 All right, good morning, everyone. I think because we've got four presenters this morning, we'll try to get things started relatively on time. So I'm going to try to also, in the interest of time, just introduce everyone right up front, just because I want to make sure that Lauren has time to talk about what she wants to talk about at the end and that she doesn't get cut short. So in the order that they are presenting, we have Ryan Constantine here, who is one of our own here from the University of Utah. He's an MD, PhD, and he's going to be talking about UNC 119, which does not stand for University of North Carolina, I just learned, but it stands for Uncoordinated, which is quite different. We have Christina Lippi from Penn State, who we'll be speaking next. She's a Big Knit and the Lion fan. Don't mention Joe Paterno in her presence. She gets angry. And then Max Padilla, another one of our own, we'll be speaking after that. And then we have Lauren Imbornoni from the University of Arizona. She seems to like both Arizona and Arizona State, so you won't make her angry by mentioning either one of those schools. So those are our presenters this morning and we'll let Ryan get started. So in response to that, I will do my best to cram about four years of research into 10 to 15 minutes here. As Dr. Bell just said, UNC actually stands for Uncoordinated because the protein was initially identified in C. elegans in which it was knocked out, the worm became Uncoordinated and was unable to localize and find food. I'm gonna talk about UNC 119 in response to its effect in rods, more specifically mouse than anything, and how it affects trafficking. So to orient ourselves first, we're just gonna look at photoreceptor cells. Here's a schematic of a rod. We have the outer segment, the inner segment. Outer segment houses all of the phototransduction machinery. Phototransduction, obviously, as most of us know here, is the process by which we convert light into an electrical signal. The inner segment houses all the biosynthetic machinery. It makes all the proteins that are used in phototransduction, which then have to be transported through a connecting psyllium into this outer segment. If we look at this blown up area of an outer segment, we see all these membranous disks. These are the disks that house the phototransduction machinery. These cells are exceedingly biosynthetically active in that each of these disks is renewed every 10 days. They move in this sort of vertical fashion, so on day one, this disk is newly formed. By day 10, it's up here at the distal end. It's phagocytose by the retinal pigment epithelium cells, and it's all renewed, including all the proteins involved. So it's a really biosynthetically active cell. The other thing to keep in mind is a lot of these phototransduction components are membrane-associated, and while there's a whole slew of proteins involved in this cascade, I want you to really just focus on transducin for the purpose of this talk. Transducin in the dark consists as a heterotrimer with an alpha subunit bound to GDP and a beta subunit and a gamma subunit. In response to light, rhodopsin's activated, causes transducin to dissociate into an alpha subunit and a beta-gamma subunit, and then it also translocates or swaps GDP for GTP. You see here, most of these proteins are either integral membrane proteins or membrane-associated. Membrane association happens with prenal moieties, either a foreign acyl or a gerinal-gerinal, or acyl moieties, either laurel or meristoil. In the case of transducin, the alpha subunit has an acyl moiety on it in order to tag it into the membrane. The gamma subunit is prenalated to tag it into the membrane. So keep that in mind. And when it's a heterotrimer with both of those lipid moieties inserted into the membrane, it's very difficult to remove that. One of the significant trafficking procedures that takes place in these cells is something called light-driven translocation. In the case of transducin, when the rod cells are in the dark, all 80 to 90% of transducin is located in the outer segment. In response to light, all that transducin is driven to the inner segment. And so this was sort of this, one of these unknown sort of biological processes and what we were actually studying. The interesting thing is, when this process, when light hits these rods, it takes about five minutes for 80 to 90% of transducin to be driven to the inner segment. However, to return to the outer segment in response to return to the dark, it takes three hours. So the question was, why is there this difference? And what is the mechanism behind this? Is it active transport? Probably not, because active transport is one-dimensional. It's done by molecular motors. One-dimensional active transport would be overwhelmed by the amount of transducin that needs to be transported in this amount of time. So we were believing that it was diffusion and we were trying to understand why and how this actually takes place. This was sort of an unresolved question in photoreceptor trafficking biology. This is just an example. In Xenopus, you