Syndecan-4/PAR-3 signaling regulates focal adhesion dynamics in mesenchymal cells - PubMed (original) (raw)

doi: 10.1186/s12964-020-00629-3.

Alejandra Valdivia 1 2 3 4 5, Marianne Brenet 6 7, Horacio Maldonado 6 8 9, Milene Kong 6 7 10, Jorge Díaz 6 7, Keith Burridge 11, Pascal Schneider 12, Alejandra San Martín 13, Rafael García-Mata 14, Andrew F G Quest 6 7, Lisette Leyton 15 16

Affiliations

Syndecan-4/PAR-3 signaling regulates focal adhesion dynamics in mesenchymal cells

Alejandra Valdivia et al. Cell Commun Signal. 2020.

Abstract

Background: Syndecans regulate cell migration thus having key roles in scarring and wound healing processes. Our previous results have shown that Thy-1/CD90 can engage both αvβ3 integrin and Syndecan-4 expressed on the surface of astrocytes to induce cell migration. Despite a well-described role of Syndecan-4 during cell movement, information is scarce regarding specific Syndecan-4 partners involved in Thy-1/CD90-stimulated cell migration.

Methods: Mass spectrometry (MS) analysis of complexes precipitated with the Syndecan-4 cytoplasmic tail peptide was used to identify potential Syndecan-4-binding partners. The interactions found by MS were validated by immunoprecipitation and proximity ligation assays. The conducted research employed an array of genetic, biochemical and pharmacological approaches, including: PAR-3, Syndecan-4 and Tiam1 silencing, active Rac1 GEFs affinity precipitation, and video microscopy.

Results: We identified PAR-3 as a Syndecan-4-binding protein. Its interaction depended on the carboxy-terminal EFYA sequence present on Syndecan-4. In astrocytes where PAR-3 expression was reduced, Thy-1-induced cell migration and focal adhesion disassembly was impaired. This effect was associated with a sustained Focal Adhesion Kinase activation in the siRNA-PAR-3 treated cells. Our data also show that Thy-1/CD90 activates Tiam1, a PAR-3 effector. Additionally, we found that after Syndecan-4 silencing, Tiam1 activation was decreased and it was no longer recruited to the membrane. Syndecan-4/PAR-3 interaction and the alteration in focal adhesion dynamics were validated in mouse embryonic fibroblast (MEF) cells, thereby identifying this novel Syndecan-4/PAR-3 signaling complex as a general mechanism for mesenchymal cell migration involved in Thy-1/CD90 stimulation.

Conclusions: The newly identified Syndecan-4/PAR-3 signaling complex participates in Thy-1/CD90-induced focal adhesion disassembly in mesenchymal cells. The mechanism involves focal adhesion kinase dephosphorylation and Tiam1 activation downstream of Syndecan-4/PAR-3 signaling complex formation. Additionally, PAR-3 is defined here as a novel adhesome-associated component with an essential role in focal adhesion disassembly during polarized cell migration. These novel findings uncover signaling mechanisms regulating cell migration, thereby opening up new avenues for future research on Syndecan-4/PAR-3 signaling in processes such as wound healing and scarring.

Keywords: Cell polarity; Cytoskeleton; Focal adhesion turnover; Mesenchymal cell migration; Wound healing.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Fig. 1

