[MUSIC] So a reminder about what we've learned last time. So we've learned that lipid synthesis take place very often far away from the site where lipids are required inside the cell. And this is metabolism and signaling. And we have seen as well that some lipid are even required at two different places inside the cell. For example, in the plasma membrane for the phosphatidylserine and also in the mitochondria. So they're really logistical challenge for the cell to dispatch those lipids, at the proper place and also at the proper time. And we have seen that series of very specialized protein, lipid transfer protein can also assist with some of those functions. And we have also seen that those proteins often localizing membrane contact site in very specialized organelles. So what we see here is a schematical representation of a lipid transfer protein that bridges two membrane, the one of the endoplasmic reticulum and the Golgi. This oxysterol binding protein contains several domains that can tether simultaneously both the ER And the Golgi, and this the ph domain and also a ffat domain. So this leads the lipid transfer domain free to swing around and to transfer lipid from the Where cholesterol is synthesized to the Golgi, where cholesterol accumulates. And what is very interesting in this case is that on the way back from the Golgi to the ER lipid transfer domain does not travel alone, but instead carries another lipid and this is phosphatidylinositol 4-phosphate. And then this lipid is very abundant in the Golgi and when it arrives in the endoplasmic reticulum, it gets hydrolyzed into phosphatidylinositol. So in this case, this is this hydrolysis of phosphatidylinositol 4-phosphate that drives the directionality of the transfer. So we've seen that the transfer of cholesterol in this case is coupled to the transfer of another lipid, phosphatidylinositol 4-phosphate. And we've seen as well that the subsequent step in metabolism is what drives directionality, and by this mechanism, this lipid transfer protein can create lipid gradient. So we've seen that lipid transfer protein can have many different functions. We have seen that they are able to transfer lipids across membrane. We have seen that in some cases, they can couple metabolic pathway. For example, cholesterol synthesis with phosphatidylinositol phosphate synthesis and it can also contribute to the formation of lipid gradient inside the membranes. And this is not all, some of those transfer proteins can also transfer their cargo to other enzyme, and they can work as lipid chaperone. They can also transfer their lipid to other lipid transfer proteins and work in small pathway networks, or transfer the lipid to a transcription factor that will then translocate to the nucleus to activate transcription. Or transfer the lipid to a transmembrane membrane transporter for the secretion of those lipid. Another important observation is that those lipids transfer proteins sometimes also contain signaling domain like kinase domain or phosphatase domain, and that open binding of their cargo. This will lead to the activation of the signaling pathway who best than the transporter to sense the metabolic state or the presence or absence of a lipid in the membrane. So how can we measure the activity of this lipid transfer protein? So many biochemical assay have been developed to measure many biochemical properties of those proteins. Essentially, the way they can shuffle lipids across membrane, and also the way they can sense biological membranes. We have seen that this is very important for their biology. So first a few words about the lipid transfer assay for this unit 2 liposome to artificial membrane, one that contains the lipid of interest. The yellow lipid in this case, phosphatidylserine and one that doesn't contain that lipid and instead contains a fluorescently labeled lipid, phosphatidylethanolamine in this case, a couple to a green dye. And we can then measure the accumulation of phosphatidylserine in this liposome using a probe for phosphatidylserine. In this case, this is a protein, Annexin V, that is fused to a red fluorescent dye. So when phosphatidylserine is transported to the acceptor liposome, the accumulation of phosphatidylserine is detected and the liposome becomes red. So this is just to see how this works in a real assay. So you see, on the upper part, an acceptor liposome, fluorescently labeled phosphatidylethanolamine and on the lower part. You can see the efficient transfer of phosphatidylserine from the donor liposome to the acceptor one when both donor liposome and Osh6 to transport on our present. As a read out, we could also have an unbiased way to do it and this is based on mass spectrometry-based lipidomic. The advantage here is that it doesn't know require a probe for your lipid and it's completely unbiased. We can really see any lipid that is being transported and you can see that in this case, we also see that phosphatidylglycerol is transported. So there are so many assays that have been developed to measure the ability of protein to sense membrane and recognize lipid. Many of them are high throughput so available for screening. So there are prerequisites or that should be applicable to well-based assay. They should also be very versatile in terms of both protein and lipid. Ideally, they should present the lipids in a way that is very close to biological membranes, so that its physiological. And also very important, as I say, should be quantitative so that we can monitor the effect, for example, of a mutation on this binding activity. So several assays have been developed at fulfill some of those criteria. One is a lipid overlay assay. Here, we immobilize, we spot lipid on a solid surface. And then we overlay this lipid array with recombinant protein and we can detect where the recombinant protein interact with lipid or it is deposited using antibodies, for instantly, labeled antibody. The advantage of this assay is that it has been very widely used and there are commercially available membrane of lipids that can be purchased. The main disadvantage is that the lipid here are really presented as bulk lipids. They don't form a biological membrane, so they're not in native state, so this assay is prone to artifact and is not quantitative. Another assay is based on protein array. Here the protein is immobilized on a solid surface and overlay with a solution containing artificial membrane and liposome that is fluorescent. So here, the assay is more physiological, the lipid are presented in a more physiological condition in membrane bilayer. The main problem here is that the liposome are difficult or tedious to produce that we cannot store them for a very long time. And that's very difficult to very multiplex and test many different types of artificial membrane in parallel. So in the group, we have developed a new assay where we could immobilize liposome, an absolute surface and then measure the recruitment of recently labeled protein on the surface of those artificial membranes. And this assay really overcome many of the issue that we have discussed, they are really multiplex. We can measure tens of thousands of different membranes in parallel, and they're quantitative that is based on, for instance, microscopy. [MUSIC]
