 I'm pleased to introduce Alicia Gross who's our guest speaker today. She's of course disappointed that she's not out here skiing and doing doing all the winter things here, although she was telling us they did have a snow day yesterday in Alabama they got a whole inch of snow and her kids got the day off. So, but we have a bit more snow here finally this winter. So, Alicia is from New England and she did her bachelor's degree in biochemistry at University of New Hampshire, and a PhD in biochemistry at Brandeis University. She did a postdoctoral fellowship with Ted Wenzel at Baylor College and then has been in Alabama since about 2006 I think and has worked at worked her way up through the ranks, where she's currently an associate professor with tenure at the at the University of Alabama Department of neuro neurobiology, and I also see she's an assistant dean for faculty onboarding and I don't know what that really means but I guess I'll learn about that when I talk with her later today. I'm like the welcoming committee. Okay, all right. So, the reason, obviously, as we're going to learn Alicia does some really great work on neurobiology of the retina. Our connection goes back almost 10 years when we were Alicia and I were on the program committee for Arvo in the biochemistry section so we work together. We work together for about three or four weeks every year right around ruining our Christmas breaks doing, figuring out and making a good program reading through hundreds of abstracts and trying to formulate a good Arvo. So, with that, I will let Alicia take, take the lead and give us a great talk this morning, and she will also be giving a noon more basic science seminar at that time. Thank you. Thank you Paul for inviting me to come again I'm sorry I can't be there in person more than you can imagine because I would love to be there and see some good friends that I've known for throughout the years. I'm going to try to, I have this on two screens so if I look over here it's because I'm trying to look at your faces. So if, if you have any questions please interrupt me, I have no problem with just straight up. I'm pushing the space bar and shouting out a question so I would really welcome that. So my lab is interested in the molecular mechanisms of retinal degenerations and what happens on a protein level when, when rod cells are formed and when rod cells degenerate what happens on subcellularly. And we have, we have several models of different, different retinal degenerations in the lab we work with transgenic tadpoles. These guys have baseball bat size photoreceptors you can express whatever you want transgenically under the rod option promoter really easy and in two weeks we have beautiful retina you can see here. I'm not going to talk about tadpoles in this talk I will in the next seminar. The work that I'm going to be talking about is on the backs of these guys these are these are Trent knock in and knock out mice that we have made and generated for these projects. So, in case you decide to fall asleep in this talk. How do I. Trying to advance. Okay, if you do fall asleep in the middle of this talk, these are the take home points. So I'm hopefully going to be convincing you that the protein bbs five is required for cone cell health and retinal function in cones but not so much in rods. And MK a six is critical for for rod cell health and homeostasis, but not so much in cones. Okay. So let's take a little step back. And in this crowd it's kind of, hold on, it's kind of silly to talk about. I can't advance my slides. How did I do it last time. I'll do it the old fashioned way just click on that tile. It seems silly to talk about where photo receptors are but humor me for just a minute so this is my cartoon version of a sagittal section taken of a vertebrate eye and you can see that light comes in through the cornea and gets focused on to the lens where it further gets to the back of the eye cup here on the neural retina in the outer retina sit these rods and cone photoreceptors and so I these are the the two cell types that my lab is mainly interested in. If you were to turn those over, you can see that they here in this scanning electron micrograph image of a mouse retina that I took at Woods Hole some years back. And you can see how gorgeous the retina is. Right. So we have all of the outer segments of the photoreceptors lined up these are the, this is the portion of the cells that that capture a photon of light. Here's the inner segment region, the outer nuclear layer, the synapse and then the inner retina is down below there and this is a chunk of retina that is split so you can see down in that the kind of crack down in there to see that the, synapses, but here you can see this is the top of a chunk of retina, you can see all of those photoreceptor cells they are the outer segments look like shag carpeting. And these big RPE cells sit right on top of about 50 of those sit on top of outer segments, and they provide they have a multitude of jobs. So look at these, these photoreceptor cells in this cartoon format here, we know that photoreceptor cells are neurons they have a synaptic ending. They've got a nuclear region here, all these, all these nuclei there, and then an inner segment region which contains which houses all of the cell