Okay welcome back to this ISEV Basics of Extracellular Vesicles. So I'm going to carry on talking about mechanisms of extracellular vesicle uptake. So, there I am, that's me again. If you missed part one I'm Dave Carter, I'm from Oxford Brookes University in the UK. Right then so in part one we talked about, we had a little introduction and we talked about measuring EV uptake, and we talked about mechanisms of EV uptake. Now, I'm going to talk about the roles of proteins and lipids in EV uptake. So we're going to talk a bit about protein-protein interactions. We'll talk a bit about lipids. We'll talk a bit about target specificity and we'll talk about EV uptake heterogeneity and some of the other issues and considerations surrounding this topic. Cool okay so the role of proteins in EV uptake. So we know that if we strip proteins from EVs, this reduces their uptake. So here's a nice picture of an EV with proteins and RNA content and various other things and we know that there are membrane proteins that can be displayed on the surface of the EVs. And if we treat those EVs with enzymes like proteinase K, which strip away proteins on the surface. It reduces uptake. So clearly this suggests that proteins are required in some way, in EV uptake. So that's how those various proteins are involved. So we are beginning to understand this idea that if we have proteins on the EV surface, they can presumably interact with some kind of proteins on the cell surface. And that this specific interaction is part of what mediates. The uptake of the EV and presumably, possibly also some of the specificity in the uptake in terms of cell to cell specificity. So there's that interaction going on right there. No we we start in to find this out by using a variety of methods and one of the main methods that can be used is the use of antibodies. So if we use antibodies which can interact with the specific proteins we think are involved, this then can prevent the interaction between proteins on the EV and proteins on the cell surface and reduces uptake. We can also combine this with the use of other techniques like RNAi and inhibitors and so on to start to build a picture of how EVs are taken up by cells. So let's talk about some of the different classes of proteins that are involved. So the first one I'm going to talk about is the Tetraspanins. Now presumably most people watching this video will already know what tetraspanins are. They are a class of membrane proteins, they're involved in a variety of processes including salatician, motility, regulation of proliferation, amongst others and here I've put examples of what are the most commonly used or known ones in the EV field, CD63, CD81 and CD9 and we think of them as enriched in EVs they're often cited as EV markers. Now they may well be involved in uptake and certainly one thing that we often see is that they are enriched in these tetraspanin enriched microdomains which may act potentially as landing platforms for EVs coming in. So they certainly seem to be involved in the uptake of EVs. [COUGH] For example CD9, CD81 antibodies prevent EV uptake and there is a good body of work that's growing, that they are involved and that they may also be involved in tissue specificity. So for example, magozolos group have done a good work looking at how the different tetraspanins may be involved in cell type specific interactions and uptake. Integrins and immunoglobulins are also involved. So these have important roles in cell to cell interactions and cell signaling. And there's evidence that they may well also be involved in EV uptake. So they immunoglobulin-integrin interactions also may affect EV uptake. So for example the integrin CD11a may interact with the membrane bound ICAM-1 and mediate uptake of vesicles under certain conditions similarly some of the other integrins, CD51 and CD61 may also be involved and blocking these integrins. Using antibodies for example can reduce EV uptake. So, we're going to come back to integrins a little bit later in terms of cell types specific uptake. But they do certainly seem to be involved. Proteoglycans are a type of protein carbohydrate combination, so here we have a protein core. So we have our protein core with these carbohydrate decorations with these modifications on the side of the protein. So these are important in cell signaling and also seem to be involved in EV uptake. Now one example that I'm going to focus on is heparin sulphate proteoglycans, the HSPGs, as these do seem to also be quite important in EV uptake. So EVs colocalized with glypican which is one of the key heparin sulphate proteoglycans. And this actually work that I showed in part one from Belton's group where they showed that exosomes labeled with PKH26 overlap with vesicals marked glypican one, so that suggest that this overlap when you merge these images suggest that there is quite the degree of overlap and so there may be some interaction. There's also other evidence that these HSPGs are involved. So, heparin sulphate for example can block uptake but acting essentially as a deco by interacting without the proteins. Blocking HSPG synthesis also block uptake of EVs, so again evidence that HSPG are involved or HSPGs are involved in uptake. Also