The fibronectin synergy site re-enforces cell adhesion and mediates a crosstalk between integrin classes
- Universitat de València, Spain
- Ludwig-Maximilians-Universität München, Germany
- Klinikum der Universität München, Germany
- Eidgenössische Technische Hochschule Zürich, Switzerland
Figures
Figure 1 with 2 supplements
Normal tissue development and prolonged bleeding in Fn1syn/syn mice.
(A) Cartoon of FN and the nucleotide point mutations disrupting the function of the synergy site. (B) Representative images of 3-months-old Fn1 +/+ and Fn1syn/syn heart sections stained with H and E …
Figure 1—figure supplement 1
Strategy used to generate the Fn1syn/syn mice and tissue and platelet analysis.
(A) Scheme of the FN gene and the targeting vector to generate the mouse with a dysfunctional synergy site. The synergy region is located in the FNIII9 and encoded in exon 28, shown in red. (B) The …
Figure 1—figure supplement 2
FN levels in platelets and blood from Fn1syn/syn mice and platelet aggregation assays.
(A) Western-blot to estimate FN levels in non-activated, washed platelets from Fn1+/+ (n = 6) and Fn1syn/syn mice (n = 6). (B) Western-blot to calculate FN and fibrinogen (Fg) levels in blood plasma …
Figure 2 with 2 supplements
The FN synergy site is dispensable for FN fibrillogenesis, cell adhesion and spreading.
(A) Fn1-Knock-Out (Fn1-KO) fibroblasts grown in 1% plasma derived from either Fn1+/+ or Fn1syn/syn mice, fixed at the indicated times and stained for FN (green), F-actin stain (with Phalloidin; red) …
Figure 2—figure supplement 1
Integrin surface levels and plasma FN purification and glass coating.
(A) Integrin levels on the surface of Fn1-KO fibroblasts analyzed by flow cytometry (graphs show representative results of 3 independent experiments). (B) Coomasie blue staining of purified pFNwt …
Figure 2—figure supplement 2
Captures of life-time microscopy videos of Fn1-KO fibroblasts spreading on pFNwt or pFNsyn.
https://doi.org/10.7554/eLife.22264.007
Figure 3 with 1 supplement
The FN synergy site is required to establish tensioned FN-α5β1 bonds.
(A) Quantification of adhesion strength. 7 × 105Fn1-KO cells attached onto purified, full-length (fl) pFNwt or pFNsyn or FNIII7-10wt or FNIII7-10syn and spun with a spinning disk device (n = 7 …
Figure 3—figure supplement 1
Representative spinning disk experiment showing the cell distribution profile against the shear force.
The shear force was calculated according to the method described by Friedland and Boettiger (Friedland et al., 2009). The force corresponding to the inflexion point of the curve is the τ50.
Figure 4 with 1 supplement
α5β1 integrins require the synergy site in FN to induce cell spreading.
(A) Adhesion of pKO-β1, pKO-αv and pKO-αv/β1 fibroblasts seeded on pFNwt or pFNsyn for indicated times (n = 3 independent experiments; mean ± sem). (B) pKO-β1, pKO-αv and pKO-αv/β1 fibroblasts were …
Figure 4—figure supplement 1
Captures of life-time microscopy videos of pKO-β1 fibroblasts spreading on pFNwt or pFNsyn.
https://doi.org/10.7554/eLife.22264.013
Figure 5 with 1 supplement
Fn1syn/syn;Itgb3-/- mice suffer from severe hemorrhages and fail to separate the blood and lymphatic vasculatures.
(A) E12.5 Fn1syn/syn;Itgb3-/- embryos display hemorrhages in the jugular and axilar areas in the left side (arrowheads). Scale bar, 50 mm. (B) Representative images from E15.5 littermates embryos …
Figure 5—figure supplement 1
Blood vessel formation in Fn1syn/syn; Itgb3-/-embryos.
