Research ArticleCell Biology

CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, β1 integrin recycling, and cell migration

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Science Signaling  30 Apr 2019:
Vol. 12, Issue 579, eaav5938
DOI: 10.1126/scisignal.aav5938
  • Fig. 1 Cell-ECM adhesion, cell spreading, and directional migration are substantially reduced in the absence of CD13.

    (A and B) Cell-ECM adhesion was assessed in MEFs [WT, CD13KO, and WT treated with CD13-blocking Ab SL13; (A)] and human epithelial cells [C33A cells expressing EV, HCD13, or a phospho-tyrosine mutant CD13 (Y6F); (B)] plated in the presence of FN for the indicated time periods. Cells attached to ECM were stained with crystal violet dye, and absorbance was measured at 595 nm. OD, optical density. (C and D) Cell spreading was assessed in MEFs (C) and C33A cells (D) plated on FN and left alone to spread for the indicated time periods. Cells were then stained with antibody against paxillin and imaged using confocal microscopy. Spreading was measured in cells of similar nuclear size as the area circumscribed by paxillin staining (total cell body) using Fiji software. (E and F) Cell migration was assessed using an in vitro scratch assay, in which monolayers of MEFs (E) or C33As (F) grown on FN for 6 hours (h) were allowed to migrate into the cleared space. For MEFs, the number of cells migrating into the wound area was counted, whereas for C33A cells, the distance traveled by the leading front was measured by a phase-contrast microscopy and Fiji software. Data are means ± SD of three independent experiments. **P < 0.01 and *P < 0.05 by two-tailed Student’s t test.

  • Fig. 2 Reduced β1 integrin recycling in MEFs lacking CD13.

    (A to C) Recycling of surface β1 integrin (green) was tracked in pulse-chase assays in WT and CD13KO MEFs grown on FN. Cell surface β1 integrin was tagged with unlabeled monoclonal antibody (mAb) clone 9EG7 for 30 min on ice, followed by incubation at 37oC (Pulse) for 1 hour to allow internalization. After pulse, cells were acid-stripped and washed to remove remaining surface antibody and chased for 2 or 4 hours in antibody-free medium (Chase/2h or Chase/4h). After chase, FITC-labeled secondary Ab was added to either fixed cells to detect recycled surface integrin or to permeabilized and fixed cells to detect both recycled and internalized 9EG7 by immunofluorescence analysis (A), quantification of fluorescence intensity of 9EG7 by Fiji software (B), and flow cytometry (C) in the presence of vehicle (V) or the recycling inhibitor primaquine (PQ; 1 μM). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 5 μm. (D and E) Biotin-labeled surface integrin was measured by capture enzyme-linked immunosorbent assay (ELISA) using 9EG7 mAb to determine % internalized 9EG7 (D) and % recycled 9EG7 (E) over time in WT and CD13KO MEFs. Data are means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test. A.U., arbitrary units; MFI, mean fluorescence intensity.

  • Fig. 3 β1 integrin recycle to the surface in epithelial cells expressing CD13 but not in the absence of CD13 or cells expressing CD13 phospho-tyrosine mutant.

    (A) In a pulse-chase assay, human β1 integrin was labeled with Ab clone 12G10, and expression of β1 integrin (green) was tracked in antibody-pulsed cells for 1 hour (P/1h) followed by chase in antibody-free medium for 2 or 4 hours (C/2h or C/4h) by immunofluorescence analysis. Cells were counterstained with DAPI (blue), fixed, and imaged by confocal microcopy. Scale bars, 5 μm. (B) Quantification of fluorescence intensity of 12G10 staining shown in (A) using Fiji software. (C) β1 integrin receptor surface recycling was measured in a pulse-chase assay with flow cytometric analysis of human β1 integrin Ab clone 12G10 in C33A cells expressing EV, HCD13, or Y6F in the presence or absence of recycling inhibitor primaquine (1 μM). (D) Equivalent level of CD13 expression in C33A expressing phospho-tyrosine mutant Y6F compared to HCD13 indicated by Western blot analysis. Images (A) are representative and data (B to D) are means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test.