machinery necessary for its function right we've got mitochondria and Golgi and microtubules, all in the outer section here, in that inner segment is separated from the outer segment, via a connecting psyllium or a transition zone. And so this is the primary psyllium it's highly modified in photoreceptors they're about in rod cells they're about 2000 membranous discs that are that are vesicles swish down like pancakes, but 2000 of those are stacked on top of each other. So these are post mitotic cells, but by no means are they are they static, these are very, very dynamic cells we know that RPE cells have phagocytose the distal tips they come in and they they plop off about 10% of the other segment discs, every single day, and that causes a turnover about a million molecules of rhodopsin gets expressed in the inner segment, transported through this, this connecting psyllium region here, and then packaged into that doesn't happen with high fidelity, and astute precision, then the cell undergoes apoptosis and takes out all photoreceptors with it. Okay, so my lab is interested in the molecular mechanisms of that process, and of the proteins that help sort. Who goes where. Okay, so if we were to take a look. A very common mutation in rhodopsin, one of my favorite proteins. We know that rhodopsin is expressed highly in the outer segment so this is a cryo section taken from a mouse and stained in red for rhodopsin you can see it's really highly enriched in the other segments very little in the inner very very little if any down in the synapse and in the nuclear layer, but look what happens if you mutate just the last five amino acids of rhodopsin. Here you can see massive protein this localization q344 tur rhodopsin is found in human patients with retinitis pigmentosa. So really does this protein miss localized this mutant rhodopsin but it takes the wild type along with it. Okay, and so, so part of my lab is interested in this process the molecular process of determining how rhodopsin gets to the outer segment. I'll talk to you a little bit about that in the next seminar. But I'm also interested in the proteins that are involved in the connecting psyllium region here. So let's take a look a cartoon look at of that region. You've heard some excellent. I'm sure you've, you've followed Wolfgang's work and his he's done some excellent work on rabate and Rob 11 in mice. And in that's in the transcolging network and we're not going to talk about Rob 11 today, although my lab is played with that as well. What I am going to focus on though is the baby zone in the transition zone. And so these are, this is a longstanding collaboration that I've had with Dr Brad Yoder who is here at UAB. He's, he's at the chair, he's the chair of cell biology right across the street over here. I'm interested in the transition zones of primary cilia in particular in the kidney. And so we, we fit well together. He has helped. Let's see. We have a series of mice that we have made mutating or knocking out these proteins to help figure out what their roles are. And I want to focus right now on bbs five. So bbs five is a found associated with Barty beetle syndrome. So Barty beetle syndrome is is a so leopathy. It's very rare. It's only about one in about 100,000 worldwide. It varies from population to population. It also varies within family. The severity of the disease varies within families. It is. Here's a patient with Barty beetle syndrome. You can see this, this young man is obese is polydactyly. He has hyper gonadism, polycystic kidneys learning disabilities, brain loss, but important for my research and for this talk, retinal dystrophy. So with Brad, we made a knockout mouse, a congenital knockout, we have a flux knockout that I'm not going to be talking about today, where if you were to take, take tissues from this animal, you can see that in a wild type retinas, there's a blood here showing really intense staining of bbs five in the retina and bbs five in the kidney, but it's lacking in the knockouts in both bbs five retina and kidney knockouts. You can also see that it's that it's recapitulating some of the phenotypes seen in humans right this this mouse is obviously obese these are h matched litter, h matched animals. Okay, so we wondered. Is there any phenotype in the eye and one thing we can do quite easily is just histology to look to see if the retina is degenerating and this was done by a former graduate student in my lab. Katie Bale she's now doing a postdoc with Michelle Purdue at at the VA and at Emory right now so she just left my lab just a little bit ago but this is her dissertation work. So the other with Katie and Brad, we looked at the two month old animal and a nine month old animal and so you can, we can judge the overall health of a retina by simply counting the number of nuclei and the other nuclear layer. They do stack on top of each other and so you can just count up and see, see that in wild type animals here in red. You can see a nice healthy retina. As you as you pan across the retina itself there's the optic nerve right here. We see a slight decrease in the numbers of nuclei in the two month old bbs five knockout. Now these animals are hard to eat they're really hard they're difficult to raise. But you can see that the retina is slightly