Protease-activated receptor signalling initiates α5β1-integrin-mediated adhesion in non-haematopoietic cells

Abstract

Haematopoietic cells and platelets employ G-protein-coupled receptors (GPCRs) to sense extracellular information and respond by initiating integrin-mediated adhesion. So far, such processes have not been demonstrated in non-haematopoietic cells. Here, we report that the activation of protease-activated receptors PAR1 and PAR2 induce multiple signalling pathways to establish α5β1-integrin-mediated adhesion. First, PARs signal via Gβγ and PI3K to α5β1-integrins to adopt a talin- and kindlin-dependent high-affinity conformation, which triggers fibronectin binding and initiates cell adhesion. Then, within 60 s, PARs signal via Gα13, Gαi, ROCK and Src to strengthen the α5β1-integrin-mediated adhesion. Furthermore, PAR signalling changes the abundance of numerous proteins in the adhesome assembled by α5β1-integrins, including Gα13, vacuolar protein-sorting-associated protein 36, and band 4.1-like protein 4B or 5, and accelerates cell adhesion maturation, spreading and migration. The mechanistic insights describe how agonist binding to PAR employs GPCR and integrin-signalling pathways to initiate and regulate adhesion and to guide physiological responses of non-haematopoietic cells.

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Fig. 1: PAR activation increases fibroblast adhesion force to FN after 60 s contact time.
Fig. 2: Activation of PAR1 or PAR2 shifts β1-class integrins into high-affinity conformation.
Fig. 3: Signalling proteins involved in PAR-mediated adhesion regulation.
Fig. 4: PAR2 activation changes early adhesome composition of α5β1-integrin adhesion sites.
Fig. 5: PAR signalling affects spreading and migration of fibroblasts.
Fig. 6: PAR activation regulates early cell adhesion to FN.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank C. Grashoff for providing talin-reconstituted fibroblasts, as well as N. Beerenwinkel, M. Bharadwaj-Arora and J. Helenius for fruitful discussions. We thank M. Huber for assistance in creating Gα13-depleted fibroblasts and R. Newton for virally transfecting fibroblasts. We thank R. Böttcher for the vector pRetroQ-eGFP-hRap1aDN(S17N), B.M. Gaub for his support with the Ca2+ imaging protocol, M. Yu for teaching SCFS, and the single cell facility for advice regarding cell imaging and flow cytometry. This work was supported by ETH Zurich (grants nos. ETH-03 14-1 and ETH-20 17-2) and the Swiss National Science Foundation (SNF; grant no. 31003A_182587/1).

Author information

P.M.S., N.S., R.F. and D.J.M. discussed and designed the project. P.M.S. and N.S. performed SCFS, flow cytometry and Ca2+ and timelapse cell spreading and cell migration imaging experiments. P.M.S. and Z.S. designed and performed the adhesome proteomics experiments. Z.S. and P.M.S. discussed and analysed the proteomics data. N.S. performed confocal imaging. All authors wrote and discussed the paper.

Correspondence to Daniel J. Müller.

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Extended data

Extended Data Fig. 1 AFM-based single cell force spectroscopy (SCFS) to study the effect of PAR-activation on early cell adhesion.

a, Illustration of AFM-based SCFS. A single fibroblast is attached to a tipless concanavalin A-coated cantilever. Subsequently, the cantilever-bound fibroblast is brought into contact with an extracellular matrix (ECM) protein-coated support. After a predefined contact time allowing the fibroblast to establish adhesion to the ECM substrate, the cantilever is retracted to record the force needed to detach the fibroblast from the support in a so-called force-distance curve. b, Exemplary force-distance curves recorded after contact times ranging from 5 to 240 s show distinct features: The retraction force-distance curve (black) records the adhesion force of the fibroblast, which represents the highest force required to separate the fibroblast from the ECM substrate. In total we have recorded 3’500 force-distance curves. c, Exemplary force-distance curves detecting the (un-)binding of single integrins upon separating a fibroblast from a fibronectin fragment (FNIII7-10)-coated support. To record single integrin (un-)binding events with SCFS, fibroblasts are approached to the FNIII7-10-substrate (red curve) until reaching a contact force of ~ 200 pN for a contact time of ~ 150 ms. After this, cantilever and fibroblast are retracted (black/green curve). The green force-distance curve shows a single adhesion event while the black curve shows no adhesion event. In total, we have recorded 12’203 force-distance curves in binding probability assays. d, e, Influence of procedure detaching the fibroblast from cell culture flasks on the adhesion measurement by SCFS. Parental fibroblasts were detached either with (d) 0.25 % (w/v) trypsin or (e) 12 mM EDTA from culture flasks and then attached to the AFM cantilever for SCFS. Dots represent adhesion forces of single fibroblasts, < n(cells) > the number of fibroblasts tested, and black bars median values. Statistical significances (P-values) were determined using two-tailed Mann-Whitney tests and are given if P < 0.05. In the first 30 min after trypsin-induced detachment from culture flasks the adhesion of fibroblasts to FNIII7-10-coated supports increased significantly. 30–60 min after detachment the fibroblasts fully recovered. Adhesion to FNIII7-10ΔRGD or collagen type I was not affected by trypsin detachment. The contact time of fibroblast and ECM-coated supports was 60 s. Consequently, for all SCFS experiments in this work the fibroblasts were allowed to recover from trypsin-induced detachment from culture flasks for at least 30 min, before being attached to the cantilever. f, Experimental assay used for SCFS. Fibroblasts detached from culture flasks using 12 mM EDTA were allowed to recover for 30 min in SCFS media prior to peptide exposure and SCFS (Methods).

