Start the simulator. Once, the initial biochemistry of a simulation has been set you can use the simulation description file (.sdc) to start the simulation. Click cancel to proceed and create such a file.

Click "set model file".

Select the file with the model Click "Open".

Click "set geometry file". The select "TwoCellContact_static.geo" and click "Open".

Click "Accept".

Now, we can populate the regions of the cells we defined with molecular complexes that are not inter-cellular and do not contain "Don't care" tags. In the case of the Ecad model, we defined Ecad_unbound as the only complex fulfilling both conditions.

Set a concentration of 50 molecules per square micron of Ecad_unbound for both cells. Leave the "Maximum Complex Size" at 6. This means that the complexes in the automatically generated reaction networks of our model will contain up to 6 molecular components. Click OK.

For the simulations with static morphology, we will not simulate Potts Model dynamics. Disable the "Enable cellular potts model" checkbox or set the "Time between Potts steps" to a value larger than the expected duration (in simulated biological time) of the simulation you want to perform. Click OK.

Now save the simulation description you just generated in a file, e.g. "Ecad_static.sdc". After you clicked "Save" the main simulation interface will appear.

In the "Simulation Window" you will see the two cells in contact.

You can move the cells with the mouse.Click into the display area to activate it, then use the "x" or "z" key to zoom out or in.

In the menu list click "Slice Views" -> "Slice View of Entire Region".

The "Slice Selection Window" will now contain the cells and will allow you to define slices through the simulated space and select molecule complexes for concentration profile display. Note that you may initially only see the blue layer representing the slice of volume elements selected by the initial position of the selection plane. In that case, move the mouse within the 3D display to obtain a different aspect. Again, you can zoom in and out by pressing the "x" or "z" key after having clicked in the display area.

Click "Align Cut" XZ", select "Surface Concentration", "Molecules" in the combo box, select "Ecad" and click "Update Display", then click "View Slice".

The "Slice View Window" will display the color-coded concentration profile of Ecad. Since all membrane elements initially contain the same concentration of Ecad the color code will be homogeneous. Click on one of the membrane containing volume elements to see the local concentration. It should show 50 molecules per square micron.

Back in the main "Simulation Window" click the "Time Step:" button and set a time step of 10 seconds. Click OK.

Click "Simulate for X Seconds" then set 600. Click OK.

The simulation will perform 60 update steps of 10 seconds and then pause. Depending on the performance of your computer this may take a couple of minutes.

Click "Update Display" and then "View Slice" in the "Slice Selection Window". The "Slice View Window" should now show a significant accumulation of Ecad molecules at the edges of the cell-cell contact.

Click "Volume Elements intersected by line" and then draw a line along the cell-cell interface. Click OK.

A window showing the concentration profile along the line will appear. The concentration plotted corresponds to the 2D concentration of molecules that are directly anchored in the membrane belonging to the volume elements we drew the line over. The total amount of molecules at the interface -- as seen by confocal microscopy -- would be twice higher (the sum of two membranes).

In order to obtain a concentration profile that fully includes inter-cellular complexes we can switch to a slice plane parallel to the cellular contact. Click "Align Cut" ZY" and move the slice into the contact. This can be monitored by zooming out in the Slice Selection Window until the selected slice becomes visible.

When the slice is in the cell-cell contact the molecular contents between the cells becomes visible. Click "Launch/Update 2D View" again. Note that the surface density is calculated per volume element intersected by or touching the slice selection plane by averaging over all membrane elements of the volume element. This means that the volume elements at the periphery that have one membrane in contact with the other cell and some others without contact will show a considerably lower Ecad contents. To exclude the membrane elements not parallel to the selection plane select 'Exclude Orthogonal Membrane Elements'.

In the "Slice View Window" draw a line along the cell-cell interface again. Click OK.

Now, the concentration profile includes all molecules that are bound to the membrane the plane is closest to. This means that trimers and tetramers contribute 3 or 4 Ecad molecules, respectively, leading to concentration peaks of more than 110 molecules per square micron. Note that this is not simply twice the single membrane peak concentration (which was around 65 molecules per square micron) since those molecules that are only attached to the opposite membrane (such as monomers and cis-dimers) are not counted.

Start the simulator. Click "Cancel" -- since we have no Simulation Description file for the dynamic simulation yet. Select the model file Ecad.dbf.

Then select the geometry file "TwoCellContact_small_dynamic.geo". Click "Accept".

Again set initial concentrations of 50 Ecad_unbound for both cells. Click OK.

Now, set "Time between Potts steps" to 10 and set the "Membrane molecule redistribution mode" to "Reconst. keep Nb.". This redistribution mode means that when regions of the cell surface are extended (as the cell grows) the concentration of these new surface regions is copied from the original region concentrations (see documentation of the simulator for a full explanation of the options). The inter-cellular molecular bonds determining the adhesive contact strength are given by the associations mediated by the Trans Interface sites of Ecad. Set the "Fraction of surface elements used" to 0.1. Set "Area Conservation" and "Curvature (Coherence)" to 0.5. Set "Interaction Strength" to 10. Click OK.

Save this configuration as "Ecad_dynamic.sdc". Click "Save" to start the simulation.

The simulator main Simulation Window now shows the two cells with a small contact region.

Set the time step to 10 seconds and -- after clicking "Simulate for X Seconds" -- set 3600. Click OK. Simulating the dynamic morphology of the cellular interface driven by Ecad bond formation may take a while, depending on your computer's performance up to several hours.

In order to explore the contact interface select "Slice View of Entire Region" from the "Slice View" menu.

In the "Slice Selection Window" click "Align Cut:ZY" then zoom out by pressing the "x" key (you may have to click once in the display area to activate the focus) until the slice is visible. Select "Molecules" and then Ecad and click "Update Display".

Move the slice until the contact becomes fully visible. To exclude the membrane elements not parallel to the selection plane select 'Exclude Orthogonal Membrane Elements'. Click "View Slice".

Adjust the color gradient wheel until the concentration profile is well resolved. Click "Volume Elements intersected by line" and then draw a line across the interface. If the line is parallel to the grid lines you may choose a line width to average over more than one volume element perpendicular to the line. If the line crosses grid lines its width will be automatically 1.

The contact shows a relatively even accumulation of Ecad with a slight central preference.

To see how the diffusion coefficient of Ecad influences the structure of the cell-cell interface you can set a 10-fold lower diffusion coefficient of 0.001 square microns per second (a value typically found for larger transmembrane structures).

Re-running the simulation for 3600 seconds shows a preferential accumulation of Ecad at the periphery of the contact.

The concentration profile can again be explored quantitatively by drawing a line across the interface as described above (11).