 Good morning and welcome everyone it is 8 o'clock I want to welcome everyone to our virtual grand rounds will be turning the time over and just a moment. To Dr Wolfgang bear for the introduction of our speaker today. Just a couple of updates, we are virtual for the foreseeable future, we will make an announcement if we are returning to in person grand rounds and in person educational events at the end of the month. I'm outside of that wish and hope everyone is healthy please. If you do have questions about the current protocol for if you test positive or have an exposure. Check out the link on pulse. That's where you'll find the most updated recommendations, and given that it's been a bit of a moving target. Well, recommend you check out the latest there with that. We'll turn the time over to you Dr bear for introduction. Good morning. I'm very pleased to introduce Max Naguri University of California and San Francisco. He received a PhD from UC Berkeley and University of Paris or sorry in 2001. His topic is this is topic was in nuclear transport and my daughter's spindle assembly. This position in 2007 with Peter Jackson at Stanford University and China tech. He started to work on primary Celia. Subsequently, he was assistant professor at the Department of Molecular and so the biophysiology Stanford University. And since 2017 is associate and full professor Department of Ophthalmology, University of California San Francisco. He was a mentor Peter Jackson and China tech. He started working on the body Beatles and home characterised by obesity when the generation polydactyly and polycystic kidneys. In the last 15 years, Max established established himself as an expert in the babies home primary Celia so your chances. He has published the series of excellent papers in the last 20 years, topped by a recent solution of the native babies home structure. Today, he will present a talk on the molecular basis of the battle Beatles at home. Welcome Max, we're happy to be here. Thank you, Jeff. Thank you all for being here. I'm very sad that we're not in person. I really was looking forward to my first trip in whatever 20 months since the beginning of 2020 so I had that on my calendar as something to look forward to stepping on the plane and seeing people. But yeah, it looks like it's going to still take a few more months for all of us to see each other. So, with that said, I think that it was definitely, I mean, I'm also happy I don't have to travel, given the current circumstances. So, I hope that we'll be able to get some good science exchange today. And what I would say is that I'm not, not very used to giving talks to clinical audience I tried as much as possible to, to make this presentation engaging accessible to everyone. But please, if there's anything at any point that doesn't make sense. Unmute yourself interrupt me. I'll try to monitor the chat so feel free to post in the chat I may not always sees the chat being lit up so really don't be afraid to unmute yourself and and tell me if you have any questions. Alright, so with that said, I'm going to start this grand round presentation on the molecular basis of bothered be those syndrome. I've started by telling you that I have no disclosure whatsoever, and the outline of this presentation is as full. So, I will start to give you a short history of the solopathy is something that, guessing many of you have heard, but might be new to some the meat of the stock will be on bothered build syndrome and this will be on this journey from the history of genes to the unveiling of the molecular mechanism. And finally, I'd like to finish on what is a current topic in my lab, which is the maintenance of the photoreceptor outer segment protein. This is what I believe all of us are interested in and leave and breeze to understand and improve which is vision. How do we see things and for us human seeing is one of is probably the most important sensory function where we're lousy at hearing at smelling, but we're very good at seeing things and so it's that our, our brain inputs are really dominated by vision. And the way that vertebrates see is thanks to the retina in the back of their eye. And if you look at the retinas, the cell type that really receives the photon and transduce transduces them is the photoreceptor. The architecture of the photoreceptor started to get described in the 1950s in the golden age of electron microscopy. And Eddie de Roberto is the elder, his son is a famous embryologist, but Eddie de Roberto is an Argentinian electron microscopist, described in 1956, the structure of that connected what he called the inner segment to the outer segment. And he was struck by the organization of the structure and you can see here he's, he's rendering of his results in that this disconnection between inner segment and outer segment contains these bundles of microtubules. Okay, some things that had been hinted at but he was really the first one to see with such precision. And that led him to realize that this entire outer segment of photoreceptor was a psyllium. And a psyllium is a structure that you see on the surface of cells that projects from the surface of the cell. And that consists of a bundle of nine doublet microtubules, we call it the