An Arf6- and caveolae-dependent pathway links hemidesmosome remodeling and mechanoresponse

Abstract

Hemidesmosomes (HDs) are epithelial-specific cell-matrix adhesions that stably anchor the intracellular keratin network to the extracellular matrix. Although their main role is to protect the epithelial sheet from external mechanical strain, how HDs respond to mechanical stress remains poorly understood. Here we identify a pathway essential for HD remodeling and outline its role with respect to α6β4 integrin recycling. We find that α6β4 integrin chains localize to the plasma membrane, caveolae, and ADP-ribosylation factor-6+ (Arf6+) endocytic compartments. Based on fluorescence recovery after photobleaching and endocytosis assays, integrin recycling between both sites requires the small GTPase Arf6 but neither caveolin1 (Cav1) nor Cavin1. Strikingly, when keratinocytes are stretched or hypo-osmotically shocked, α6β4 integrin accumulates at cell edges, whereas Cav1 disappears from it. This process, which is isotropic relative to the orientation of stretch, depends on Arf6, Cav1, and Cavin1. We propose that mechanically induced HD growth involves the isotropic flattening of caveolae (known for their mechanical buffering role) associated with integrin diffusion and turnover.

FIGURE 1:

Hemidesmosome integrins are located in Arf6-intracellular compartments containing Cav1. (A) Confocal plane of HaCaT cells at the level of the basal membrane (BM) or +1 µm above stained for ITGA6 and ITGB4. Scale bar 10 µm. (A′) Magnification of the area boxed in A. Scale bar = 1 µm. (B) Quantification of the various markers found at ITGA6-containing intracellular compartments. Number of ICs Arf6 = 46, EEA1 = 16, Rab11 = 22, Cav1 = 43 cells from three independent experiments. (C–G) Close-up on ICs in a confocal section +1 µm above the basal plasma membrane of cells coimmunostained for ITGA6 and (C) expressing EGFP-Arf6(wt); (D) for endogenous Rab11; (E) expressing RFP-Cav1(wt); (F) for endogenous EEA1; and (G) for endogenous Cav1 and expressing EGFP-Arf6(wt). Scale bar = 1 µm. (H, I) Immunoelectron micrographs of HaCaT cells after staining with antibodies against ITGA6 (15-nm gold beads) and Cav1 (10-nm gold beads) showing colocalization of ITGA6 and Cav1 in vesicles 500 nm above the plasma membrane (arrowhead). The dotted square in H shows the magnified region in I. Scale bar = 50 nm. (J) Quantification of the localization of ITGA6 and Cav1 in intracellular vesicles from EM micrographs (177 vesicles from 30 cells) from three independent experiments. Fischer test: ITGA6+ vs. ITGA6– in Cav1+ or Cav1– ICs.

FIGURE 2:

ITGA6 localizes close to caveolar and noncaveolar Cav1 at the basal membrane. (A) HaCaT cell immunostained for ITGA6 and Cav1. Left panel: TIRF image, scale bar = 10 µm; right panel: GSD-TIRF image (pixel size = 20 nm), scale bar = 1 µm. Arrowheads highlight colocalization. (B) Mean correlation of both channels measured in single GSD images for each pair of markers. Values are normalized to the mean correlation coefficient of ITGA6/ITGB4 GSD images used as positive control and compared with the correlation of ITGA6 at focal adhesions marked by βPIX or PAK1 taken as a negative control from a least four independent experiments. Mann-Whitney tests: R(ITGA6/Cav1) vs. R(ITGA6/βPIX(FA)), R(ITGA6/PAK1(FA)), or R (ITGA6/Cav1 randomized). (C) Immunoelectron micrographs of the plasma membrane of HaCaT cells after staining with antibodies against ITGA6 (15-nm gold beads) and Cav1 (10-nm gold beads). Scale bar = 50 nm. (D) Quantification of the localization of ITGA6 relative to caveolar and noncaveolar Cav1 (noted caveolae and membrane, respectively) from EM micrographs (N = 30) from three independent experiments. Fischer test: d < 100 nm vs. 100 < d < 500 nm in caveolae or at the membrane.