getting thinner, although it's not statistically significant at this at this age. So look what happens in the nine month old animal. We definitely see retinal degeneration we see the number of nuclei markedly decreased statistically significantly decreased in the nine month old animals. We know that the outer segment lengths are getting smaller so we can stain the retina with wheat germaglutinin which stains the sheets of photoreceptors we can then measure the outer segments, and we see that the outer segments are getting smaller. So we know in cells, when a cell is dying or trying to save itself one of the first things it does is retracted psyllium. There's no, there's no need to have a psyllium sensing the environment. If it's dying it needs to save itself so it takes that that psyllium back in retracts it and then and tries to save tries to save itself. Here we can also see that we can look for cell death by staining with by doing tunnel staining on these on these retinas. We do see some tunnel staining in the two month animals but we see markedly increased cell death as shown here in red but with the tunnel staining. So in the, this is a very slow degeneration right this isn't this isn't a rapid degeneration we're not talking about an already one mutation where the retina is dead and degenerated in just a couple of weeks. This is slow but it is degenerating. And so that tells us that BBS five is critical for at least the overall health of the retina right of the photoreceptor cell layer. We know that proteins translocate across that that psyllium in different light levels. So if you take an animal and dark adaptive for some time 30 minutes an hour in complete darkness. But typically red ops is going to be in the outer segments right it's a red ops and isn't as a transmembrane protein it lives in those disks, it's not going to move. Transducent is in the outer segments as well it's it's right up kissing up next to the disk membranes, ready and rating it's poised for a full time of light to come through and activate a red ops and so we can, it can activate photo reconstruction. In contrast, arrest in is down in the inner segment region. And that's because we think that's because transducent and arrest in are about one to one molar ratio, they're really concentrated and there's not a lot of space in other segments so you really want arrest in out of the way in the dark. Let's see. But when you light adapt the animal, you see this massive translocation of, of pro of these proteins arrest and actually doesn't get out of the cell this was just an average arrest in over there, but we see arrest and traffic up to the outer segments and it's down in the inner segments. And so we wondered if this protein movement in the light and in the dark. It holds true in the absence of bbs five. Okay, so we, we looked at this and that in the bbs five knockout animals. And these are the two month old animals that we have dark adapted red ops and is where it's supposed to be. Right, we see a little bit of red ops in this localization not so much. See just a little red ops in this localization. Transducent is where it's supposed to be in the outer segments and arrest in is where it's supposed to be in the inner segments and down by the synapse and throughout the the cytoplasm. Oh, I wish I could just use my clicker. Oh, there it is. Oh, hold on. Technology so much fun right in the light. Red ops and is where it's supposed to be and transducent is where it's supposed to be. Look what happens with arresting. So we see arrest in miss localization. We see arrest in down at the scent down at the synapse through the new auto nuclear layer. We see some in the inner segments. We don't know these are just static pictures we don't know if arresting has gone up and is leaking back down, or if it's having a heck of a time you've been getting up through the that connecting psyllium. So that's, that's studies that were actually using photo switchables tagged arrestings to be able to answer those questions. So what about function, does this miss localization of arresting have anything, does it affect the function, or does the absence of bbs five affect the function of rod cells, and we can measure overall retinal health and function by use it by by a lecture, and so we've done that here so this is just dark adapting an animal and scotopic lights, we've placed a contact, not a contact lens electrode just a electrode onto the corny and you can measure the overall signaling upon very low light levels in here this is very low light levels we've delivered on to on onto the eye. And then you can see a wild type animal has a nice a wave, and it can come in b wave that's the that that's mostly due to the bipolar cells here. Look what happens in the bbs five nulls in purple, a wave is markedly decreased. Okay. It's even more prominent and in underscore topic but brighter light levels you can see that that that the amplitude of the a wave that is due to the rod photoreceptors is diminished. Okay, as is the b wave. So we can analyze those data here. So the a wave amplitude in wild type is significantly higher than it than it is in the bbs five knockouts, as is the b wave so we know that the rod cells are not functioning very well under scotopic conditions. We can also measure the time to