axonim, that's something a term I may use today. And it is within a sheath of the ciliary membrane, membrane that itself is continuous with a plasma membrane. Then at its base, there is a structure that consists of a triplet now of microtubules as a basal, but so here you go. That's your run of the meal primary psyllium and now that is your photoreceptor outer segment and this structure Eddie de Roberto's named it the connecting psyllium. But the important realization here is that the entire outer segment is the equivalent of a primary silly. Psyllium are things that you see on a variety of cell types and you can see mode actually, for example, on your airway Psyllium. They perform very important functions in a community, if you need to fight COVID particles that are getting in your airway. The first line of defense is simply mucus moving trapping these particles and moving them out of the airway. But it turns out that you also have primary Syria and this primary Syria are non motile Syria they don't have the structures all these little elaborations here that that you can see in these motile Syria cross section, they don't exist in primary Syria. So this primary Syria, they cannot move, but they're present on nearly every cell in your body and here is a summary you have them on olfactory receptor neuron you have them on kidney cells, you have them on developing ear cells, nearly every cell, and in your brain neuron has a primary psyllium. But for the longest time, and this is taken from a book from the 1980s that was really a classic of cell biology by Don Fossett who was a chair of cell biology at Harvard. And this really gives you a sense of what the appreciation for primary Syria was at the time. And it was not even called primary Syria. In fact, it was called an aberrant solitary psyllium. People like Don Fossett, these things was absolutely beyond the scope of any scientific endeavor. There was nothing interesting about it. And the term aberrant solitary Syria could be exchanged for an abortive psyllium. It's literally some things that didn't even deserve to be on the cell. It's something that probably was a remnant of evolution when all cells were moving around, and give it a few million years and we should now lose it. So now you can fast forward to 2010. And my former institution, Stanford had a little magazine there, where now they were talking about the cell tower, the rise of the cell. So how did we get from Don Fossett calling the psyllium an abortive structure to people talking about the cell tower of the psyllium? Well, there are really two aspects that transformed our appreciation of the psyllium. And the first one should be no surprise to any of you in the audience, is that the psyllium is important for signaling. And in particular, in the case of vision, that really forms a paradigm for how Syria facilitates signaling. You have the entire photo transduction casting, all the way from the light sensing G protein coupled receptor rhodopsin down to the cyclic nucleotide gated channel that will change membrane polarization and transmit a hyper polarization from this outer segment all the way to the inner segment and to the synaptic terminal so that now the cell can trigger the release of neurotransmitter that can be further interpreted within the brain. But this paradigm of Syria sensing the outer environment is not exclusive to photoreceptors, a very similar paradigm applies to olfactory receptor neurons, where, again, you have a G protein coupled receptor. In this case, that's a GPCR that senses chemical. It's present on the surface of Syria and these olfactory receptor neuron, and it will instruct the entire olfactory transduction cascade down to again a cyclic nucleotide gated channel. The same principle applies the cell is on your own, ultimately, the sensing of an odorant triggers the release of a synaptic messengers. However, the the breakthrough in our appreciation of the importance of Syria came from the work of Catherine Anderson in 2003, who is through forward genetics in mice realize that hedgehog signal, which is a developmental pathway that patterns your limbs patterns, your neural that these developmental pathway also required the primary silly. And the reason there is a paradigm that was developed in for the receptors that was developed in olfactory receptor neuron applies again in the cells that are transusing hedgehog. And the paradigm is that you have signaling receptors that are present on the surface of Syria and that transduce signal. There is a little twist there, however, and you can appreciate the twist here on this diagram in that you have a dynamic organization of these signaling cascade. So you start out with a certain complement of signaling molecule you have this receptor for hedgehog named patched one, you have a G protein copper receptor gpr 161. That's in the basal state. Once you activate the cascade now you completely switch the complement of signaling molecule and now you have a positive regulator of the past waiting smoothen that enter seal. So, now, slight twist on the paradigm, but the important thing is that Syria organized signaling tests, and that happens in sensory signaling in developmental signaling happens also in homeostatic