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First published online 26 February 2008
doi: 10.1242/jcs.024877

Journal of Cell Science 121, 877-886 (2008)
Published by The Company of Biologists 2008
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Fibrogenic fibroblasts increase intercellular adhesion strength by reinforcing individual OB-cadherin bonds

Philippe Pittet1, Kyumin Lee2, Andrzej J. Kulik2, Jean-Jacques Meister1 and Boris Hinz1,*

1 Laboratory of Cell Biophysics, Institute of Physics of the Complex Matter, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland
2 Laboratory of Nanostructures and Novel Electronic Materials, Institute of Physics of the Complex Matter, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland


Figure 1
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  		Fig. 1. Adhesion of differentiated myofibroblasts is stronger than that of proto-myofibroblasts. Adhesion forces are measured with AFM between cadherin dimer-coated cantilevers and similarly coated substrates (A-C), between cadherin dimer-coated cantilevers and myofibroblasts (D,E), and between myofibroblasts grown on cantilevers and myofibroblasts grown in monolayers (G-I). In all conditions, cantilever approach-retraction velocities were set to 0.1 µm/second, loading force to 3 nN and contact time to 2 seconds. (B,E,H) Typical force-distance curves under different conditions are displayed for each configuration; arrows indicate positions where bond rupture occurs in a `jump'. Red profiles indicate typical interaction between OB-cadherins or differentiated myofibroblasts in the different set-ups, green lines represent controls in the absence of extracellular Ca2+ (EGTA) and pink lines show controls performed with IgG-coated cantilevers (B,E) or contacts formed in the presence of OB-cadherin-blocking peptides (H). (C,F,I) Rupture forces displayed as histograms (n>=5000 in each condition), normalized for the total number of rupture events in every configuration and fitted with Gaussian curves. Results obtained with proto-myofibroblasts and N-cadherin are displayed in blue, whereas results with differentiated myofibroblasts and OB-cadherin rupture forces are indicated in red. Note the different scale in H, which allows for the significantly higher bond strength in the cell-cell set-up compared with set-ups in B and E. Cadherin specificity of interactions was controlled by coating cantilevers with human IgG (C,F, pink), by using EGTA (green) and by applying anti-cadherin peptides (I, dashed lines).

 

Figure 2
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  		Fig. 2. De-adhesion of two cells occurs with multiple rupture events. Histograms summarize the number of rupture events (jumps) preceding the complete detachment of the cantilever from the touched (put into contact) respective substrate from all force-distance profiles (Fig. 1B,E,H). Different interaction setups were tested. Recombinant cadherin dimers grafted to AFM cantilevers were put into contact with carpets of the respective cadherin dimers (A,B) and with myofibroblasts grown in monolayer (C,D). Proto-myofibroblasts were put into contact with N-cadherin (C) and OB-cadherin (F) and differentiated myofibroblasts were put into contact with OB-cadherin (D) and N-cadherin (E). In the cell-cell setup, proto-myofibroblasts (G) and differentiated myofibroblasts (H) were spread on tipless AFM cantilevers and put into contact with the same cell type grown in monolayer. Cantilever approach-retraction velocities were set to 0.1 µm/second, loading force to 3 nN and contact time to 2 seconds.

 

Figure 3
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  		Fig. 3. Last rupture event analysis in force-distance profiles is similar to analysis of all rupture events. Differentiated myofibroblasts grown on tipless AFM cantilevers were connected with differentiated myofibroblasts grown in monolayer for 2 seconds with constant approach velocity (0.1 µm/second) and loading force (3 nN). (A) Results obtained by including only the last rupture events in force-distance profiles (supplementary material Fig. S2) are displayed as Gaussian curve fits of histograms, normalized to the total number of last rupture events (n>1000). Force distribution is comparable to the histogram obtained after analyzing all rupture jumps in force-distance profiles (n>=5000) (Fig. 1I).

 

Figure 4
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  		Fig. 4. Intracellular association with F-actin increases extracellular cadherin binding strength. (A,B) Differentiated myofibroblasts were grown on tipless AFM cantilevers in control conditions (A) or treated for 30 minutes with 1 µM cytochalasin D to depolymerize F-actin (B). Cells were immunostained for {alpha}-SMA (green) and nuclei (blue) and images were taken with a confocal microscope. (C,D) AFM was used in imaging mode to probe the topography of monolayer cells before (C) and after cytochalasin D treatment (D); false-color intensity increases with cell height. (E) Differentiated myofibroblasts grown on tipless AFM cantilevers were put into contact for 2 seconds with myofibroblasts grown in monolayer using 3 nN loading force and an approach velocity of 0.1 µm/second (n>1500). All measured rupture forces are accumulated in histograms that were fitted with Gaussian curves and normalized to the total number of events. Contact was performed in the presence of cytochalasin D (solid red line) and compared with histograms obtained by putting into contact differentiated myofibroblasts in control conditions (dashed black line) (Fig. 1I) and by putting into contact recombinant OB-cadherins (solid black line) (Fig. 1C). Note that actin depolymerization reduces intrinsic binding of native myofibroblast cadherins to the level of recombinant OB-cadherin dimers. Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. The bond strength of native OB-cadherins in differentiated myofibroblasts increases with increasing contact time. Differentiated myofibroblasts grown on tipless AFM cantilevers were put into contact with differentiated myofibroblasts grown in monolayer using a constant approach velocity (0.1 µm/second) and loading force (3 nN). (A) All obtained rupture forces are displayed as histograms (n>=4000 per contact time), normalized for the total number of rupture events in every configuration and fitted with Gaussian curves. (B) With increasing contact time, the probability of obtaining three distinct force peaks increases; probability is calculated from the area under each individual Gaussian peak and divided by the total curve area (see supplementary material Fig. S2). (C) The average number of rupture jumps (±s.d.) preceding total cell detachment was determined in force-distance profiles for each contact time. (D) The total work needed to completely detach two differentiated myofibroblasts is displayed as a function of contact time (2-60 seconds).