peak to look at the overall kinetics. So we know that there is some bbs. There is some arresting that is up in the connecting psyllium of rod cells clay Smith showed that bbs five holds on to a small little amount of of arresting in the outer segment. And we think that it's, it's, it's kind of the Sentinel arresting that immediately shut off the the signaling cascades it's. We have such fast turnover of of the photo transduction cascade. So we wondered, we can look at the latencies that the times to peak their spot on so that the in the absence of bbs five, we're not seeing any kinetic changes of the photo transduction cascade. Okay. So what about bright lights, bbs five nulls really have non functioning cones. So you can see this is a nice a nice a and a b wave in in wild type animals but the pink here the bbs five nulls there they're almost flat line. Okay, we can measure the a wave amplitudes and the b wave amplitudes here. And you can see that there's there's really just non functioning cones. So what about the cone risk, the cone photo transduction proteins right there are some differences between rods and cones and the expression of of different photo transduction components. So arrest and forest found only in cones it's not found in rods and and arrest and should be in the outer segment and bright lights and you see some in the inner segments. And that this is we've stained with the help of Cheryl crafts amazing antibodies we've stained cone arrest and for here in green. You can see really nice outer segment expression inner segments you see some at the synapse. But look what happens in the bbs five nulls you see a market increase at the synapse it's really not properly localizing Similarly, we can stain for the options. So the medium wavelength option the green cone option is expressed in mice and we can stain for it is really nicely in these outer segments the culprit those cones are and but look what happens in the bbs five nulls. So we see a M cone option miss localization to the inner segments and down to the synapse. Okay, so the M cone options are miss localizing as are the S cone options. So we see here stained in purple. Instead of seeing as cone option in the outer segments and some in the inner segment region, but none at the synapse we're seeing M cone as cone the short wavelength blue cone options down at the synapse. So the market miss localization of this of this for photoreceptor in cones. Okay, so the cones also express a different alpha sub unit of cone translucent. It's so we can we can selectively look for cone out cone alpha. So we can selectively look for cone translucent or Jeanette to here in the outer segments it should be under bright light conditions very little in the inner segments synapse. But look you see it littered throughout the entire cell in cones in the bbs five knockouts. Something else to look at is the alpha sub unit of the cyclic nucleotide gated channel that's only expressed in cones and it should be up in the outer segments we think that this green here is background. You can really nicely see the outer segments. Look at the staining here it's almost punk tape the outer segments morphologically may be looking different on the ultra structural level. So we were curious about that as well. There are proteins that are that are not trafficked the same way that the options are so preferring to localizes the same between between wild type and bbs five knockouts and that tells us that the peripheral and pathway to get to the other segments is really unaffected in in bbs five knockout animals. So to take a better look at this ultra structure of these other segments we ran some transmission electron microscopy. And so I'm showing you here. Some images taken from ultra thin sections of a three month old wild type of photoreceptor layers so you can see the outer segments look really nice. But look at these in the bbs five nulls. We see discs that are that are abnormal in their, in their juxtaposition relative to each other. So we're looking to to, we are presently doing scanning electron microscopy serial blockface scanning em, and these animals and then some other animals that we have in our, in our lab with time we're going at University College London to be able to really see what the outer segments look like in these, in these, in these cones to get a better picture of a 3D rendering of it. So hopefully I've convinced you in this protein in the bbs five mouse model at two months we see deep increased cell death in the bbs five nulls, significantly shortened outer segments and significant cell death by nine months. So it's a slow degeneration that we see, but it is degenerating in. Let's see. Mislocalization of arrest in, in both rods and cones during light adaptation, but the cone phototransduction components are mislocalized markedly, and they have abnormal disk disk orientation. I have another bullet point. Oh, there it is. I've shown you that the scotopic and a and b wave amplitudes are significantly decreased and there's, there's an absence of a cone response in these animals. So let's move from the bbs five here in the BB zone up to the transition zone itself. So let's, let's take a look at mks six. So this transition zone is thought to be a molecular said that allows proteins through and, and