signaling. So, some things that has broad physiological importance. The second reason why silly I started to become appreciated as being important in physiology and health and disease is because of the discovery of cellopathy. This term cellopathies was coined in 2006 by Nico Katzanez in a visionary review where he realized that a host of diseases with common symptoms, likely all had their root cause in dysfunction of the primary silly. The symptoms that are seen in silly opossies are kidney malformations in particular kidney cyst retinal degeneration skeletal malformation open manifesting as polydactyly extra digits extra toes. And frequently you see obesity in in silly opossy patients. And you can get a heterotaxi change of body plant symmetry to various extent. You often get close of the sense of smell and infertility. Really, by far the vastest class of silly opossies are the retinal silly opossies in almost every silly opossy, you do see an indication of retinal degeneration. Overall, and here it's a diagram that's extracted from a review from four years ago. So well over 120 different genes that cause at least 10 different diseases that are all and and compass under this umbrella of silly opossies. One silly opossies that I became interested in now 15 years ago is bordered beetle syndrome bordered beetle syndrome is the name for two physician George Bardet, who was a medical student in fact at the University of Paris. And so, patients who presented with obesity polydactyly and retinal degeneration at the time they didn't appreciate the kidney indication. And the disease is named also for Arthur beetle, who is by many considered to be the father of modern endocrinology. And so these two gave the names to these disease bordered beetle syndrome. So, what is exactly bordered beetle syndrome, as I described to you you have four cardinal symptoms retinal degeneration polydactyly obesity and kidney cyst. And I just wanted to say a few things about specifically the indication in the ophthalmology field. What you see in patients is that you have you start to detect the RG abnormalities at about five year old. And from that point on, you have a rapid progression of the retinal degeneration that leads to blindness typically by age 15 or early. The progression of retinal degeneration is rather unusual compared to classic cases of fraternities pigmentosa, in that you see a degeneration of cones, either before, or at the same time, as well. This is also the more recessive disorder. It's a very rare disease in the American populations of frequency is one in 150,000 lifers. And it's genetically very heterogeneous. So this is something that was quite surprising when the disease was was first analyzed by human geneticists is that you don't have a handful of gene, you have over 20 different genes and once you want that has been identified, they are likely many more because they are about 20% of patients that do not have mutations in known genes. And sadly, there is absolutely no treatment when ophthalmology see bbs patients, the only things that they can tell them is that the vision will be lost, and that they should prepare themselves for that. But at this point, there is really nothing we can do to combat either the degeneration of the photoreceptor or obesity in these patients. So telling you a little bit more about retinal degeneration and these are studies here from mouse models. And in this case, this is a bbs type knockout, but pretty much all the bbs knockout behaving exactly the same way. What you see is that at two months of age, you have compromised a rod photoreceptor function, but you still have some function of rod photoreceptor. However, for the cones, the cone function is almost completely absent. And again, this largely recapitulates the human disease. At the ultra structural level, however, what you see is that these these rods, even though they appear largely functional in ERG ultra structurally, they're a complete mess. What you see there are these instead of having these nice stacks of this, you have this highly elongated ribbons. So the disk instead of nicely closing and being enclosed within the membrane of the other segment, they just keep growing, which which gives rise to these long worlds of and you see a similar things in cones where again, you have hyper elongated disk and instead of stacking in a normal plane to the axis of photoreceptor, you see them often time in a horizontal parallel to the main axis position. So large ultra structural defects and some clear defects at the functional level for cones, some milder defects for rods. Okay, so the questions that I really was faced with when I started to get interested in bordered middle syndrome was how is this genetic heterogeneity. What does it correspond to as a molecular what is a molecular mechanism that underlies bordered middle central. I've been trained as a biochemist cell biologist. So, for me, the problem and the way I was seeing this, this problem that was that emerged from human genetics is really that genetics gave us a part to this list. So genetics identify genes which when defective gave rise to these disease. That's all the parts that makes the engine so it told you about a specific m four by 20 screw. But