heparinase, which constrict this carbohydrate modifications, heparinase treatment can block uptake of EVs. So again this suggests that the interaction is involved in EV uptake. Although interestingly. They suddenly seem to work. And again, this is working in. They suddenly seem to work if you use the heparinase on the recipient cells and not the EVs themselves suggesting that at least in this instance that it's the presence of the proteoglycans on the cell which is important in this particular interaction. But they certainly do seem to be involved although we need to do more work to find out exactly how they're involved. Another class of protein that seems to be involved is the Lectins. So, lectins are proteins that bind to glycoproteins. And again the attraction between these glycoproteins and this specific lectin proteins seems to be important. I'm going to give a couple of examples. The first example is the attraction between the C type lectin, DC-SIGN and the glycoprotein, MUC1. We're going to come back to this in a minute, but antibodies against DC-SIGN reduce EV uptake suggesting that, that interaction is important. Another C type lectin DEC-205 also seems to be involved in EV uptake. Antibodies against it, that bind to it can block EV uptake, as can mannose. And treatment of the cells with mannose which may bind to the lectin, reduce uptake and thus, when the mannose is binding to the lectin, it might act as a decoy and prevent interaction with the EVs. So again suggesting that lectins are involved. Lipids do also have a role in the EV uptake. So lipid composition of EVs is quite different to normal plasma membrane. There's an enrichment in a variety of different lipids like cholesterol and ceramide and other single lipids. Which suggests that they are potentially different. It's hard to tell whether that difference in lipid content is important for its uptake or simply reflects their particular biogenesis pathways that EVs are made by. But interestingly, the treatment of cells with Filipin, which is a cholesterol sequestering agent, does reduce EV uptake, suggesting that it is potentially important. Certainly, cholesterol is important somewhere in EV uptake. So more work needs to be done in this particular area, I would suggest. Okay, so, some important questions in EV research. So targeting and tissues specificity, is the uptake of EVs, cell type specific, or is it not? And I'd say for now, the answer is possibly, it's hard to tell. Some experiments seem to suggest that all cell types can take up all EVs by a variety of different routes. Whereas other publications seem to suggest that EV uptake is very cell type specific, and may be context-specific, and also perhaps uptake mechanism specific. So I quite like, I think the idea of context and tissue specific uptake is quite cool, and it's quite interesting, so I'm going to give a couple of examples of this. But we have to bear in mind that some of these differences in what different labs observe may come down to the differences in isolation, technique, or the different kind of EVs that are being looked at. So we need to do more work on this, but I'm going to give you a couple of cool examples. Okay, so cool example number one is the interaction between MUC1, as we talked about earlier, and DC-SIGN. The C type lect in DC-SIGN. So there's a nice example in the literature of milk EVs. So EVs extracted from milk seem to contain a high proportion, or high degree of this like protein, MUC1. And this specific kind of dendritic cell in this publication that's isolated, interacts very strongly and very specifically with milk EVs containing MUC1. So this interaction here mediates the up of these EVs. EVs from other sources may lack MUC1. They don't seem to have MUC1 and so they're not taken up as efficiently, suggesting that a nice sort of example, potentially, of specific uptake. If we inhibit integrin-mediated uptake using RGD, peptides for example, then we see differences in what different cell types are blocked in terms of their EV uptake capacity. So we see that, when we use these kind of blockers we reduce EV uptake in dendritic cells and macrophages, but not so much in microglia. So that suggests that these different cell types may take up via different routes, or may use different protein-protein interactions in their uptake. One example which I also, one last example which I also want to mention, which I haven't actually written down in the slide. But I think it's a really, really cool example, was recently published in Nature by David Liden's group. And they really beautifully showed that EV's have an important role to play in metastasis in cancer cells. So cancer cells, different cancers seem to have a different tropism, if you like, for what different kinds of organ they like to metastasize to. And this may come down to the protein content of the EVs. So their group showed in this really nice publication. They showed that the different cancer cell lines have different kinds of integrins, so the different integrins in the EVs that they release. So it seems that the different integrins act almost as like an address book, deciding where these EVs can go to, what cells will take up these EVs. And once they