Extended Data Fig. 2 PAR2-activating peptide and PAR2 agonist increase adhesion forces of parental fibroblasts to similar levels.

a, Adhesion force of parental, pKO-β1 and pKO-αV fibroblasts, adhering to FNIII7-10-coated (dark colors, black bars) or FNIII7-10∆RGD-coated (light colors, grey bars) supports. Experiments were conducted in the absence and presence of 1 µM control peptide. The control peptide did not influence the adhesion force to FNIII7-10 or FNIII7-10∆RGD measured after 60 s contact time for any fibroblast line. b, Adhesion force of parental fibroblasts adhering to FNIII7-10-coated (dark colors, black bars) or FNIII7-10∆RGD-coated (light colors, grey bars) supports after incubation with different concentrations (as indicated) of control, PAR1-activating, or PAR2-activating peptide. The experiments, which measure the adhesion force after 60 s contact time to FNIII7-10-coated supports, validate that the peptide concentrations used in our experiments are sufficient to elicit a full cellular response. Additionally, the experiments show that the small molecule PAR2 agonist AC264613 increases fibroblast adhesion to FNIII7-10 similar to as detected for the PAR2-activating peptide. Top or lower n(cells) give the number fibroblasts tested on FNIII7-10-coated or FNIII7-10∆RGD-coated supports, respectively. Dots represent adhesion forces of single fibroblasts and bars median values. Statistical differences analyze fibroblast adhesion to FNIII7-10-coated supports in the presence of 0.1 and 1.0 mM peptide or 25 µM PAR2 agonist using the two-tailed Mann-Whitney test. P-values are given if < 0.05.

Extended Data Fig. 3 PAR-activation induces calcium signaling in fibroblasts.

a, Parental or pKO-αV fibroblasts plated on full-length bovine FN-coated WPI dishes were loaded with 1 µM fluorescent calcium indicator (Oregon GreenTM 488 BAPTA-1, 06807, Themo Fisher Scientific) for 2 h and imaged for 10 min before adding PAR1-activating, PAR2-activating or control peptide to a final concentration of 0.1 mM. A representative image frame of spread, calcium-indicator loaded fibroblasts is shown. Five independent samples with at least five fibroblasts were analyzed per condition. Scale bar, 40 µm. b, Fluorescence intensity of fibroblasts monitored over time. The fluorescence intensity traces of four representative fibroblasts (outlined by grey line in a and labeled 1–4) are shown. Representative fluorescence intensity characterized before and after adding PAR1- or PAR2-activating peptide. This indicates a fast calcium increase, common for GPCR signaling. c, Intensity of calcium signaling approximated as the increase of indicator fluorescence intensity after adding control or PAR-activating peptide. In case of adding control peptide, no calcium peak was observed and the calcium level following the peptide addition was taken. Each point represents this calcium ratio for one fibroblast and bars their median. n(cells) gives the number of fibroblasts characterized. Two-tailed Mann-Whitney tests were used to determine P-values between PAR1-activating or PAR2-activating and control peptide. Parental fibroblasts, as well as pKO-αV fibroblasts both show calcium signaling after PAR1- or PAR2-activation.

Extended Data Fig. 4 PAR-activation shifts β1-class integrins into high-affinity conformation.

Suspended parental fibroblasts were incubated first with 0.1 mM control, PAR1- or PAR2-activating peptide, or 1 mM Mn2+ for 20 min, then incubated with monoclonal antibodies either detecting the high-affinity conformation of β1-class integrins (9EG7) or detecting all β1-class integrins (KMI6). Subsequently, fibroblasts were incubated with the same secondary antibody and analyzed by flow cytometry. Each dot represents the median of one flow cytometry measurement (n(exp) = 12 per condition), normalized to the median fluorescence intensity of fibroblasts in the absence of peptide and Mn2+. Black bars indicate the mean of all flow cytometry experiments. Two-tailed Mann-Whitney tests were used to determine P-values between activating peptide or Mn2+ and control peptide.

Extended Data Fig. 5 TKO and KKO fibroblasts establish higher adhesion forces to FNIII7-10 than to FNIII7-10ΔRGD.

a, b, Adhesion forces of (a) KKO and (b) TKO fibroblasts to FNIII7-10ΔRGD-coated (light color) and FNIII7-10-coated (dark color) supports after 60 s contact time. Fibroblasts were incubated with 1 mM control, PAR1-activating, and/or PAR2-activating peptide. Peptides used are indicated above each condition. n(cells) represents the number of fibroblasts tested, dots the adhesion forces of single fibroblasts, and black bars median values. All P-values were determined using two-tailed Mann-Whitney tests.