it didn't really tell you what the specific component did it didn't tell you how you went from this part list to a functioning molecular machine. And that was really the, if you want to the detective words that was needed was to go from here is a little screw that we've identified to there is a machine that is screw functions. And aspect that I decided to, to leverage was to work in culture cells and a lot of the work I presented today has been done in culture and mammalian cells. And the reason for that is that Celia are present in culture and mammalian cells, and they're present in specific conditions so cells in culture will cycle so they'll divide constantly in terms of the right nutrient. But if you withdraw growth factors cells enter what's called quiescence. So they, they enter a state that's not quite a dormant state they're there they're waiting for growth factor to come in this state of quiescence is the cells start to grow Celium and show you can see one such instance of a cell in culture, and the Celium is stained with this marker acetylated tubulin, and you can see the Celium is positioned fairly close to the new case. So these is the type of system that I'll be presenting you. Quite a bit today. Okay, so when I started working on bbs around 2005 2007, we had 11 bbs genes that had been identified by human geneticists. And really, none of these gene product gives you any idea about what the molecular basis of bbs could do. One thing that I did was to purify the protein complexes that these bbs gene product where components of, and that led to the discovery of this protein complex that I named the bb zone, and that contains a different bbs protein. So I just pushed the question, one level further which was okay, what is this bb zone doing. Now we have a molecular entity that exists in the cell. What does this molecular entity function in. Here more biochemistry led to the realization that another bbs gene product which is a product of the bbs three gene and codes for a protein that's thing all six. And what this protein does is that it binds to nucleotides to GTP and GDP. And when it is bound to GDP, it is able to recruit the bb zone to members. This this this product the product of the bb zone recruitment to membrane is a formation of a code of a polymer on the surface of membrane. So this realization that the bb zone forms a code now gave us an ID, which is that code complexes had been described by many talented investigator, including the Nobel Prize winner range checkman Jim Rothman. And these codes, their function in the cell is to move proteins from one compartment to another. And here you can see this diagram of the major codes in the cell clustering cup one cup two, and all these codes are going to move protein from one compartment to another. So, what was the bb zone code doing in Syria. There, what we found is that all six and the bb zone are localized in little patches. Okay, that are flanking this microchip all accident and our interpretation here is that these patches are the correspond to polymers of bb zone and all six that are stuck that that are opposed to the membrane of the cell. Okay, so we also see and that's not now by super resolution, but we could see that our six sense of bb zone did co localize in these discontinuities along the primary cell. One thing I should mention that's very important is that there are no basic calls inside the primary series. So everything that I'll be describing in terms of movement of patches movement of protein. This is always patches or proteins that are laterally moving or literally stuck to the silly remember there are no little basic calls that are moving up and down. And speaking of movement. This is what I wanted to show you, which is that the bb zone, these bb zone patches, they are moving up and down within the ceiling, and they're moving thanks to a system called intra flagellar transport or IFT that was discovered by Joel Rosenbaum in the late 1990s. And that's a system that encompasses a set them in a couple of large protein complexes and molecular motors. So there you have it you have a you have these little machines these microchip motors that are moving these IFT bb zone trains is what they're called in the field up and down inside the primary cell. Okay, so now we had understood some of the cell biology of the bb zone. But the question is, what's it really moving. What are the cargos that the bb zone code is moving. And here what we discovered is that the bb zone recognizes some of the cytoplasmic determinant of the signaling receptors that are localized to Syria. So in particular, in this case, this receptor named gpr 16 one, that is responsive to heads up. And what had been described by sci cat macopadi and Peter Jackson is that gpr 16 one. And you see that in cultured cells here. So here you have the merge and here what I did was just to shift the channel between the Celia marker acetylate tubulin and gpr 16 one ever so slightly so that you can well appreciate the green signal being present here in untreated cells and disappearing once the cells stimulate these signal dependent exit of the gpr gpr 16 one no longer happens when either all six or bb zone function is compromised. So there you have it. That