown in the presence of either PalmB or vehicle control (DMSO). Input lanes show cell lysates before pulldown. Samples without hydroxylamine (−HAM) were negative controls for the ABE reactions. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, t test (E and F) or analysis of variance (ANOVA) (C, D, and G to J). Error bars indicate SD.

  • Fig. 2 Palmitoylation and APT1 activity drive Numb localization.

    (A) Sequence comparison of the N terminus of Numb from fruit fly (D. melanogaster), zebrafish (Danio rerio), mouse (Mus musculus), and human (Homo sapiens). The phosphotyrosine domain (PTB) is highlighted in green, and the putative palmitoylated cysteines are highlighted in yellow. Conserved residues are indicated by an asterisk (*). (B) Immunoblot showing transgenically expressed wild-type Numb (NumbWT) or the NumbAAA mutant and endogenous β-catenin in U2 OS cell lysates after purification of palmitoylated proteins. Cells were metabolically labeled with palmitic acid azide or treated with DMSO (vehicle control), and then lysates were subjected to click chemistry to convert the palmitic acid moiety to biotin, pulled down on streptavidin beads, and used for immunoblotting. Input was taken from cell lysates before pulldown. (C) Time-lapse images of dividing U2 OS cells coexpressing NumbWT-YFP (yellow), APT1WT-CFP (blue), and mCherry–Histone H2B (red). Fluorescence pixel intensity was measured along the division axis (dashed line), and the corresponding pixel values of Numb (yellow line) and APT1 (blue line) along the division axis were plotted on graphs. Red arrowheads on images and graphs indicate the peak Numb and APT1 pixel intensity at the membrane or cytokinetic midbody. Time is shown in minutes (min). a.u., arbitrary units. Scale bar, 15 μm. (D) Quantification of the number of dividing U2 OS cells showing asymmetric localization of NumbWT-YFP (black bar) and NumbAAA-YFP (gray bar) when each was coexpressed with shAPT1. Cells expressing a scrambled (Scr) shRNA sequence were used as a negative control for APT1 knockdown. (E and F) Time-lapse images of dividing U2 OS cells coexpressing either APT1WT-CFP (E) or APT1S119A-CFP (F) with mCherry–Histone H2B (red). Fluorescence pixel intensity was quantified as in (C). Scale bars, 15 μm. (G) Quantification of the number of dividing U2 OS cells showing asymmetric APT1WT-CFP or APT1S119A-CFP localization. Cells expressing an empty green fluorescent protein (GFP) plasmid (Vector) were used as a negative control. n = 102 to 143 cells scored for each group from three independent experiments. *P< 0.05 and **P < 0.01, t test and ANOVA. Error bars indicate SD.

  • Fig. 3 Palmitoylating enzymes and depalmitoylating enzymes are asymmetrically partitioned during cell division.

    (A) Images of dividing MDA-MB-231 cells stained to show endogenous APT1 (red), DHHC20 (green), and nuclei (blue). (B and C) Images of dividing MDA-MB-231 cells stained to show endogenous APT1 (B) or DHHC20 (C), caveolin, and nuclei. Asymmetric localization is indicated by arrowheads (A to C). (D) Images of dividing MDA-MB-231 cells treated with PalmB or DMSO and stained to show biotin-labeled palmitoylated proteins (red), acetylated tubulin (green), and nuclei (blue) by ABE immunofluorescence. Samples without HAM (−HAM) were negative controls for the ABE reaction. (E) Distribution dot plots showing the difference in mean fluorescence pixel intensity of biotin-labeled palmitoylated proteins. The distribution of the percentage differences of all quantified cells was plotted, and cells with a difference of >20% (dotted line) were scored as asymmetric. n = 91 to 101 cells scored for each experimental group. Each dot represents a single cell. Asterisks indicate statistically significant differences between the indicated groups. (F) Quantification of the number of dividing MDA-MB-231 cells showing asymmetric palmitoylated proteins after treatment with PalmB or DMSO control. (G) Confocal images of ABE immunofluorescence in nondividing MDA-MB-231 cells showing all palmitoylated proteins (green), APT1 or DHHC20 (red), and nuclei (blue) by ABE immunofluorescence. White dotted boxes indicated magnified areas shown directly below (zoom). Samples without HAM (−HAM) were negative controls for the ABE reaction. Scale bars (including zoom), 15 μm. **P < 0.01, ***P < 0.001, and ****P < 0.0001, ANOVA. Error bars indicate SD.