Extended Data Fig. 6 Talin and integrin expression levels of engineered fibroblasts.

a, b, TKO + talin1 and TKO + talin2 fibroblasts were labeled with PE-conjugated antibodies against integrin subunit (a) α5 or β1 and (b) αV or β3. c, YFP signals of talin1 YFP constructs and talin2 YFP constructs used to reconstitute talin into TKO fibroblasts were quantified by flow cytometry to compare talin expression levels. Single points represent the median fluorescent intensity of 20’000 fibroblasts, bars the means and error bars the s.d. d, Verification of Gα13 depletion in pKO-β1 fibroblasts engineered by the CRISPR/Cas9 technology. Western blots, which were repeated three times giving the same results, were used to verify the depletion of Gα13 in pKO-β113-KO fibroblasts and talin1 was used as loading control. As reference pKO-β1 fibroblasts were used.

Extended Data Fig. 7 Representative morphologies of pKO-β1 and pKO-αV fibroblasts adhering to FNIII7-10.

Serum-starved pKO-β1 and pKO-αV fibroblasts were allowed to adhere for the indicated time on FNIII7-10-coated dishes at 37 °C, washed and fixed. The experiment was done in presence of 0.1 mM control or PAR2-activating peptide. Representative cells are exemplified. Scale bars, 25 µm. For adhesome proteomics we had to wait 10 min until pKO-β1 fibroblasts and 15 min until pKO-αV fibroblasts immobilized on the dishes before crosslinking. Representative pKO-β1 and pKO-αV fibroblast morphologies after different adhesion times. The first time frames correspond to the time point fibroblasts were used for adhesome proteomics analysis. At least 6 fibroblasts were analyzed per experimental condition.

Extended Data Fig. 8 Comparison of label-free quantification intensities (LFQ) in pKO-αV and pKO-β1 fibroblasts.

The adhesome composition experiments resulted in 119 proteins significantly changing abundance at the adhesion site in pKO-β1 fibroblasts. Out of 119, 32 proteins were not biosynthesis related, nuclear or mitochondrial and therefore worth further investigation. The 32 proteins are summarized and classified according to whether their levels increased or decreased in pKO-β1 fibroblasts (control peptide in dark blue, PAR2-activating peptide in dark pink) and compared to how they behave in pKO-αV fibroblasts (control peptide in light blue, PAR2-activating peptide in light pink). Label-free quantification (LFQ) intensities are based on the (raw) intensities before imputation of missing values. They are normalized at multiple levels so that their profiles across samples accurately reflect the relative amounts of the proteins. For better representation in this graph LFQ values were additionally normalized to the highest value observed for each protein. In total four measurements were done per experimental condition. Each dot represents one measurement and bars the median. Statistical significances (P-values) were calculated applying two-tailed t-tests comparing the abundance of specific adhesome proteins in pKO-β1 fibroblasts incubated with control peptide and PAR2-activating peptide.

Extended Data Fig. 9 PAR2 activation affects fibroblast spreading and adhesion maturation on the longer term.

a, Integrin adhesion maturation and spreading of a representative fibroblast, as seen by monitoring recruitment of the adaptor protein paxillin to adhesion sites over time. Parental fibroblasts in suspension were exposed for 15 min to 0.1 mM PAR2-activating or control peptide prior to experiments. Subsequently the fibroblasts were seeded into FNIII7-10-coated glass-bottom wells containing 0.1 mM PAR2-activating or control peptide in 100 µl DMEM (without FCS). Timelapse confocal microscopy was used to image (frame time 34 s) paxillin-GFP. Time between images shown here is 2.8 min. Scale bar, 20 µm. b, Classification result of a representative fibroblast, at one time point (40.2 min). After imaging, the spreading of single fibroblasts could be analyzed. For each single fibroblast and in every image (one per 34 s), each pixel was evaluated using the object classification program ilastik. Eight fibroblasts (4 per condition) were used as training set for the classification. We trained the algorithm to recognize background, cytoplasm, nucleus and adhesion site by intensity, edges and texture. The classification was then applied to all fibroblasts (n = 42 and n = 51, for PAR2-activating or control peptide, respectively). Finally, the output for each pixel is the probability of being background, cytoplasm, nucleus or adhesion site (total probability of 1). The analysis and classification procedures were used to determine the fibroblast spreading area, total adhesion size shown in Fig. 5a–c. Scale bar, 20 µm. The experiment was repeated 10 times with at least four cells analyzed per repeat and condition providing the similar results.

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Spoerri, P.M., Strohmeyer, N., Sun, Z. et al. Protease-activated receptor signalling initiates α5β1-integrin-mediated adhesion in non-haematopoietic cells. Nat. Mater. 19, 218–226 (2020). https://doi.org/10.1038/s41563-019-0580-4

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