FIGURE 3:

Arf6 is essential for proper HD organization. (A, B) Confocal images of the basal membrane and associated quantification of the amount of HDs (from ITGA6 staining) at the basal membrane of cells transfected with control or (A, B) or Arf6 siRNAs and then immunostained for ITGA6 and CK14 (A). The Rescue column (B) corresponds to cells transfected with Arf6 siRNAs plus a plasmid encoding wild-type RFP-Arf6 plasmid mutated to be Arf6 siRNA-resistant (Rescue). Scale bar = 10 µm. Data are from three independent experiments; number of cells = 40, 46, and 45. Student’s t tests: siCTL vs. siArf6 or Rescue. (C) Confocal images of the basal membrane of cells expressing EGFP-Arf6(wt) or (T27N) immunostained for ITGA6. Scale bar = 10 µm. (D) Quantification of the amount of HDs (from ITGA6 staining) at the basal membrane of cells expressing mutant forms of Arf6 from three independent experiments; number of cells = 37, 42, 61, and 63. Student’s t tests: Arf6(wt) vs. NT, Arf6(T27N). or Arf6(Q67L). (E) Confocal images of the basal membrane of cells expressing EGFP-Arf6(Q67L) immunostained for ITGA6 and CK14. Scale bar = 10 µm. (E′) Magnification of the boxed region in E (magnification ×3). Arrowheads highlight the colocalization of Arf6 (red) and ITGA6 (green) at the basal plasma membrane. (F) Quantification of the presence of Arf6 in ITGA6-enriched intracellular compartments in cells expressing mutant forms of Arf6 from three independent experiments; number of cells = 45, 44, and 46. Student’s t tests: Arf6(wt) vs. Arf6(T27N) or Arf6(Q67L).

FIGURE 4:

Caveolae proteins are essential for proper HD organization. (A–D) Confocal images of the basal membrane and associated quantification of the amount of HDs (from ITGA6 staining) at the basal membrane of cells transfected with control (A, B) Cav1 siRNAs or (C, D) Cavin1 siRNAs then immunostained for ITGA6 and CK14 (A, C). The Rescue column (B) corresponds to cells transfected with Cav1 siRNAs plus a plasmid encoding wild-type RFP-Cav1 mutated to be Cav1 siRNA resistant. Scale bar = 10 µm. Data are from three independent experiments with (B) number of cells = 50, 65, and 46 and (D) N = 31 and 31. Student’s t tests: siCTL vs. (B) siCav1 or Rescue and (D) vs. siCavin1. (E) Confocal images of the basal membrane of cells expressing wt or mutant forms of RFP-Cav1 immunostained for ITGA6 and CK14. Scale bar = 10 µm. The white arrowhead highlights integrin accumulation at the edge of a Cav1(Y14D) expressing cell. (F) Quantification of the amount of HDs (from ITGA6 staining) at the basal membrane of cells expressing Cav1 mutant forms from three independent experiments; number of cells = 29, 59, 58, and 59. Student’s t tests: Cav1(wt) vs. NT, Cav1(Y14F), or Cav1(Y14D). (G) Quantification of the presence of Arf6 in ITGA6-enriched intracellular compartments in cells expressing mutant forms of Cav1 from three independent experiments; number of cells = 27, 38, 33. Student’s t tests: Cav1(wt) vs. Cav1(Y14F) or Cav1(Y14D).

FIGURE 5:

Arf6-dependent traffic and Cav1 regulate HD integrin dynamics. Time-lapse acquisition of EGFP-ITGB4 (green) and (A) mCherry-Arf6(wt) (red) extracted from Supplemental Video 2 or (B) RFP-Cav1(wt) (red) extracted from Supplemental Video 3 (see arrowheads). Time is shown in seconds. Scale bar = 1 µm. (C, D) FRAP analysis of EGFP-ITGB4 in HDs of cells expressing (C) m-Cherry-Arf6 or (D) RFP-Cav1 constructs (MF: mobile fraction) from three independent experiments, number of cells (C) N = 24, 16, and 17 (D) and 16, 18, and 20. Mann-Whitney tests: (C) Arf6(wt) vs. Arf6(T27N) or Arf6(Q67L); (D) Cav1(wt) vs. Cav1(Y14F) or Cav1(Y14D). See Supplemental Figure S5.