(A) E11.5 embryos of indicated genotype. Scale bar, 2 mm (B) E11.5 whole mount embryos of indicated genotype stained with PECAM. High magnifications indicate that Fn1syn/syn;Itgb3-/- embryos have …
Figure 6
Shear flow exposed platelets fail to adhere to pFNsyn.
(A) Cartoon showing the platelet integrins that can be ligated to the different substrates used in the experiments. The color intensity of the integrin denotes whether the integrin is active or …
Figure 7
Malformed blood vessels in Fn1syn/syn;Itgb3-/- embryos.
(A) PECAM-positive endothelial cells (red) and α-SMA-positive smooth muscle cells (green) in dermal whole mounts from E15.5 Fn1+/+;Itgb3+/+ and Fn1syn/syn;Itgb3-/- littermate embryos indicate veins …
Figure 8
The major role of the FN synergy site is to re-enforce cell adhesion.
(A) Hydrodynamic shear force-exposed fibroblasts seeded on a FNwt-coated surface form catch-bonds that strengthen α5β1 integrin-mediated adhesions to FN and trigger phosphorylation of Y397-FAK …
Videos
Video 1
Life-time microscopy video of Fn1KO fibroblasts on pFNwt.
https://doi.org/10.7554/eLife.22264.008
Video 2
Life-time microscopy video of Fn1KO fibroblasts on pFNsyn.
https://doi.org/10.7554/eLife.22264.009
Video 3
Life-time microscopy video of pKO-β1 fibroblasts on pFNwt.
https://doi.org/10.7554/eLife.22264.014
Video 4
Life-time microscopy video of pKO-β1 fibroblasts on pFNsyn.
https://doi.org/10.7554/eLife.22264.015Tables
Table 1
Progeny of Fn1syn/+;Itgb3+/- x Fn1syn/+;Itgb3+/- intercrosses.
| Age | Num. | Fn1syn/syn Itgb3+/- | Fn1syn/syn Itgb3+/+ | Fn1syn/syn Itgb3-/- | Fn1+/syn Itgb3+/- | Fn1+/syn Itgb3+/+ | Fn1+/syn Itgb3-/- | Fn1+/+ Itgb3+/- | Fn1+/+ Itgb3+/+ | Fn1+/+ Itgb3-/- |
|---|---|---|---|---|---|---|---|---|---|---|
| E11.5 | 36 | 6 (16.7%) | 1 (2.8%) | 1 (2.8%) | 10 (27.8%) | 6 (16.7%) | 4 (11.1%) | 5 (13.9%) | 2 (5.6%) | 1 (2.8%) |
| E14.5 | 23 | 2 (8.7%) | 2 (8.7%) | 2 (8.7%) | 5 (21.7%) | 5 (21.7%) | 1 (4.3%) | 1 (4.3%) | 4 (17.3%) | 1 (4.3%) |
| E15.5 | 121 | 12 (9.9%) | 5 (4.1%) | 3 (2.5%) | 39 (32.2%) | 5 (15.4%) | 12 (9.9%) | 14 (11.6%) | 17 (14%) | 4 (3.3%) |
| E16.5 | 16 | 2 (12.5%) | 1 (6.25%) | 0 | 5 (31.5%) | 1 (6.25%) | 1 (6.25%) | 3 (37.5%) | 1 (6.25%) | 2 (12.5%) |
| E17.5 | 16 | 2 (12.5%) | 0 | 0 | 6 (23%) | 3 (19%) | 2 (12.5%) | 2 (12.5%) | 1 (8%) | 0 |
| P 21 | 245 | 33 (13.5%) | 32 (13%) | 0 | 57 (23%) | 46 (18.7%) | 13 (5.3%) | 35 (14.4%) | 17 (3.9%) | 12 (4.9%) |
| Mendelian Distribution | 100 | 12.5% | 6.25% | 6.25% | 25% | 12.5% | 12.5% | 12.5% | 6.25% | 6.25% |
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Table 1—source data 1
Progeny of Fn1syn/syn;Itgb3+/- x Fn1syn/syn;Itgb3+/- crosses
- https://doi.org/10.7554/eLife.22264.017