Fig. 1

Thy-1 treatment induced association of PAR-3 with Syndecan-4 in DI TNC1 astrocytes. a Cell lysates obtained upon FBS or (b) Thy-1-Fc-Protein A (Thy-1) stimulation were precipitated using biotinylated peptides consisting in the 10 amino acids of Syndecan-4 cytoplasmic domain (SDC4) or control peptides, in which the last 4 amino acids (EFYA) were replaced by glycine. Samples were immunoblotted for PAR-3. Syntenin was used as a precipitation control since it interacts directly with the Syndecan-4 EFYA sequence. c Syndecan-4-HA transfected DI TNC1 cells were stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) as a negative control. Cell lysates were immunoprecipitated with the anti-HA antibody and immunoblotted for PAR-3, FAK, Syndecan-4, or Syntenin. d Association between PAR-3 and Syndecan-4 was evaluated by Proximity Ligation Assay (PLA). DI TNC1 cells were transfected with Syndecan-4-HA or with the empty plasmid. Subsequently, cells were serum starved for 30 min and stimulated or not (NS) with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL), fixed and processed for PLA using antibodies against PAR-3 or HA. Pictures are representative of PLA puncta observed for each condition as indicative of the interaction between PAR-3 and Syndecan-4-HA. Lower panels show magnification of the indicated area. Magnification bar = 10 μM. e Graph shows the number of PLA puncta per cell. ***P < 0.001

Fig. 2

Fig. 2

PAR-3 knockdown impaired astrocyte migration and adhesion, but not Golgi polarization induced by Thy-1. a Immunoblotting for PAR-3 isoforms in cells transfected with PAR-3 siRNA compared to control siRNA. Actin was the loading control. Migration was evaluated using the wound-healing assay. Evaluated were DI TNC1 cells with silenced PAR-3 or control, stimulated or not (NS) with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL). Pictures show overlapping images of wound closure at time zero (red) and after 24 h (green). Dotted line shows the wound-closure boundary. Graph shows wound closure in relative units. b DI TNC1 astrocytes, transfected as in (a), were stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) for 7 h. After fixation, cells were stained for nucleus (DAPI) and Golgi apparatus using an anti-giantin antibody. Golgi (red) positioning within an angle of 120° in front of the nucleus (blue), facing the wound, was considered polarized (1). Non-polarized cells are labelled as (2). Arrow shows the direction of migration. One hundred cells were monitored per condition. Graph shows the percentage of polarized cells. Scale bar = 10 μm. c DI TNC1 astrocytes, transfected as in (a), were stimulated for 15 min. FAs were visualized by immunofluorescence using an anti-vinculin antibody (red) and DAPI for nuclei (blue). Scale bar = 10 μm. Image analyses were performed with the Analyze Particles Tool in the Fiji image processing software [44]. Values in the graphs represent the mean ± s.e.m. from 3 independent experiments (n = 3) determined from at least 50 cells in each experimental condition per experiment. Significant differences are indicated as *P < 0.05

Fig. 3

Fig. 3

PAR-3 silencing impaired Thy-1-accelerated microtubule-dependent focal adhesion disassembly. a DI TNC1 cells treated with Nocodazole and washout were stimulated or not (NS) with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) for different time periods. FAs were detected by immunofluorescence staining for vinculin (red) and nuclei (blue). Scale bar = 10 μm. Graphs show quantification of the FA number per cell (left panel) and average area of FAs (right panel) observed in DI TNC1 astrocytes after the indicated treatments. Values in the graphs represent the mean ± s.e.m. determined from at least 50 cells in each experimental condition per experiment. b DI TNC1 astrocytes co-transfected with mCherry-vinculin and siRNA (control or PAR-3) were treated with Nocodazole and washout. Cells were then stimulated as in (a), and a 30-min time-lapse microscopy video was recorded. Values in the graphs represent the mean ± s.e.m. determined from scoring at least 10 mCherry-positive focal adhesions per experiment. c Model depicting the interaction of αvβ3 integrin and Syndecan-4 with Thy-1, which triggers signaling events that include activation of FAK and PI3K generating PIP3. An unidentified Rac1 GEF, is then recruited to the plasma membrane, where it activates Rac1 and leads to cell migration. d DI TNC1 astrocytes, transfected as in (a), were treated with Nocodazole and washout, and cells were stimulated for different time periods (0–15 min). Cell lysates were immunoblotted for p-FAK or total FAK. e Graph values are compared against the normalized time 0 and represent the average numbers obtained from the ratio of p-FAK/FAK densitometric data analysis. All graphs show the mean ± s.e.m. of n = 3. Significant differences compared to control conditions are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 4