get taken up by those specific cells at the specific location, for example, it might be the liver, it might be the brain, or the bone. So, depending on what intergrins the EV has determines which cells, which of these cells take up the EVs and once the EVs go in, they seem to create a pre-metastatic niche. So, they're kind of seeding the soil, if you like, preparing the ground for the later uptake of cells, and thus colonization of this secondary site by that cancer cell. So it's really, really quite cool work, and it shows that there may well be a degree of tissue specificity. But for now, I'll leave that and say, it's uncertain, there's evidence for both cell type specific and more generic uptake. And actually, I think this is a really cool area of research. Okay, another really important question is to do with the heterogeneity of EV uptake, so an important question, which is the prevalent pathway? So, I put a load of different paths, I haven't put them all on here, there are various different pathways. I talked to you in part one, about the different uptake mechanisms. I talked about endocytosis, which I've included in here, I've talked about lipid raft uptake, mediated uptake. I've talked about macropinocytosis, and I talked about phagocytosis. So I'm just going to put these here for now. But the point I want to make in this particular side is can all types of EVs be taken up by all of these different pathways? I think it's fair to say that most methods of preparing EVs produce a degree of heterogeneity in the kinds of EVs that are present. As nice and specific as we'd like to think these preparation techniques are, there is a degree of heterogeneity. And so these different EVs may contain different proteins, may be of different sources. I think it's quite hard to tell at the moment, it's one of the areas we really need to work on as a community. So a question then is if we have these different kinds of EVs in a heterogeneous mix, so we have all these different EV's everywhere with different proteins, maybe different sources. Can they all go in by all the different methods? So can they all, for example, go in by endocytosis? Can all of these different EVs go in by endocytosis? Can all of these different types of EV go in by lipid rafts. Can they all go in by phagocytosis? Can they all enter the cell by macropinocytosis? And again, this links to the heterogeneity of EVs. We just, at the moment, I would say we don't know whether this is the case, whether all uptake mechanisms can take up all the different kinds of EVs. So it could be like this, or on the other hand, it might be slightly different. So, it might be that the heterogeneity in uptake of EVs, is reflected, I suppose, in the different specificities of the different pathways. So, it might be that, for example, let's say we have a class of EVs within this heterogeneous mix, which is blue. Obviously it is not really actually blue, but it might contain a certain protein. So it might be that within the heterogeneous mix, the ones which have this specific protein go in by endocytosis. And another group, the yellow ones here, might go in by lipid rafts. And the green ones might go in by phagocytosis. And the red ones might all skirt around the cell until they can find a way [SOUND] where they can go in by macropinocytosis. And actually, it's hard to distinguish between these two methods. Whenever you use an inhibitor against any of these given pathways, you never completely lose uptake of EVs, which can suggest then, it's harder to tell if that's because if you block a single pathway. You are preventing the uptake of all of these vesicles by that particular pathway, but all of the other pathways can still take up EVs. Or whether you're blocking the uptake of a specific subportion of the EVs. And the problem is that we don't have enough EV markers. We need, desperately, more EV markers. I think this has to be an important push for the EV field to start to look for these different kinds of markers so we can start to get a handle on what the different mechanisms of uptake are with regards to the heterogeneity of EVs. So more work. Another consideration is the use of pharmacological inhibitors. Now, these are great. These are brilliant for starting to unravel the mechanisms of EV uptake. But some are not as specific as we would like them to be, and well this makes life quite difficult. So we here have our picture of the different kinds of pathway. So if we use for example cytochalasin D which inhibits actin polymerization, this is often used as a mark or as a tool to block endocytosis. But it doesn't tell us what kind of endocytosis it is, and to be honest, if you're inhibiting actin polymerization, you potentially are going to have really quite pleiotropic effects across the cell, and we may affect other uptake mechanisms. Dynasore, I still love that name dynasore. Dynasore is also used to block endocytosis. It's a dynamin-2 inhibitor, but dynamin-2 is involved involved in caveolae independent endocytosis and clathrin-mediated endocytosis. So we can't necessarily tell the difference in terms of what dynasore is affecting. So if we see a reduction in uptake of EVs, we don't necessarily know which is the prevalent