is now what we understand as being the function of the bb zone, which is in the signal dependent exit of signaling receptor from Seattle. And this is something that Fanny, who has a postdoc in the lab was able to directly visualize as a single molecule level. So here what fan did was to use a quantum dots a very bright fluorescent entity that he coupled to a single molecule of gpr 16 one in the Celium. And he could visualize a movement of this single molecule of gpr 16 one diffusing then rapidly moving back to the base, and then exiting silly. So, this really is a basis. Here. This is a basis of the models that I'm giving you here, which is that when signaling receptor in the Celium get activated to become recognized by the bb zone. And the bb zone forms this code that become coupled to these intraflagellar transport train that's moved from the tip to the base of the Celium and fairy these molecules is signaling receptors outside of the cell. So that really leads now to a more general model for bounded build syndrome, which is that most of the symptoms that are observed in bbs patients are caused by defects in removing signaling receptors from cell. And there are many signaling receptors that we now know are in Celia that need to function in Celia, and that probably need to exit Celia in order for signaling reaction to be properly propagated. However, in the case of retinal degeneration, there wasn't such an obvious signaling receptor that needed to exit Celia via the bb zone mediated pathway. So, what is the function of the bb zone in photoreceptors? Did all this work that was done in cell culture in model organism give us insights about what the function of the bb zone was in photoreceptors. So here, I'm showing you the entire phototransduction cascade, just to indicate that within this cascade, none of the membrane proteins are undergoing signal dependent exit. Opso never exit the celium to go back to the inner segment. And no other membrane protein is known to go from the outer segment back to the inner segment. So what exactly can the bb zone do in this cell type? So here I need to present you the foundational work of Song Jin Seo at the University of Iowa, who conducted a heroic purification of outer segment from bbs mice and compared them to wild type mice. What you could see by doing the proteomic analysis is that there were over a hundred proteins that accumulated in the outer segment of these bbs mutant mice. What he proposed is that the flux of rhodopsin that's going from its place of synthesis in the inner segment to its place of function, the outer segment, this tremendous flux of synthesis that's been estimated to be as high as 1000 rhodopsin per second in frog photoreceptors. This flux of rhodopsin is going to be like the Niagara force. It carries every little rock and grain of sand from the upstream location into the downstream location. So that accidentally you get proteins that belong to the inner segment that accidentally enter the outer segment. And here is one such instance in this protein syntax in three, which in Song Jin work was shown to accumulate in the outer segment of bbs photoreceptors. So this now leads to the following model, which is that in photoreceptors, what the bbsm does is not to remove proteins on demand as it does in other cell type, it's to remove protein constitutively is to remove what I call pollutant things that don't belong to the outer segment, but that accidentally get into the outer segment. So this gives you a now unifying theory if you want of bbs, which is that the bb zooms job is to remove unwanted proteins from Syria. But one question that remained unanswered is, how are these unwanted protein specifically recognized as being unwanted and as being prospective bb zooms. How does the bb zoom know which protein to remove and which protein to leave behind in the ceiling. And here, a few years back, we started to think about this post translational modification name ubiquitin ubiquitin is a 76 amino acid polypeptide it's a very small protein. It's a protein that becomes attached to substrates, and it's become attached to an isopeptide bomb onto lysine residues. What's fascinating about ubiquitin is that it is not, it's not simply that you attach ubiquitin you're done with it, you can form chains of ubiquitin, and these different chains are completely different. There's not just one flavor of ubiquitin, you have an entire code of ubiquitin modification, and depending on the specific linkage that's formed when these chains are elongated and here I just show you that on ubiquitin itself. These are the proteins that are highlighted. But if one of the lysine is spec lysine 48 that targets proteins to this degradative machine called a proteasome that is a multi protease machine that will chop off polypeptides that need to be digested. But if you choose another lysine, which is now lysine 63. Now that gets a membrane protein to be targeted to the lysosome. And I'm just showing you two functions here, they are myriad of function. There are seven different linkages. So there is a very rich complexity to ubiquitin modification. And there were a few indications that ubiquitin may function in Syria, which led Swapnil Shinde a postdoc in the lab to