denies other proteins from entering the psyllium and mks six is one that we also worked with with Brad Yoder, and this is a devastating disease. It's, it's rare. It's it on average is about the prevalence of about one to 100,000 in the world. It depends on population subpopulations and in specific countries. It is a recessive lethal silly apathy. This is an embryonic lethal disease. It's a major contributor to syndromic neural tube defects. These, these humans have poisons to kidneys polydactyly sinus inverters. These are almost every single homework of a silly apathy that you could get and including retinal dystrophy. And so we wanted to model. We wanted to figure out what mks six was doing, but we wanted to sidestep that pesky embryonic lethality. And so how we designed it is we had a flux to Leo event mks six, and we can knock it out after induction we know that this protein gets degraded, this small bit of protein gets degraded right away. And so it's, it's, it's, it's deemed a knockout and we can, we can take these animals that have the flux to Leo and inject through tamoxifen to induce the knockout at P seven. And so that's, we can wait two more weeks and and and we can euthanize at P 21 to look at early, early generation of the retina of photoreceptor cells this way, or we can see, or we can inject in adults and so we can see the contributed contribution of mks six and fully mature retina by simply injecting at P 56 once a day for five days. We can euthanize at five months old. So we've got an early knockout and an older knockout. Okay, so we can look at the differences in those two models. So here are those fun spider grams again so Katie was back at doing counting nuclei and the outer nuclear layer. This is now a three week, three week old juvenile induced knockout of mks six. And we can see the air bars are there they're just under these big symbols. We see a statistically significant decrease in the retinal thickness in the in the juvenile animals, and a market decrease in the degeneration in the adult induced. So this tells us that mks six is critical for the overall homeostasis of these cells, because when when the retina is fully formed and happy, right, healthy cells, and you induce the mks six knockout and fully formed retina. It's still undergoes a degeneration. Okay, so it is, it is a contributing factor to to the overall health of the photoreceptors. So we can stain with tunnel to look for apoptotic cells. And, and we can see that in mks six knockouts we have market tunnel staining. So that's, oh shoot my graphs. So this is, this is wild type. And this is the mks six nulls. And we can see that there's a lot more apoptotic nuclei and the mks six animals. Sorry about that. So what about the protein local is. Oh goodness me. So we have sorry about that I keep. I have truck electronic stuff sometimes translucent properly traffics in the mks six knockouts. So we see translucent localization and to the outer segments in the, in the flocks to lealed animals same as wild type. So here in the mks six nulls, we see, we see translucent in the light down here where it should be as the same in the mks six. The red ops in looks to be miss localized in mks six nulls instead of being in the outer segments like you're seeing here in green. We see red ops and miss localized to the inner segments, and into the outer nuclear layer in the juvenile and induced animals. In light we see we see red ops and still miss localized there's no massive movement of red ops and not surprisingly, look at arrest and so arrest in here is should be in the inner segments in dark adapted animals, it is in mks six nulls. But look what happens in the light, we see massive arrest in this localization. So it's it's all over the place any place in the side of plasma rest in is there. And same thing with cone arrest and it seems to be localized the way it should be in just arrest and for looking at in cone arrest ends. So we don't see a market change in the localization we don't see those those brightly green punctate staining of arrest and down at this at the cone synapses. And so this is looking more and more like it's it's a rod phenotype instead of a cone phenotype which frankly I find remarkably interesting. So rods and cones are very, very similar but they do have some subtle differences and I think we're uncovering some of the subtleties between rods and cones. Now, what about the functionality of them so we were so Katie ran ergs on these animals. These are one week post induction. Okay, so in the adult induced so these are the adult animals that we've induced to knock out mks six and now we're tracking it over time tracking the, the, the ergy signatures over time and while type here, or actually this is the flux to deal but it's the same as well type. Green deflection of the corneal voltage that's the a wave due to rods and a corneal positive B wave. Same with the mks six right so this is the, the, the knockout adult induced knockout we see an a wave and a B wave almost looks like it's it's Well, the kinetics are about the same between the two at both light levels right at low light level and a brighter light scotopic ergs. Here's five weeks. It's coming. There it is. Here's the five week post injection. Right. So we have an in purple we have a nice a wave