look at these rods in the dark. We're standing for transducin. In response to light, you see that it goes to the inner segment. So one of the proteins that our lab had studied was a protein called PDE Delta. And this is a known Prenyl binding protein. So some old data from the lab before I started, if we look at a PDE Delta knockout mouse, you can see in wild type, it stands very nicely here in the outer segments. When we take the knockout, you can see a lot of the staining is now found in the inner segments, indicating that there was some problem with transport after the biosynthesis of, in this case, PDE. So this is a known Prenyl binding protein, but if we look at cone T gamma, we see that in the PDE knockout, and remember that gamma is Prenylated, it seems to be transported just fine, trafficked from inner segment to outer segment without a problem, okay? So there must be some other Prenyl binding protein that we don't know about. If we look quickly, you can, this is a structure of PDE Delta. It's got this beta sandwich fold with this kind of cavity in the middle. When we overlay it with another known PDE, or Prenyl binding protein called BroGDI, with this Prenyl moiety in its pocket, you can see that this pocket can accommodate a Prenyl binding protein. So the question is, why do some proteins make it from inner segment after biosynthesis to outer segment? But others don't, even though they're also Prenyl moieties. So we look for a new binding candidate, and we aligned a protein that we were interested in called Unq119 with PDE Delta, and in this alignment we see that they're 30% similar, and if we also look at this dendrogram, we can see that they're also biologically related. Here we see PDE, Unq119, another protein, Unq119B, which we're not gonna talk about today, but they are biologically related if we follow the dendrogram back. So we found this protein, Unq119, we were interested in studying. So we did a simple protein pull down, we used GST tagged human Unq119, we ground up a whole bunch of mouse retina, and we identified this new band, sent it off for mass spec, mass spec identified this band as transducent alpha. Keep in mind transducent alpha is isolated, not Prenylated. However, the transducent gamma subunit is Prenylated like I mentioned. So do we know if we have the heterotrimer or if we have the dissociated transducent alpha or beta gamma? To identify if we were interacting with the alpha subunit or the gamma subunit, we did another pull down, probed for transducent alpha, here's our GST Unq119, and here we identified that we're interacting with the alpha subunit. Probed for transducent gamma, we don't see anything. So most likely Unq119 seems to be interacting with the alpha subunit in the acylo moiety that it's attached to. Then we took a transducent alpha knockout mouse to further identify if we were accurate and you see in the alpha knockout we're not pulling down transducent alpha obviously or still not pulling down gamma, which identifies that Unq119 does appear to be interacting with the alpha subunit between these two experiments. In order for these proteins to be isolated, they need a glycine at position two. We mutated that glycine to an alanine, repeated the pull down, doesn't act. So that goes to show that that acylo moiety is important. It needs to be there. Then we were wondering what the interaction was like. Since it's a lipid tail interacting with Unq119 as we expected, we thought we might be able to disrupt it. Here we're showing the interaction, SC389 is the antibody for transducent alpha, Unq119 interacts with transducent alpha. We had detergent which interrupts lipid interactions. We no longer see that interaction. So we were pretty confident at that point that we had a lipid interaction. Furthermore, we made a synthetic, laurelated, these are the first 10 residues of transducent alpha, peptide. We added it into our pull down. Now we're not seeing the interaction with transducent alpha. And that's because we added so much excess of this, we're able to out-compete the endogenous interaction with transducent alpha. Furthermore, to quantify the binding capacity and the association constant, we took the same peptide with either the laurel or myri-stoyl, laurel is 12 carbons, myri-stoyl is 14, and we used something called isothermal titration calorimetry. Don't worry about the blue line right now. And we see a really nice binding curve showing that Unq119 will interact with these synthetic peptides. In the absence of the lipid tail, we see a flat line indicating no interaction. While this was going on over the course of about three years, and I would say greater than 10,000 crystallization conditions, we were able to actually crystallize, this is Unq119 and we see the same beta sandwich fold, and inside green is the laurel tail of transducent alpha and in gray, we have those first 10 residues. So we co-crystallized that synthetic peptide in the Unq119 cavity. This shows beautifully how this lipid tail inserts into that cavity and the rest of transducent sort of spins out in an alpha helical fashion. Interestingly, the