  • Fig. 4 The reciprocal interaction between APT1 and CDC42 establishes and maintains asymmetric protein partitioning during cell division.

    (A and B) Quantification of the number of dividing MDA-MB-231 cells showing asymmetric localization of APT1 when CDC42 was knocked down with shCDC42 (A) or PARD3 was knocked down with shPARD3 (B). Cells expressing a scrambled (Scr) shRNA sequence were used as a negative control for knockdown. (C and D) Quantification of the number of dividing U2 OS cells showing asymmetric APT1WT-CFP or APT1S119A-CFP in cells expressing constitutively active (CDC42V12 and CDC42F28L) or dominant-negative (CDC42N17) forms of CDC42 (C) or expressing isoforms of CDC42 that are prenylated (CDC42Pren) or both palmitoylated and prenylated (CDC42Palm) (D). (E and F) Time-lapse images of dividing U2 OS cells coexpressing CDC42Palm-YFP (yellow) and either APT1WT-CFP (E) or APT1S119A-CFP (F) (blue) and mCherry–Histone H2B (red). Overlapping CFP and YFP signal in the merge appears green. Fluorescence pixel intensity was measured along the division axis (dashed line), and the corresponding pixel values of CDC42 (yellow line) and APT1 (blue line) along the division axis were plotted. Red arrowheads on images and corresponding graphs mark the peak asymmetric CDC42 and APT1 accumulation at the membrane or cytokinetic midbody. Time is shown in minutes (min). Scale bars, 15 μm. n = 152 to 288 cells scored for each group from four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, t test (A and B) and ANOVA (C and D). Error bars indicate SD.

  • Fig. 5 APT1 restricts Wnt and Notch transcriptional activity to one daughter cell.

    (A and B) Images of cytokinetic MDA-MB-231 cells stained to show the expression of the pGF1-Notch GFP reporter (A) or pGF-1 TCF/Lef1 GFP reporter (B) (red), acetylated tubulin (green), and nuclei (blue). Arrowheads indicate asymmetric localization. Scale bars, 15 μm. (C and D) Distribution dot plots showing the difference in mean fluorescence pixel intensity of pGF1-Notch reporter (C) or pGF1-TCF/Lef1 reporter (D) across dividing cells. Cells expressing an empty pGF1-mCMV GFP reporter were used as a negative control for the reporters. The distribution of the percentage differences of all quantified cells was plotted, and cells with a difference of >20% (dotted line) were scored as asymmetric. n = 784 to 822 cells scored for each experimental group. Each dot represents a single cell. Asterisks indicate statistically significant differences between the indicated groups. (E and F) Quantification of dividing MDA-MB-231 cells showing asymmetric localization of pGF1-Notch GFP reporter (E) or pGF1-TCF/Lef1 GFP reporter (F) (black bars) after treatment with PalmB or DMSO vehicle control. Cells expressing an empty pGF1-mCMV reporter (gray bars) were used as a negative control for reporter expression. (G and H) Quantification of the number of dividing MDA-MB-231 cells showing asymmetric localization of the pGF1-Notch GFP reporter (G) or pGF1-TCF/Lef1 GFP reporter (H) (black bars) when coexpressed with shAPT1, shCDC42, shAPT1 and shCDC42 (DKD), and shDHHC20. Cells expressing an empty pGF1-mCMV GFP reporter (gray bars) were used as a negative control for reporter expression, and cells expressing a scrambled (Scr) shRNA sequence were used as a negative control for knockdown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001, t test (between reporters and pGF1-mCMV) or ANOVA (C to H). Error bars indicate SD.

  • Fig. 6 Altering APT1 expression changes β-catenin and Notch gene signatures in MDA-MB-231 cells.