pathway here. Wortmannin two, sorry excuse me, wortmannin is a drug which is used to inhibit PI3-kinase. And we think that PI3-kinase signaling is involved in phagocytosis. But the thing is, PI3-kinase is also involved in macropinocytosis. So, again, if we use Wortmannin to block PI3-kinase, we don't necessarily know whether it's because it's blocking phagocytosis or because it's blocking macropinocytosis or because of some other pleiotropic effect. Methyl beta-cyclodextrin again, is thought to inhibit lipid raft mediated uptake, but there's also evidence that it may inhibit caveolin-independent endocytosis. So again, this basically means that whatever inhibitor we're using we can't be 100% sure about its degree of specificity on how specific it's blocking that pathway that we're interested in. So we have to interpret some of these results with a little bit of caution. And I suppose for maximum power, if you'd like, it's best to use these inhibitors, but then also combine them with other techniques like RNAi. One final slide of consideration, that I just want to put on there is, could uptake actually be due to natural recycling of the plasma membrane? Could it be this sort of passive endocytosis, if you like? So if we have EVs which are attaching, they're flying around and then they attach to the cell, could it be that the uptake of these EVs? It's not because of some active mechanism which is reeling them in, but could it actually be that they are passively taken up when the plasma membrane is naturally recycled. So the plasma membrane is recycled on a constant basis. The membrane is brought in and is replaced with fresh membrane. So it could be that as the membrane is naturally recycled in, that the EVs that happen to be sitting on that bit of membrane are passively brought into the cell. But this actually seems quite unlikely because of the rapid speed of EV uptake suggest a much more directed and active process. And also the number of different examples of antibodies blocking specific uptakes suggesting that it's a directed active process. So it seems unlikely, I'd argue. Is uptake actually needed? Well, this is kind of full circle now because in one of the first slides in part one, we gave an example of how sometimes you don't need uptake. Sometimes EVs can have their effects by interacting at the cell surface and then activating certain cell signaling pathways. Different vesicles, different mechanisms, microvesicles, exosomes. Could they be going in via the same route, or by a different route? And again, the answer is we don't really know. Most of the studies being done are being done with exosomes. But it'll be really interesting to find out whether microvesicles and exosomes take different routes in. It'll be pretty cool to find out. Okay, so summary for part two, then. I hope I've convinced you that understanding EV uptake is really, really important and also really cool. It's important for a number of different reasons that we've talked about earlier. It's important because EVs are involved in health. They're involved in homeostasis and regulating all these different biological processes. That's what we're discovering and why it's so interesting and cool. They're also involved in disease. If they're involved in health and when they go wrong, they're bound to be involved in disease and that is, indeed, what people are finding. They're also important therapeutically, therapeutic targets and therapeutic delivery vehicles. So really, really important to understand uptake. Protein interactions are involved and lipids may also be involved. There is some evidence for tissue specificity and I gave some examples. EV heterogeneity gives us a big headache and complicates things but also give us a really interesting opportunity because there is load of experiments and cool stuff we can find out. We need to do more experiments and we also need more tools. One of the great difficulties with working with EV's is the low amounts of IMP arterial that we have, but again these are all issues which are fun to work on. There's so many different questions that we can ask. It's such a nascent fields is what makes it so exciting. Okay, it will all be worth it though, because EVs are really cool and fun. And that's why we're all here and doing this stuff, because it's cool. I hope I said the word cool enough. Okay so acknowledgments in case you missed my slide from part one. So I'd like to thank ISEV for asking me to give this nice talk and also for all the cool work that they're going. I'd like to thank everyone in my lab, all the people who are in the lab now and all the people who've left the lab, have worked in the lab in the past. Laura Mulcahy who has just submitted her thesis, well done, Laura, and helped to write the review on exosome uptake. I'd like to thank my funders over the last few years. And also my lovely collaborators whom, sorry I can't fit everyone in because of the font size. But I couldn't fit everyone in. But it's a pleasure working with everyone. And also if you like that Twitter sphere thing, then there's my Twitter thing. So thank you all very much for listening and have fun doing exosome and EV work because it's cool. Thank you very much. See you around. Bye.
