Great, so the essay that we are going conduct is called complementation. Genetic complementation, okay? And the way, how it works is most of the time, we are doing the genetic screening and you are mutating the gene. Most of the gene, the phenotype will be recessive, okay? That is, if you have this hit alone, if the is a deployed, [FOREIGN], deployed a species. If you only have one heat, on here, okay, then this east will not display any thing of type, because of this mutant. Most of the time it is recessive, okay? Inch and two bit, okay? So, now wait to observe [INAUDIBLE] we will self across the east, to obtain the homocytis. Okay? So, so you identify this as a mutant, okay? And if you have another Newton, potentially, you might locate in a different locus other than this locus for this east, okay? But how do you know? Indeed, they are located in a different locus. Well, you can somehow cross them, okay? If they are indeed in the same locus, or if they are not in the same locus, what you observe is going to be, let's just put it this way. Here we put it as a T. A mutant as a T, and this mutant is x. If they are not innocent lookers, once they cross these two, you obtain the mutant, the progeny. This is that, the progeny will be. Something right here. Okay. Then, here you have a well type copy of x and here, you have a well type copy of t. Okay. And both of them are well type. They will be functional. And single built an allele. Because it's recessive it will have no phenotype okay? So in this way if you observed the phenotype from a progeny it's that okay. You say they can complement each other. This is a world type phenotype. Okay? Then you know, they are different complementation group, okay, they are heating different locals, different genes, but if the T happened to be just in the same gene here, then this progeny will no longer have the wild-type phenotype okay. They will have the phenotype close to this one or maybe close to the this one or intermediate, but it will have a phenotype. Okay? So this is a classical complementation asset. So even before you are mapping the gene, which even though today is much easier still takes a lot of effort, you want to classify the different mutations into different groups. For example, after you identify that they are in the same compilation group. Maybe they have slightly different mutation reasons. Some of them will cause frame shift. Some of them will cause a premature stop codon. Okay? Some of them might mutate in a key residue. That is important for the protein function, okay? But again, we are all the same gene, and making this gene less functional, okay? And then you can observe it, okay? So, in this what they did is, after the group, and they could guess. One might be the phenotype because in that case, they were just conducting the electron micrograph analysis, okay, Analysis. And in those case you can see in the wild type or in the mutant, in the optimal temperature, they have the same genetic background. But this is a good temperature that is just happy. And now you can switch to a [INAUDIBLE] permissive temperature, in this case 37 degree, for two hours. And you can observe this great difference in terms of the morphology of the circulatory pathway so in the wild-type-like pathway you can see that this is a nucleus and then this is a vacuole, relatively small, but if you switch to a none of temperature okay. And while you observe there is a huge number of its okay. And this is the mutant sect mutant sect four. And then you can do that with the rest of these 23 groups. That has 23 different gene mutations. And again what I observe is interestingly you observe a lot of different phenotypes. For example this one that seems to have this long extended maybe it's the Okay, not seeing in the regular condition. And this one has this larger [INAUDIBLE], okay, and this one is yet very different from this Extension. And this one has a very small vesicles. Okay? So, for most mutants then you can guess the gene encoding or this essential protein. Maybe involved in the process related to this different trafficking event. For example, keep trafficking out to this organelle and not trafficking to the plasma membrane will lead to a larger vacuole, okay? And this seems to be accumulate with a lot of things in the Right? So you can sort of try to guess where the protein might function. And again, later the development of the genomic approach. People can clearly map mutation. And in some of the genes are just Homelock is full of snare compacts that we mentioned before okay. So in the early 1990s, with the identification of snares, biochemical, tossology approaches. And then people find out, well the sense require for transmitter release, actually it's almost [INAUDIBLE] to the essential genes for east to secrete. They are a similar set of proteins, they are [INAUDIBLE] and the whole field become very excited, because this is showing the evolution conservation of the same set of molecules that get to use or specialize further to control transmitter release and that lead to the identification of smears with a transmitter release. Wait a moment. These are different mutants and different Structures that you can see. There's ERs and vesicles and even names like Berkeley bodies, because there are some structures that you have no idea, you cannot see in so they name it Berkeley bodies. So, without we want to conclude the past that are related to identification of [INAUDIBLE] molecules of using machinery. But that's actually important works from [INAUDIBLE] Richard Chater, they all contribute significantly into understanding the release process. For example, Tom also got his Nobel Prize recently. And he, with help from his colleagues, collaborators, identified synaptotagmin. As the that can bind to calcium entwined with snares and D2 transmitter release. And here's a question for you. How do you demonstrate synaptic [INAUDIBLE] indeed is the calcium sensor mediating transmitter release during synapse transmission. Okay? You can say, I'm just going to note it out synaptic. Then the question becomes, for example if you note it out the that's much a reduce of this fast synchronous transmitter release. Does it prove that it's an? Is it indeed a calcium sensor media transmitter release? Well if you are looking out that we mentioned, there's also no transmitter release. But we can say that is a calcium sensor? Okay? So, we are going to ask you guys to tell why you think, how do you design experiment to prove it's not [INAUDIBLE] it's the calcium sensor for transmittal release. And during the vesical release we didn't have time to discuss in detail, but was the vesical filled with plasma membrane? The plasma membrane eventually will get recycled back. Okay, internalize recycle back to generate new empty vesicles and those vesicles will be refilled with transmitter release with different neurotransmitters, okay. So there is also very essential protein machineries that it will regulate that process. So the synapse transmission involves both of the release of transmitters and the recycling of those past membrane components. Because if you think about it, if you only release this [INAUDIBLE] cycle, eventually our [INAUDIBLE] time depends on nerve activity. All the [INAUDIBLE] will get released [INAUDIBLE] minor terminals that they have normal transmitters indeed it flies people identify key molecules for example the so called mutant which encodes for GTPase called dynamic that is essential for internalization of fission of this internalized vesicles. To generate new vesicles, and those are also a integral part of exocytosis transmitter and then there's also the additional question, how do those machineries know that there is already exocytosis and they need to internalize. Right, because if you think about it this process also need to be regulate. If they don't regulate it they just keep internalizing and then probably you don't have that much membrane left. Or you get Internalize, become extra vesicle which doesn't happen. And in a regular nerve cells they need to be in balance to keep enough plasma membrane and enough supply of synaptic vesicles, okay?