but it's not as deep that that the amplitude of the a wave is not as is not as big as it is in wild types. Same thing. Well, looking here at the 12 week post induction to really the rod photoreceptors are dead. So it took. It took a few weeks for for the rods to be completely dead in these adults induced animals. Okay, what about the photopic response, photopic response. It to was attenuated, although early on, you don't really see a market difference in the A and the B wave is slightly amplitude is actually a little bit stronger in the knockouts and that was statistically significant, but it does start to diminish at this point. The retina is is is pretty much dying at this point. So hopefully I have told you, you have convinced you that in our mks6 mouse model in the juvenile induced we see a significant increase of cell death at three weeks. Adult induced we see a significant decrease in the outer nuclear layer at five months. In the adult induced mks6 nulls, we see a mislocalization of rhodopsin in the dark in dark and light and mislocalization of arrest in and light at a adapted animals. The ERGs and the adult induced animals exhibit a progressive loss of retinal function and all the function, all the retinal function was diminished by 12 weeks post induction and the adult animal models. So in these two projects. We found that the connecting psyllium component roles are different between rods and cones and to me that is that is quite striking. Both complexes involved arrest and arrest in one trap trafficking issues. So regardless if it was a BBA zone component or the transition zone component arrest and trafficking was affected in both photoreceptors. There could be potential interactions between the complexes that lead to the different differences in disease severity so remember there are a lot of proteins in those areas and in. We all have subtle mutations along the way and one thing that could be happening is that we're seeing that that bbs5 may be affected by other mutations and that could cause the changes in phenotypes that we see, not only within populations but within families. We're interested in the arrest and mislocalization we don't know if it's getting to where it needs to go and is linking back or if it's, if it's never not quite getting up there and so like I had mentioned before. One of our, one of our projects that we're doing now, we're, we're tagging with with photo switchable constructs dendritu and photoactivate balls to be able to see older populations of arrest and versus newer populations of arrest and to be able to see if these proteins are actually leaking back down or not getting to the other segments. And so that's still projects that are ongoing in the lab. My next slide is to thank everybody this is Katie bells shoes the rock star that did the vast majority of the work here's Brad Yoder right there in one of our. We, we do fun outings when it's not COVID time so this was one of our painful sessions, but Katie's now now doing a postdoc. So I'd like to thank you for your attention, and I would love to take any questions if anybody has any. Okay, all right. Thank you very much. Alicia, and I don't know, I can at least start with the questions I don't know if people want to do chat or how we work with that. But a couple questions just as you map out, you know your projects and what you're doing. How do you choose you know when you've got nine, whatever nine plus bbs proteins. How do you choose bbs five what what what attracted you to that protein or was that or did you try many different proteins try to figure out your project. We're trying to be the answer is, is anything that that is anything that we can uncover we we we try to knock it out. And, and that's mainly Brad's arena right he's he's the, the mouse generator. And he is critically interested in what's happening with polycystic kidneys and and I will always argue with Brad and I do constantly that the retina is the best place to look for silly apathies simply because the, almost all of these silly apathy of patients that have a silly apathy, have retinal disease. And so it's, it's better to be looking in the retina than it is to be looking in other other organ types. But there are some there are a bunch of a bunch of people in in that are doing research on these different, different proteins and so we're basically taking the animals that haven't, that haven't had knockouts yet and making those and trying to map them better. This is a pretty act of pretty. This, this cartoon was based on on data from the literature so these we know that, for example mks6 interacts with mks135 and php5 and sub 290 bbs5 were interacts with bbs2 mainly, hardly with bbs7 but not with bbs6. They all have slightly different roles. But we, we have a series of, of animals and let's see mph before we've got running around in the lab mphp1. It's kind of a hand waving answer to it, other than we're trying to go for broken uncovered really the, the molecular composition of the transition zone it's a really small area and all these proteins are so stuck together that it's, it's pretty difficult and be fun to figure it out. And with patients with bbs5 how, you know, do, do their ERGs correlate with your mice? What correlations or differences do you see? So they do. We, we are