 

Figure 6
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  		Fig. 6. The bond strength between recombinant cadherin dimers increases with increasing contact time. (A) AFM cantilevers coated with recombinant OB-cadherin (10 µg/ml) were put into contact with similarly coated surfaces, using a constant approach velocity (0.1 µm/second) and loading force (3 nN). All measured rupture forces are summarized in histograms that are fitted with Gaussian curves for contact times of 2, 5, 10 and 60 seconds (n>=5000 in each condition). Gaussian curves obtained after 5, 10, and 60 seconds contact time are normalized to the total number of events obtained after 2 seconds of contact time for direct comparison. The dashed lines represent Gaussian curve fits of single peaks that are included in each total data set. (B) Overlaying all Gaussian fits obtained for each contact time demonstrates increasing formation of a third force peak with increased contact time, at decreasing amplitude of the first peak. Inset in B shows the average number of rupture jumps (±s.d.) that precede complete separation of two recombinant OB-cadherin bonds after different contact times. (C) AFM cantilevers coated with 10 µg/ml recombinant OB-cadherin were put into contact with surfaces that exhibited recombinant OB-cadherin coatings in decreasing concentrations of 20, 10, 8, 5, 2, and 1 µg/ml (n>=5000 in each condition), using a constant approach velocity (0.1 µm/second), loading force (3 nN) and a contact time of 60 seconds. Inset in C shows the average number of rupture jumps (±s.d.) that precede complete separation of two recombinant OB-cadherin bonds as a function of cadherin density. Note that lowering cadherin concentration does not reduce the number of force peaks but rather decreases the average number of rupture events leading to complete bond separation.

 

Figure 7
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  		Fig. 7. Single-molecule bond strength of native cadherins in living myofibroblasts increases with increasing loading rate. (A) Differentiated myofibroblasts grown on tipless AFM cantilevers were put into contact for 2 seconds with myofibroblasts grown in a monolayer at 3 nN loading force and with approach velocities ranging from 0.1-1.0 µm/second (n>5000 per condition). All measured rupture forces are summarized in histograms that are fitted with Gaussian curves for approach velocities of 0.1, 0.2 and 1.0 µm/second, normalized to the total number of events. Dashed lines represent Gaussian fits of single peaks that are included in each total data set. (B) Overlaying all Gaussian fits obtained for each loading rate demonstrates a right shift in all Gaussian-fitted force peaks but no change in the number of peaks per histogram. (C) After fitting the data with Bell's model, force peak position is displayed as a function of the loading rate for each approach velocity.

 

Figure 8
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  		Fig. 8. Possible models for AJ reinforcement in differentiated myofibroblasts. In our experiments, the reinforcement of OB-cadherin bonds using either recombinant cadherin dimers or intact differentiated myofibroblasts, follows a sequence of maturation steps, always revealing three distinct force states. Different models can explain these states. (A) In the `zipper' model, the EC1 domains of cadherin monomers or dimers (here presented for dimers) of two cells trans-interact. Multiple force states are created by increasing the number of laterally (cis-) associating cadherins in the plasma membrane. (B) The interdigitation model predicts that three force states are achieved at the single-molecule level. Here, consecutive and homotypic interdigitation of the EC domains 1-3 increases binding strength: being weak when EC1-EC1 interact, of medium strength during interaction of EC2-EC2 and strongest when the inner EC3-EC3 domains interact. (C) Models A and B are in conflict with structural data obtained from monomeric C-cadherin favoring a trans-`strand-dimer' model. Bent cadherin monomers trans-interact with their EC1 domains through binding of a flexible Trp residue to a hydrophobic pocket (not displayed). In addition to this trans-interaction, hydrophobic domains in EC1 and EC2 can cis-interact. To explain three distinct force states with this model, one has to assume multimer formation. (D) The synthesis of models B and C can explain both structural and functional data. Bent cadherins (here presented for dimers) trans-interact laterally through a sequence of homotypic EC domain interactions. With increasing alignment, this model predicts increasing single-bond strength and shortening of the junction. Owing to the rigid conformation of the cadherin molecules, more than three force states may be energetically unfavorable, but still possible. In all models, EC domains that contribute to trans binding are highlighted in red.

 

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© The Company of Biologists Ltd 2008