    (A to C) Gene set enrichment analysis (GSEA) of a top-scoring β-catenin overexpression transcriptional signature in MDA-MB-231 cells when APT1 was knocked down and compared against control cells [false discovery rate (FDR) q value, 0.073] (A), a Notch inhibition signature when APT1WT was overexpressed and compared against control cells (FDR q value, 0.302) (B), and a mammary stem cell signature when APT1WT was overexpressed and compared against shAPT1 cells (FDR q value, 0.0001) (C). (D) Heat map of leading-edge genes obtained from the mammary stem cell signature shown in (C). Data are grouped by APT1 knockdown (kd) cells, APT1WT-overexpressing (oe) cells, and control (wt) cells. (E) Images of cytokinetic MDA-MB-231 cells stained to show the pGF1-SRR2 GFP reporter (red), acetylated tubulin (green), and nuclei (blue). Arrowheads indicate asymmetric localization of pGF1-SRR2. Scale bar, 15 μm. (F) Distribution dot plots showing the difference in mean fluorescence pixel intensity of the pGF1-SRR2 reporter across dividing MDA-MB-231 cells. Cells expressing an empty pGF1-mCMV GFP reporter were used as a negative control for the reporters. The distribution of the percentage differences of all quantified cells was plotted, and cells with a difference of >20% (dotted line) were scored as asymmetric. n = 959 cells scored for the experimental group. Each dot represents a single cell. Asterisks indicate statistically significant differences between the indicated groups. (G) Quantification of dividing MDA-MB-231 cells showing asymmetric localization of the pGF1-SRR2 GFP reporter after treatment with PalmB or DMSO control (black bars). Cells expressing an empty pGF1-mCMV reporter (gray bars) were used as a negative control for reporter expression. (H) Quantification of the number of dividing MDA-MB-231 cells showing asymmetric localization of pGF1-SRR2 GFP reporter (black bars) when coexpressed with shAPT1, shCDC42, shAPT1 and shCDC42 [double knockdown (DKD)], or shDHHC20. Cells expressing an empty pGF1-mCMV GFP reporter (gray bars) were used as a negative control for reporter expression, and cells expressing a scrambled (Scr) shRNA sequence were used as a negative control for knockdown conditions. *P < 0.05, ***P < 0.001, and ****P < 0.0001, t test (between reporters and pGF1-mCMV) or ANOVA (H). Error bars indicate SD.

  • Fig. 7 APT1 and CDC42 maintain tumorigenic cell populations in MDA-MB-231 colonies.

    (A and B) Quantification of the average number of colonies formed from MDA-MB-231 shAPT1 cells (A) or shCDC42 cells (B) over three serial replatings. Cells expressing a scrambled (Scr) shRNA sequence were used as a negative control for knockdown. Each graph shows means taken from three independent experiments. *P < 0.05 and ***P < 0.001, as measured by t test. Error bars indicate SEM. (C) Representative flow cytometry analysis showing gating strategy of CD44+/CD24lo cells (red box) in cells dissociated from colonies or adherent. The population was gated off of ALDH+ cells, as shown in fig. S7. The percentages inside the red box indicate the relative proportion of the CD44+/CD24lo cell population. Cells were stained with phycoerythrin (PE)–conjugated anti-CD24 (CD24-PE) (x axis) and allophycocyanin (APC)–conjugated CD44-APC (y axis). Flow cytometry plots are representative of results from six independent experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/511/eaam8705/DC1

    Fig. S1. Scoring method for determining asymmetric divisions.

    Fig. S2. DHHC20 and APT1 localization in MDA-MB-231 cells.

    Fig. S3. Effect of CDC42 and PARD3 knockdown on asymmetric Numb and β-catenin localization.

    Fig. S4. CDC42 activity and lipidation promote asymmetric APT1, Numb, and β-catenin localization.

    Fig. S5. Validation of RNA-seq and additional GSEA analyses relating to Fig. 6.

    Fig. S6. Staining of asymmetric APT1 in mouse embryonic stem cell.

    Fig. S7. Colony counts, growth curves, and reporter expression relating to Fig. 7.

    Fig. S8. Gating scheme for ALDH+ cells on dissociated colonies or adherent cells.

    Table S1. Excel spreadsheet of RNA-seq annotated genes.

    Movie S1. Single channel of a U2 OS cell ectopically expressing APT1WT-CFP (blue).

    Movie S2. Single channel of a U2 OS cell from movie S1 ectopically expressing NumbWT-YFP (yellow).