fortunate and I don't have these data because they're, they're currently getting taken. We have a bbs5 family here. And so, and it's, we're, we're actually quite lucky because this gentleman that the father has, is, has five children, which is really not typical with, with party beetle patients. They have hyper gonadism and have difficult difficulty reproducing but this gentleman does not. He has more kids than I have. And so we're doing ERGs on them but the, the ERGs are, are, are very similar to then the same phenotype as our animals. Okay. All right, good. I, are there other questions? I can't see if people are trying to ask questions or not. But everything else you do absolutely gorgeous work. So, do you. So, so one of the things that always amazed me is, is how much protein trafficking actually happens in the psyllium just normally, right. Do you have any idea on, on the kinetics of translocation of arrest in these knockouts in light adaptation. Does it, I mean, you show beautifully how they miss localize and how they repartition and, and, but, but you have any information on the kinetics. I don't. I think Vadim Arshavsky does. Yeah, from his work previously and I don't have that I don't have the kinetics off the top of my head. It is quite fast. And these have to be bright bright bright lights and complete darkness to be able to see that massive translocation it's not regular room light. We stick a LED lamp right over their cage and take out all of their things that they can hide. You know, so that we can get full. This localization I don't know the kinetics of it off the top of my head that's a that's a good question. Yeah, are these. So I presume these are all. I'm sorry. I presume these are all pigmented animals. These are. Yeah. Yep. Gorgeous. I've got lots of questions about the synapses to. So are you. And you're sure about this hearing and are you doing the photoreceptor outer segments or are you doing the whole photoreceptor with with the synaptic terminal as well. So on these animals, just the outer segments. Okay. And the next project that I'll talk to you about doing the entire the entire beast. So the next, the next seminar I'm going to be talking about is about this protein called not see nuclear distribution protein see and it. It regulates F actin pools. It also regulates or actin equilibrium and it also regulates dining mediated movements of movement across microtubules train tracks. And, and, you know, I'm interested if there there's microtubules down at the synapse, right. If that gets disrupted. So, in that project with time we're going we're doing the whole photoreceptor cell. And it's expensive and it's, it's takes a lot of time and, you know, to be able to do those so we're just going to focus on the transition zone mutants we're just going to focus on the outer segments and that, and that connecting sewing area. Cool. We've got some kind of tomorrow. We'll chat about this. I'd love to. Thank you. So Paul this is this is Randy listen. That was a beautiful lecture, and look forward to getting a chance to chat a little further but you know love the basic science and being able to dig in into detail about all of these syndromes that I learned about, you know, in my my younger years and now understanding how the chemistry fits together anyway, congratulations. It was great to listen to this great work. And I have a question through the chat from Jeff petty which, unless he wants to speak up I'll just read it. Jeff petty says really beautiful science you mentioned the 3D TM reconstructions what do you anticipate learning from additional understanding of the overall morphology. I am absolutely fascinated by discs. Right. I, I don't understand the Delta G of making a disk a disk and not a sphere is like super high it's it's, I can't believe that we can see I can't believe that other segments look the way they do. They are gorgeous but they are so precise. And I, you know, my lab and a bunch of other labs are really really interested in how discs are formed. And we can see how the, if we can reconstruct how the discs look you know those bbs five goofy outer segments you know they're, they do have nice spacing between the discs, but some of them are this way and I don't know why and I'm going to show you some some of the next disc discs in the next talk with the Nazi knockouts f acton is is all is critical and disk formation. So I really hope that we can gain a better understanding of how these discs are formed in general serial blockface scanning does give you a really cool opportunity to 3D reconstruct that that region. And so we can see discs. That's the way we've ever seen before. You know they are fixed so it's not. It's, it's, it's not like doing tomography where you can flash breeze and then see a small portion but we still will be able to glean a lot of information from it. All right, very good. Any other questions from anyone. If not, we will finish up and we will see you at noon again for more more basic science so I hope many of you can can join us then and we, since this is a virtual visiting lectureship you get to talk with a lot of us through the day through today and I'm looking forward to it. I'm looking forward to it and thank you all for your attention and really appreciate you taking the time this morning.