  • Fig. 4 β1 integrin is directed to the lysosome for degradation in the CD13 deficient cells.

    (A) Cells were grown on FN for 0 to 60 min, and cells were fixed and stained for β1 integrin (9EG7; green) and one of three endosomal markers (red): Rab5 (early endosomal marker), Rab7 (late endosomal/lysosomal marker), and Rab11 (recycling endosomal marker). Cells were counterstained with DAPI (blue). Scale bars, 5 μm. Blots are representative of three independent experiments. (B) Images represented in (A) were quantified by MetaMorph software, and correlation coefficient (r) values of β1 integrin (9EG7) with endosomal markers determined by Pearson’s analysis are shown. *P < 0.05 by two-tailed Student’s t test. (C) Quantification of Rab5+, Rab7+, or Rab11+ puncta per cell for in WT and CD13KO MEFs. All cells in a field were counted, and five fields were counted for each genotype. (D) C33A cells expressing EV and HCD13 were treated with cycloheximide (CHX; 100 μg/ml) to inhibit new protein synthesis for the indicated time. Cell lysates were analyzed by immunoblotting for β1 integrin using the mAb 12G10. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. Blots are representative of three independent experiments. (E and F) Quantification of immunoblot analysis represented in (D) by ImageJ Pro as a ratio of 12G10 to GAPDH or 12G10 normalized to time = 0 hours. Data are means ± SD of three independent experiments. (G and H) Total RNA was extracted from C33A cells expressing EV or HCD13 treated with cycloheximide for the indicated time, and the abundance of ITGB1 (β1 integrin) and GAPDH mRNA was determined by Real-time PCR run on ethidium bromide–stained gels. Gels were analyzed by ImageJ Pro, and the ratio was plotted. Data in (B), (C), (E), (F), and (H) are means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test.

  • Fig. 5 CD13 is in a complex with IQGAP1 and active ARF6 in human epithelial cells.

    (A) Lysates from C33A cells expressing EV, HCD13, or Y6F were immunoprecipitated (IP) with biotinylated CD13 mAb or control immunoglobulin G (IgG) and probed for IQGAP1 and total ARF6 by immunoblot (IB) analysis. ARF6-GTP was detected by a pull-down assay of the immunoprecipitates using beads conjugated to the PBD of GGA3 for 1 hour at 4°C. (B) Lysates from T27N ARF6 dominant-negative mutant cells expressing EV, HCD13, or Y6F were immunoprecipitated with biotinylated CD13 mAb or control IgG and probed for IQGAP1, ARF6, or HA tag by immunoblot analysis. Active ARF6 was detected by pulldown with GGA3-conjugated beads from the immunoprecipitates. (C and D) ARF6 activity was measured in C33A cells expressing EV or HCD13 after cell-ECM adhesion over the indicated time. Using ARF6 PBD of the effector protein GGA3–conjugated beads, which specifically binds the GTP-bound form of ARF6, the subsequent pulldown of ARF6-GTP was quantified by immunoblot analysis using the ARF6-specific antibody. Blots (C) are representative of and quantified data (D) are means ± SD of three independent experiments. (E and F) β1 integrin receptor recycling was measured in a pulse-chase assay with β1 integrin Ab clone 12G10 [for C33A cells; (E)] or 9EG7 [for MEFs; (F)] in the presence of N-myristolated ARF6 inhibitor peptide. Serum-starved cells were treated with β1 integrin Ab at 4°C for 30 min, pulsed for 1 hour to induce internalization, acid-stripped, washed, treated with myr-ARF6 peptide (10 μM) for 30 min, and allowed to recycle at 37°C for 2 to 4 hours. Paraformaldehyde-fixed cells were stained with fluorescently conjugated secondary Ab, and MFI of surface β1 integrin was analyzed by flow cytometry. Data are means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test.

  • Fig. 6 CD13 recruits IQGAP1 at the leading edge in directional cell migration.