    Movie S3. Merge of movie S1 of a U2 OS cell ectopically expressing APT1WT-CFP (blue), NumbWT-YFP (yellow), and mCherry–Histone H2B (red).

    Movie S4. Single channel of a U2 OS cell ectopically expressing APT1WT-CFP (blue).

    Movie S5. Merge of movie S4 of a U2 OS cell ectopically expressing APT1WT-CFP (blue) and mCherry–Histone H2B (red).

    Movie S6. Single channel of a U2 OS cell ectopically expressing APT1S119A-CFP (blue).

    Movie S7. Merge of movie S6 of a U2 OS cell ectopically expressing APT1S119A-CFP (blue) and mCherry–Histone H2B (red).

    Movie S8. Single channel of a U2 OS cell ectopically expressing APT1WT-CFP (blue).

    Movie S9. Single channel of a U2 OS cell ectopically expressing YFP-CDC42Palm (yellow).

    Movie S10. Merge of movie S1 of a U2 OS cell ectopically expressing APT1WT-CFP (blue), YFP-CDC42Palm (yellow), and mCherry–Histone H2B (red).

  • Supplementary Materials for:

    The depalmitoylase APT1 directs the asymmetric partitioning of Notch and Wnt signaling during cell division

    Ewa Stypulkowski, Irfan A. Asangani, Eric S. Witze*

    *Corresponding author. Email: ewitze{at}exchange.upenn.edu

    This PDF file includes:

    • Fig. S1. Scoring method for determining asymmetric divisions.
    • Fig. S2. DHHC20 and APT1 localization in MDA-MB-231 cells.
    • Fig. S3. Effect of CDC42 and PARD3 knockdown on asymmetric Numb and β-catenin localization.
    • Fig. S4. CDC42 activity and lipidation promote asymmetric APT1, Numb, and β-catenin localization.
    • Fig. S5. Validation of RNA-seq and additional GSEA analyses relating to Fig. 6.
    • Fig. S6. Staining of asymmetric APT1 in mouse embryonic stem cell.
    • Fig. S7. Colony counts, growth curves, and reporter expression relating to Fig. 7.
    • Fig. S8. Gating scheme for ALDH+ cells on dissociated colonies or adherent cells.
    • Legend for table S1
    • Legends for movies S1 to S10

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.29 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. (Microsoft Excel format)Excel spreadsheet of RNA-seq annotated genes.
    • Movie S1 (.avi format). Single channel of a U2 OS cell ectopically expressing APT1WT-CFP (blue).
    • Movie S2 (.avi format). Single channel of a U2 OS cell from movie S1 ectopically expressing NumbWT-YFP (yellow).
    • Movie S3 (.avi format). Merge of movie S1 of a U2 OS cell ectopically expressing APT1WT-CFP (blue), NumbWT-YFP (yellow), and mCherry–Histone H2B (red).
    • Movie S4 (.avi format). Single channel of a U2 OS cell ectopically expressing APT1WT-CFP (blue).
    • Movie S5 (.avi format). Merge of movie S4 of a U2 OS cell ectopically expressing APT1WT-CFP (blue) and mCherry–Histone H2B (red).
    • Movie S6 (.avi format). Single channel of a U2 OS cell ectopically expressing APT1S119A-CFP (blue).
    • Movie S7 (.avi format). Merge of movie S6 of a U2 OS cell ectopically expressing APT1S119A-CFP (blue) and mCherry–Histone H2B (red).
    • Movie S8 (.avi format). Single channel of a U2 OS cell ectopically expressing APT1WT-CFP (blue).
    • Movie S9 (.avi format). Single channel of a U2 OS cell ectopically expressing YFP-CDC42Palm (yellow).
    • Movie S10 (.avi format). Merge of movie S1 of a U2 OS cell ectopically expressing APT1WT-CFP (blue), YFP-CDC42Palm (yellow), and mCherry–Histone H2B (red).

    Citation: E. Stypulkowski, I. A. Asangani, E. S. Witze, The depalmitoylase APT1 directs the asymmetric partitioning of Notch and Wnt signaling during cell division. Sci. Signal. 11, eaam8705 (2018).

    © 2018 American Association for the Advancement of Science

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