Fig. 4

Thy-1-induced astrocyte migration requires PAR-3-dependent Tiam1 activation. a PAR-3 silenced DI TNC1 astrocytes were stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) for 60 min. Cells were fixed, and active Rac1 GEF was detected by immnunofluorescence using GST-Rac1G15A and anti-GST (purple) and Phalloidin (green). The white rectangle indicates the zoomed area. The color map indicates levels of Rac1 GEF activation, going from 0 (blue) to 256 (white). Scale bar = 10 μm. b DI TNC1 cells transfected with siRNA control (siCTRL), siRNA Tiam1 (siTiam1), or dominant negative Tiam1 (Tiam1-DN). The Western blot shows levels of Tiam1 in siRNA-transfected cells. Migration was evaluated using the wound-healing assay, and cells were stimulated as in (a) for 24 h. Images show wound closure at time zero (green) and after 24 h (red). c DI TNC1 cells stimulated or not (NS) with endogenous Thy-1 from fixed CAD cells for different periods of time. Active Tiam1 was affinity-precipitated using GST-Rac1G15A coupled to GSH-agarose beads and immunoblotted for Tiam1. Numbers in the graphs represent the average fold-increase of Tiam1 activity normalized to total Tiam-1. All graphs show the mean ± s.e.m. of n = 3. Significant differences compared to control conditions are indicated as *P < 0.05

Fig. 5

Fig. 5

PAR-3-induced Tiam1 activation after Thy-1/CD90 treatment requires Syndecan-4. Syndecan-4silenced DI TNC1 astrocytes (siSynd4) or control transfected cells (siCTRL) were stimulated with Thy-1-Fc-Protein A (Thy-1) or treated with TRAIL-R2-Fc-Protein A (TRAIL) for 60 min. Cells were fixed, and stained for GST to detect GST-Rac1G15A (green) and for Tiam1 (red). The white rectangle indicates the zoomed area. The heat color map indicates levels of total Tiam1 co-localization with nucleotidefree mutant of Rac1 (GST-Rac1G15A) at the membrane, as indicative of Tiam1 activation. Scale bar = 10 μm

Fig. 6

Fig. 6

Thy-1-induced Syndecan-4/PAR-3 association is needed in FA dynamics in mouse embryonic fibroblasts. a MEFs transfected with Syndecan-4-HA were stimulated or not (NS) with Thy-1-Fc or fetal bovine serum (FBS). PAR-3 was immunoprecipitated (IP) from the lysates, and Syndecan-4 was detected with an anti-HA antibody (SDC4-HA). Syntenin and PAR-3 were immunoblotted as positive and an IP control, respectively. IgG was used as a negative IP control. b MEFs transfected with the siRNA control or PAR-3 were analyzed by Western blotting for PAR-3 expression. Tubulin was used as a loading control. c MEFs co-transfected with siGlo and siRNA control or PAR-3 were stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) for 15 min. FAs and nuclei were respectively visualized by vinculin staining (red) and DAPI (blue). Transfected cells were identified by the siGlo signal (green). Graphs show the mean ± s.e.m. for the number (d) and average area (e) of FAs from at least 50 cells. MEFs were transfected as in (c), incubated with Nocodazole and washout, and immediately stimulated as in (c) for different time periods. Graphs show the mean ± s.e.m. for the number (f) and average area (g) of FAs from at least 50 cells. Significant differences are indicated as *P < 0.05, **P < 0.01, and ***P < 0.001. (h) Cells were co-transfected with Paxillin-EGFP and siRNA (control or PAR-3). Shown are representative images for non-stimulated cells (t0) and cells stimulated with Thy-1-Fc-Protein A (Thy-1) or TRAIL-R2-Fc-Protein A (TRAIL) for 2.5 min (t2.5). Scale bar =10 μm

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