All right, times up. So now I am going to choose three representatives. Each one from each group to tell you your best design experiment, to prove that the calcium, by syntactically. And syntactically is serving as Calcium sensor to trigger transmitter release. The simple reason we said before just knocking out and then test if there's no transmitter release does not necessarily prove is required for calcium. Okay, because if you're eliminating snare so it's not a private by neurotoxin. Okay, we discuss that, there's also a note asked me to release. Okay so, the point is to prove that will be calcium to sense, calcium to triggers ask me to release. Okay, or we can do is we can change the calcium concentration and then we can imagine transmittal release. And what we can do is in the welfare condition, just using the [INAUDIBLE] right? And we measure, and we have already discussed that it's power relationship. Isn't it, right? It's a nonlinear power relationship in terms of calcium versus transmitter release. So in the linear faction, if we have a calcium concentration here and this is a transmitter release, then we are going to get a curve. And this is a power relationship curve and equal to three over four okay? Three to four. Okay? Now, so if you only always press an you expect that relationship will be the same or not? Well, you already have the has the two calcium binding domains. And each domain can actually bind three to four calcium. And we always. It still has a sent of calcium bite inside right? And the coopertivity for calcium is still the well test it was still by to the number of calcium. And therefore, when you are using your over express Wild type. The calcium dependency probably will remain the same because the cell molecule, it bind to the same number of calcium, okay. So the calcium dependency relationship will probably read as zip right? So, what will be the way to test, to prove that a synaptic Is the calcium sensor. Well, follow the idea, you can mutate, you can abolish the calcium binding sites, right? You already have these two calcium binding sites, so for example, since you are a good structural biology student, you know, this is C2A domain or C2P domain. This site is important for calcium binding, okay? So, now, you can introduce a single mutation. Okay, and from stratus study, you predict this, it's very critical for, coordinate. For binding to calcium. Okay? Then what can you do? Great. So, the way to do this, run it in, over it's present. You generally. Okay, you replace, or substitute, indogenous Okay? And before you're doing that, before you're doing that, how will you predict that your mutated cell indeed will affect calcium binding. Well, structurally, this is a probably quite critical. But, still, you don't know, right? So, before doing that, in vitro you're mutating it, then you test. Okay, so in the in vitro, biochemical asset, you're first using your vertex, so this is in vitro, in a purified protein. Okay, in vitro, you test the high calcium binding ability, so this is a okay? And then, because making takes a lot of effort, time, and money, right? So, you don't want to put all your eggs In one basket just go ahead I spend all my money all my time just to make noke and find out. Oops it has no effect right? So what you would do it would you make your mutant. Okay this is a calcium binding affinity. You have some biochemical assay in vitro in a purified protein you can test, okay. And then in your mutant single point mutation, indeed you can screen it a different number of mutants. And then you find the one. Ha! This one with the same calcium concentration. That it binds, this is a binding affinity, okay, that it get left shifted. Means that it needs more calcium to achieve the same level of binding, okay. You do that, and then maybe there are some mutants that you found. This mutant actually has no effect. Then you probably will not use that mutant to generate a mouse. And then you found this one out just fine, okay? And then you spent all the effort, one or two years, breeding mouse takes a lot of time. You make this mutant No ki. K I. Okay. Then what do you do? You know Ki net and you culture the neurons and you do that calcium biting s. Calcium dependent. In this case it's the transmitter release, okay? And then you measure a different calcium concentration, you also measure their trigger transmitter release if the relationship between the in vitro. And the in vitro are fitting with each other in terms of freedom from this reduced twofold and this also reduced twofold. And you say. Well, I have still confidence this, indeed, will be the calcium sensor. Because we have this perfect correlation by abolishing only calcium, binding, and then in vivo, you also affect the calcium dependent sensitivity. Okay, and then you also need to do the contrary experiment. Because, again, someone will argue well, maybe this mutant has less vesicles, okay. Maybe the reason that you see this reduction of this transmitted release, maybe because it has just twofold less vesicles or twofold less pre synaptic terminals in that culture neurons. How are you going to do it? Well, to detect the number of vesicles you can do the. You can see the vesicles in a nerve terminals. Right, you can do the EN's since you are the good EN's biologists anyway. So you do the EN's. You count In a in a mutant, whether you have, on average, whether you have the same number of. And whether you have the same number of the synapse. Well you can still do the control experiment. You have some vessical markers of post-synaptic markers. We already know that pre-synaptic neuron release transmitter will be sensed by the Post-snapped receptors. You can have specific marks to stain for those synapse. And you count the density of the synapse and the prediction will be if this effect is only specific to calcium, then there will be no change on average of the number vesicles and there will be no change of the number of synapse. In the control which you're looking, right? So, taking them together, then you'll reach a conclusion where I only ordered calcium binding, and did not observe one, two, three, four five, all your controls that could also. Contribute to the amount of transmitter that gets sent. And then you pull in together saying, this is pretty strong evidence that this is the sensor, only altering the calcium and altering the transmitter release. Sorry, I should have, code should be right shift. Well, in this way. This will create a super calcium sensor. So you mutate this side and then you do the screening and most of them it does not have altered calcium binding affinity, okay? And then you find one, okay? And in the beginning you need to have this calcium, only have a little response. But, in this mutant, you have this one has a super responses. So you create a super calcium sensor and you say ha, this is even greater. I can increase the calcium sensitivity and then you are able to detect a more transmitted release. Or better than that if you're screening. You have found two different mutants. You have some of the sense that it will have the right shift and some of the sense it will have left shift. And then you create two. Mice, and then you're measuring that. And in both of these conditions, if that matches with your prediction, that increases your predicting power, right, because maybe by chance you have one shot if it is, but the probability that both of them, two both fit with your predictions. It's much, much lower, the probability, right? But in reality, it might be difficult to create a super power calcium sensor. Right, because during the evolution, that the protein already evolve into specialized structures that can detect calcium. And indeed, we already know based on structures that C2B domain of this telomere, once it binds to calcium actually the whole domain will insert into the lipids and then drag in the membrane, disturb this vesical and the plasma membrane structure. And then people believe that is critical for trigger transmittal release. So nature already designed pretty fine machineries that can sense calcium and then mediate some conformational changes. So nutrient is much easier to disrupt the existing machinery rather than making better. Because evolution already have those pressures to evolve, to make things better, okay? But maybe you got lucky, right. Since you are doing this screening anyway. You got results and then if you have that Supermutant. Then this will be even more powerful. See, I can make supersensitive sensors, and then once you make an okay mice, in a special region, you say ah-ha! I have made a super-smart mice, because then you can learn better right? Who knows. There are people that as we are going to discuss in the synaptic plasticity there are people making special transgenic mice for example [INAUDIBLE] d receptor that we are going to discuss this class. And then the [INAUDIBLE] mouse in the [INAUDIBLE] and the found this mouse as smarter in a way that comparing the [INAUDIBLE]. It takes less time to remember where is the platform, so they can always spend less time to search for the platform. So that is a smart mouse, right?