    (A and B) In a scratch assay, after injury on the monolayer by creating a scratch, C33A cells expressing HCD13 were allowed to migrate to the wound and fixed with 4% paraformaldehyde at the 6-hour time point. Cells were stained with phalloidin (red; left rows) or CD13 (red; right rows) and IQGAP1 (green) and imaged using confocal microcopy; magnified inset of CD13/IQGAP1-stained C33A-HCD13 cells is shown (B). Scale bars, 5 μm. DAPI (blue). (C) Quantification of the area of F-actin and IQGAP1 accumulation at the migrating front of the cell, represented in (A), normalized to total cell area by Fiji software. Five fields were counted for each genotype, and all cells in each field were measured. (D) Percentages of F-actin+, CD13+, and IQGAP1+ cells from (A) at the leading front were measured in each of five fields for each genotype. Data are means ± SD of three independent experiments. *P < 0.05 by two-tailed Student’s t test.

  • Fig. 7 CD13 phosphorylation is necessary for maintenance of ARF6 activation.

    (A) C33A cells expressing EV, HCD13, or Y6F were treated with vehicle (Veh) or AlF (50 μM) for 30 min, and membrane fractions were purified from total cell lysates. Active ARF6-GTP in the fractions was measured by pull-down assay and normalized to total ARF6. Validation of the membrane fraction was verified by expression of plasma membrane marker E-cadherin. (B) Plots depict quantification of immunoblot analysis of ARF6-GTP, CD13, and IQGAP1 normalized to E-cadherin in the plasma membrane. Data are means ± SD of three independent experiments. *P < 0.05 and **P < 0.01 by two-tailed Student’s t test.

  • Fig. 8 Active CD13 is necessary for interaction and membrane localization of ARF6 GEF EFA6.

    (A and B) Immunoblot analysis of lysates from C33A cells expressing EV, HCD13 or Y6F, immunoprecipitated with CD13 mAb or control IgG and probed (IB) for EFA6 and CD13 (A) or ARNO and CD13 (B). (C and D) Representative images (C) and quantitative analysis (D) of the number (per field assessed) of EFA6+ puncta (green) at the leading edge of cells expressing EV, HCD13 (red, top), or Y6F (red, bottom). Scale bar, 5 μm. Data are means ± SD of three independent experiments. *P < 0.05 by two-tailed Student’s t test.

  • Fig. 9 Schematic of proposed mechanism.

    (A) In WT cells, phospho-CD13 and β1 integrin internalize into early endosomes, sort to recycling endosomes, and return to the cell membrane, enabling cell-ECM adhesion and migration. (B) However, in cells lacking CD13 or expressing an inactive CD13 mutant, whereas β1 integrin internalizes into early endosomes, it aberrantly traffics to Rab7+ lysosomes and is ultimately degraded. Mechanistically, CD13 must be present in a complex containing the scaffolding protein IQGAP1, active ARF6, its GEF EFA6, and β1 integrin at the plasma membrane to allow proper β1 integrin recycling and cell migration to proceed. In the absence of CD13, no active ARF6 is detected in the plasma membrane, and IQGAP1 is not recruited to the migrating front, thereby diminishing cell adhesion, spreading, and migration.

Supplementary Materials

  • stke.sciencemag.org/cgi/content/full/12/579/eaav5938/DC1

    Fig. S1. CD13 is necessary for cell spreading and migration.

    Fig. S2. Illustration of the β1 integrin mAb-induced pulse-chase assay.

    Fig. S3. Equivalent surface abundance of MB1.2 in WT and CD13KO MEFs.

    Fig. S4. Impaired cell adhesion, migration, and β1 integrin recycling in CD13 CRISPR human KS cells.

    Fig. S5. Aluminum fluoride treatment led to actin-rich protrusions in C33A-HCD13 cells.

  • This PDF file includes:

    • Fig. S1. CD13 is necessary for cell spreading and migration.
    • Fig. S2. Illustration of the β1 integrin mAb-induced pulse-chase assay.
    • Fig. S3. Equivalent surface abundance of MB1.2 in WT and CD13KO MEFs.
    • Fig. S4. Impaired cell adhesion, migration, and β1 integrin recycling in CD13 CRISPR human KS cells.
    • Fig. S5. Aluminum fluoride treatment led to actin-rich protrusions in C33A-HCD13 cells.

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