What we found was that both cellular geometry and enzymatic activity
of negative regulators are needed for the formation of these micro domains.
You might remember in one of the last slides of the previous lecture.
I showed you that ten dendrites, de, dendrites with the small
diameter favored the formation of sharp gradients of cyclic AMP.
As compared to thick dendrites.
So we explored this property further with thin and thick dendrites
but now adding another variable which was, reading concentrations.
Or reading levels of the negative regulator phosphodiesterase [LAUGH].
Also called PDE or PDE4 in this case, varying levels of the phophodiesterase.
So when there is no phosphodiesterase or negative regulator there's a lot
of cyclic AMP can be formed and then loss signal flows through.
So there are no gradients really observable.
At moderate levels of phosphodiesterase, one starts to see nice sharp gradients.
And at high levels of phosphodiesterase all the cyclic AMPs
dissipate, and all the gradients get to be dissipated, as well.
Now, you can see that the, the key in, this happens,in the case
of the thin dendrites, but not so well in the case of thick dendrites.
Although at one particular concentration of phosphodiesterase
there is some gradient that's seen here.
This effect of the phosphodiesterase which degrades cyclic AMP.
on, on which is out here, on MAP kinase, which is down here
it's sort of distill prediction of how this network is configured.
And so, we try to use this prediction to test the whether one could see
this in real life experimentally and for this we used brain slices.
Stimulated the slices with a bit [INAUDIBLE] receptor ligand.
And ask the question, does, is their MAP kinase
activity in the dendrites, or, or the cell body.
And as you can see that when the phosphodiesterase
is inhibited, with the chemical inhibitor, there are no gradients.
Similar to the condition here and there is for a
MAP kinase both in the cell body and the dendrites.
Otherwise, the MAP kinase is largely localized to the dendrites
and there is very little in the cell body of each.
Here are the corresponding simulations for this.
Okay.
So if these micro domains sort of require both enzymatic
activity of the negative regulators and the shape or size of the cell.
One can sort of ask the question.
Is this, are these conditions enough to transmit the
spatial information from upstream components to downstream components?
So in, in this particular set of simulations we
follow the flow of spatial information within the dendrite.
And it's this one white dendrite in this particular neuron.
And starting with cyclic AMP the signal goes to protein kinase A.
And from there it goes down to MAP kinase and to to a couple of pathways.
So you can see that the PKA gradient is similar to the MAP kinase gradient.
However, when the signal that goes to the Mac.
Neither RAF nor MEK show good gradients.
In contrast, the protein kinase A gradient,
is recapitulated by this second negative regulator, PTP.
Which is which is phosphotyrosine phosphatase, which is the
regulator of the tyrosine phosphorylation side of MAP kinase.
So you can see that the gradient, this
looks similar to this this looks similar to this.
Indicating that the flow of gradient was likely to be to this PTP pathway.
Again since although there was a cyclic
PKA gradient because there was no MEK gradient.
We tested whether this was observable in experimentally.
And we saw indeed that MEK is activated both
in the dendrites and the cell body in these experiments.
So, from these experiments what one can sort of conclude
is that the flow of information regarding the activity state.
Which means protein kinase A activates RAF.
B-RAF activates MEK.
MEK activates cyclic AMP forced through this RAF MAPK kinase pathway.
However, the flow of special information.
That is how much MAP kinase is activated at a certain location.
In response to the similar spatially
restricted activation of PKA flows through PTP.
You can again clearly see this is the same simulation as before.
The PKA gradient matches the PTP gradient that matches the MAP kinase gradient.
So what does simulation which was indeed a very surprising finding
was that the flow of spatial information is through a different arm.
As compared to the flow of information with regard to the activity.
Of course, the model of the computation predicts, the
per that PTP is going to be very important.
And we tested, therefore, the importance of PTP for loo, by looking at
what happens when you ablate the PTP by using antisense oligos.
And what we found is that when you
knock out the PTP, the MAP kanise gradient disappears.
And you get MAP kinase all over the cell body,
which is what is shown here, as well as the dendrites.