FIGURE 6:

Microtubule-dependent traffic is required for HD maintenance. (A) Confocal plane of the basal membrane of a cell stained for ITGA6 (green) and microtubules (MTs, red) and close-ups of the boxed region (arrowheads highlight colocalization. Scale bar = 10 µm. (B) Normalized signal along the dashed line in the HD channel (ITGA6 staining, green) and MT channel (α-tubulin, red). (C) Cells were incubated with vehicle (CTL) or nocodazole (Noco) before fixation and immunostained for ITGA6. Scale bar = 10 µm. Arrows highlight the differences observed. (D) Quantification of the amount of HDs (from ITGA6 staining) at the basal membrane of cells treated with vehicle or the MT depolymerizing drug nocodazole from three independent experiments; number of cells N = 15, 16, and 11. Student’s t tests: CTL vs. Noco 0.05 µM or Noco 20 µM.

FIGURE 7:

Hemidesmosome integrin endocytosis requires Arf6. (A, B) Antibody uptake assay after incubating HaCaT cells with the monoclonal antibody (mAb) GoH3 (anti-ITGA6) on ice and shifting cells to 37°C for 1 h. (A) Representative confocal images of ICs +1 µm above HDs in siRNA-transfected cells showing uptaken anti-ITGA6 antibody bound to the extracellular domain of ITGA6 (GoH3 – left panel) and total ITGB4 revealed by immunostaining of the endogenous protein after fixation (right panel). Scale bar = 1 µm. (B) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following siRNA transfection from three independent experiments; number of ICs siCTL = 30/262, siArf6 = 27/185, siCav1 = 30/218, and siCavin1 = 25/196 cells/ICs. Student’s t tests: siCTL vs. siArf6, siCav1, or siCavin1. (C) Amount of endocytosed ITGA6 and ITGB4 integrins, following cell surface biotinylation at 4°C followed by internalization at 37°C for 30 min. The amount of biotinylated α6 and β4 integrins was measured by capture ELISA with peroxidase-conjugated streptavidin, corrected for the total amount of ITGA6 or ITGB4 measured by Western blotting; ratios are expressed relative to control cells. (D) ITGA6 and ITGB4 protein levels assessed by Western blotting in HaCaT cells transfected with control or anti-Arf6 or Cav1 siRNAs. (C, D) All data from four independent experiments. Student’s t tests: siCTL vs. siArf6 or siCav1. (E) Quantification of the area covered by HDs at the basal membrane (from ITGA6 staining) of cells treated with vehicle or the dynamin inhibitor Dynasore (Dyna) from three independent experiments; number of cells = 31 and 36. Studen’ts t test: CTL vs. Dyna. (F) Ratio of internalized GoH3-ITGA6/total ITGB4 measured in several ICs of individual cells for each condition following treatment with vehicle or Dynasore from three independent experiments; number of ICs CTL = 18/105, Dyna = 18/131 cells/ICs. Student’s t test: CTL vs. Dyna. (G) FRAP analysis of EGFP-ITGB4 in HDs of cells treated with vehicle or Dynasore from four independent experiments; number of cells = 33 and 33. Mann-Whitney test: CTL vs. Dyna.

FIGURE 8:

Hemidesmosomes respond to external mechanical stimuli. (A) Scheme of the custom-built stretcher used. The motor motion (1) induces the displacement of the mobile anchor (2) driving the stretching of the PDMS membrane (3). (B, C) Quantification at the basal plasma membrane of the area (yellow), density (red), and total intensity (purple) changes of (B) EGFP-ITGB4 or (C) RFP-Cav1(wt) without strain (left), before and immediately after a 10% uniaxial strain (middle), or after 30 min of a 10% uniaxial strain (right), from 3–5 independent experiments; number of cells = 20, 12, and 12. Mann-Whitney tests: (no stretch) vs. (t0 no strain vs. t0 strain 10%) or (10% strain 30 min). (D–F) Quantification of the area (D) and total intensity (E) and density (F) of HDs (green) and Cav1 (red) in cells expressing EGFP-ITGB4 and RFP-Cav1(wt) after 5, 15, and 30 min of stretch with a 10% uniaxial strain. All values are normalized to time 0. Data are from three to five independent experiments; number of cells = 12. Mann-Whitney tests: 0 vs. 5 min, 15 min, or 30 min. (G, I) Confocal images of a cell expressing EGFP-ITGB4 (G) and RFP-Cav1 (I) under a 10% uniaxial strain after 5, 15, and 30 min. The green channel shows what disappears by subtracting from the signal in the first frame t(0) the signal at time t (5, 15, or 30 min): signal t(0) – signal t(x); the red channel shows what appears by subtracting from the signal at time t (5, 15, or 30 min) the signal in the first frame t(0): signal t(x) – signal t(0). Scale bar = 10 µm. (H) Quantification of the orientation of HD growth after 30 min of strain. Data are from three to five independent experiments; number of cells = 12. Images are related to Supplemental Video 4.