binding pocket showed these strange charged residues, glutamate 163, histidine 165, and histidine 192. And we didn't understand why there were charged residues in what we thought was a lipid binding pocket. And it turns out when we were able to look very specifically at this binding pocket, that they limit the depth to which the acyl chain can actually penetrate that pocket, and there they are forming this very intricate hydrogen bonding network, which provides the specificity for acyl groups as opposed to prenal groups. Then the question was, is GTP and Unq119 necessary for the binding? And so we repeated these pulldown experiments using mouse retina, and we show here that in the presence of Unq119 and GTP, only are we able to pull down transducent alpha. Furthermore, we repeated the experiment in light adapted and dark adapted because we wanted to know if we were pulling down the alpha subunit or the alpha subunit in combination with the full heterotrimer, which we would see in the dark. We don't see that. We see we're only pulling down the alpha subunit in the light after dissociation. We were able to use this, in this experiment, we used a radioactive GTPase assay. And this was important because transducent alpha doesn't hydrolyze GTP in the absence of rhodopsin, which is its guanine exchange factor. However, there is a minimal amount of activity, and so we depleted rot outer segment membranes from bovine retina, basically washed everything away from them, and then reconstituted the system and saw how GTP was hydrolyzed. So when we add the heterotrimer back to rot outer segments in the presence of rhodopsin, because we aren't unable to wash that away since it's an integral membrane protein, we see that GTP gets hydrolyzed over a slow period of time. However, when we add Unk119 back into the system, it's inhibiting it. Lastly, to sort of get at this question that I mentioned earlier about this rate difference, and we made a knockout mouse. We have the Unk119 knockout mouse. We dark adapted, which means we expect transducent to be all in the outer segment. We see that here in the wild type mouse. In the knockout, there's obviously a little bit in the inner segments, things are a little bit different. Then we took these mice and we shined bright light on their retinas for an hour, put them back in the dark. At time zero, you can see that in response to light, a lot of transducent gets shuttled back into the inner segment. After three hours, it's slowly in the dark, it's slowly returning to the outer segment. After six, it's almost completely there. However, in the knockout, even after 24 hours, we're still seeing transducent in the inner segment. We're basically able to take our interaction data, develop our model in vitro, then use an animal model to identify and recapitulate all of this data. Basically, we've come up with this light-induced translocation model of transducent in which in response to light, we have the transducent heterotrimer. GTP gets exchanged for GDP. It dissociates, now that it's dissociated, it's easier to pull these out of membranes, they get transported into the inner segment, recombine on any membrane really that's available, although we don't know exactly, but we assume endoplasmic reticulum is available to do this, Golgi's available to do this. Now in the inner segment, in the absence of rhodopsin, which is the guanine exchange factor that I mentioned, GTP gets exchanged for GDP, or gets hydrolyzed to GDP. This happens, transducent can do this alone, but it does so very slowly. On the order of about three hours, which is what we saw in those mouse experiments. When this dissociates in response to that exchange, UNQ-119 can then come and it basically acts as a chaperone protecting this acyl chain, which is unfavorable out in the aqueous environment of the cell and transporting it back to the outer segment so the whole process can happen again. With that, just wanted to thank people that have helped Wolfgang Bear's lab, which is on the south side of the building. I did a lot of the crystallography of Chris Hill in biochemistry. My thesis committee was instrumental. Northeast structural genomics helped with some of the crystallization. The Moran Eye Center was instrumental in some of the funding. Stanford was actually where we collected our crystallography data on their synchrotron and then the graduate program here. I'm happy to take questions. I know I flew through that. Hopefully found it interesting and it was convincing. Thank you. Questions, anyone? Is it clinically relevant? That's what you all want to know, probably. We don't know, unfortunately. There has been one patient identified with a mutation in UNC-119 who has a late onset rod dystrophy. The protein's relatively new. I think at this point no one has really done a lot of genotyping of patients to really know. So it sort of remains to be seen. We'll see. Cool scientific problem, though. Next person. What you're getting is wacky. And I think it's hard. Just in being totally off. Sure. Well, and it's hard to go through four years of work in 10 to 15 minutes.