FIGURE 9:

Cav1 and Arf6 are required for HD mechanoresponse to external forces. (A) Quantification of the area (yellow), density (red), and total intensity (purple) changes of HDs after 30 min without stretch in cells expressing EGFP-ITGB4 or after 30 min of a 10% uniaxial stretch in cells expressing EGFP-ITGB4 and RFP-Cav1(wt), RFP-Cav1(Y14D), or mCherry-Arf6(T27N) from three to five independent experiments; number of cells = 20, 12, 14, and 11. Mann-Whitney tests: upper bar (no stretch) vs. (10% strain 30 min), Cav1(Y14D) or Arf6(T27N); lower bar (10% strain 30 min) vs. Cav1(Y14D) or Arf6(T27N). (B, C) Representative confocal images of the basal membrane of a cell submitted to a 10% strain during 30 min expressing EGFP-ITGB4 and (B–B′) RFP-Cav1(Y14D) or (C) Arf6(T27N); image representation as in Figure 8G. (D) Quantification of the area (yellow), density (red), and total intensity (purple) changes of Cav1(Y14D) after 30 min of a 10% uniaxial stretch in RFP-Cav1(Y14D) from three independent experiments; number of cells = 14. Mann-Whitney tests: (10% strain 30 min) vs. Cav1(Y14D) (related to B′). (E) Confocal images of the basal membrane of cells transfected with control or anti-Cavin1 siRNAs and stained for ITGA6 and Cav1 in isotonic medium (300 mOsm) or after a 10-min hypotonic shock (30 mOsm). Brightness and contrasts are unmodified. Scale bar = 10 µm. (F) Quantification of the amount of HDs (from ITGA6 staining) and Cav1 at the basal membrane of cells in isotonic medium (Iso: 300 mOsm) or after a 10-min hypotonic shock (30 mOsm) in control cells or cells transfected with siCavin1 siRNAs to remove caveolae from three independent experiments; number of cells = 41, 32, and 30, respectively. Student’s t test Iso siCTL vs. 30 Osm siCTL or 30 Osm siCavin1 and 30 Osm siCTL vs. 30 Osm siCavin1. (G) FRAP analysis of EGFP-ITGB4 in the ICs of cells incubated with isotonic medium (300 mOsm) or after a 10-min hypotonic shock (30 mOsm) from three independent experiments; number of cells = 15 and 15. Mann-Whitney test: Iso vs. 30 Osm.

FIGURE 10:

Proposed mechanism for the role of Arf6 and caveolae components in HD remodeling and mechanoresponse. (i) In wt cells, on transient mechanical challenge (external stretch or intracellular forces generated by actomyosin), caveolae are flattened, acting as mechanical buffers by providing extra plasma membrane that is then further colonized by HD integrins either from the plasma membrane by diffusion or through Arf6-dependent trafficking. When the mechanical input disappears, HD integrins may be recycled in an Arf6-dependent manner as part of the dynamic maintenance of HDs at the basal membrane. (ii) If Arf6 activity is compromised, then HD integrins are less recycled, and therefore HDs are less responsive to mechanical signals and unable to remodel, leading to compromised HD maintenance. (iii) In the absence of caveolae, the cells are unable to respond to mechanical challenges and HDs are not remodeled. This leads to poorly organized HDs with compromised maintenance. (iv) Prolonged Cav1 phosphorylation alters HD integrin dynamics and prevents proper HD remodeling in response to mechanical cues due to larger caveolae (as suggested by Zimnicka et al., 2016), which do not flatten as effectively. See Discussion for details.