investigate ubiquitin in our system. And here what Swapnil is looking at is a G protein coupled receptor is a somatostatin receptor street. We express that in our cultured cells. And what we see and again the signals here I shifted for either a solution, but you see in science that you have the somatostatin receptor street Syria, and you add its ligand somatostatin, and it now disappears from Syria. So, again, like I showed you earlier, classic case of signal dependent X. It's the same experiment in a cell where baby's own function is compromised. And you see two things. You see that the science signal doesn't exit Syria. And you see that as these frustrated exits is getting stuck in in Syria. There is an appearance in yellow of ubiquitin signal in Syria. Now what I can tell you is that based on biochemical study, we know that these ubiquitins are attached to SSTR three are attached to this GP CR that cannot exit Syria. In addition, what Swapnil figured out is that this ubiquitin signal is specifically in the form of lysine 63 linked ubiquitin chains, which are the chains that are important for lysosomal degradation. So they swapped Neil, an ID and a model, which is that this ubiquitin, this lysine 63 linked ubiquitin chains become added to activated GP CR in Syria, and they provide a signal for the baby's own to recognize the protein that it should be selecting to remove all from Syria, so that these proteins can now get attached to the baby's own and ferried out of the cell. So based on this hypothesis. What I want to propose is that he should be able to introduce inside the Syrian some molecular scissors that cleave the slicing 63 linked ubiquitin chain. And this is a this molecular scissors, they belong to a class of enzymes that are called the ubiquitinases, their proteases that cleave ubiquitin linkages. And this is one such do ubiquitinase that is exquisitely specific for the lysine 63 linkage. So when we target this do ubiquitinase to Syria, and here is just the control I'm showing you again shifted channels. I'm going to show you that when we transfect this this dummy construct into sales, we are getting normal exit of SSTR three, even though this dummy protein is entering Syria. And now with the lysine 63 specific do ubiquitinase. Now what you see is this do ubiquitinase is in Syria, and this GP CR no longer can undergo signal dependent exit from Syria. The same experiment with other babies on cargoes other GP CR smooth and GPR 161 same result. Do ubiquitinase completely blocks dependent exit BBZM. Sorry signal dependent exit of BBZM characters. So there you have it. That's the major conclusion, which is that the slicing 63 link ubiquitin chains, they're required for exit of BBZM cargoes from so. This leads to the following models that I'm showing you here data I didn't present you and something you may have been wondering about is how are these lysine 63 ubiquitin chains added specifically on to the activated signaling receptor well here is there is a little molecular adapter that's been beta resting to that is able to recognize the conformation of the activated signaling receptor. And we believe that beta resting to recruits a what's called a ubiquitin ligase so an enzyme that adds ubiquitin chain specifically onto these activated receptors. And I should mention that another group Greg Azure has published similar finding findings together with us. So, again, we had now a model that we had arrived at from studies in cell culture. But the question was, how does that apply to photoreceptor with the game we don't have signal dependent exit in photoreceptors. Are these lies in 63 linked ubiquitin chain, also used in photoreceptor. And here, I'm going to present you some unpublished work from a student in the lab, Shredas, who started to become interested in the function of ubiquitin in photoreceptor. And so here she's looking in this retinal section and you can see that the layer of outer segment. There is no real detectable signal for ubiquitin. However, when she does the same experiment in a bbs knockout and what she can now is a massive accumulation of ubiquitin signal in this outer segment player. And as you might expect from the words that we've done in cell culture, this ubiquitin signal is in the form of lies in 63 linked ubiquitin chain. That's what you can see here with this region that she's using to specifically detect lies in 63 linked ubiquitin chains. You have a massive accumulation of signal in the outer segment of bbs knockout animal. And all of that is done fairly early at P 15 before the onset of retinal degeneration. In fact, she can even see accumulation of ubiquitin signal in the outer segment as early as PA. So, as soon as there is an outer segments that forms, it seems that you already have ubiquitinated proteins that accumulate in the outer segment of these bbs knockout mice. And the question that we wanted to answer is what are these ubiquitinated proteins that are accumulating in the outer segment of these bbs mutant animal. So here, she bravely decided to undertake a biochemical certification of these ubiquitinated products. And the schema is on paper, very simple, which is that you take these mutant retina you purify the outer segments. And then you use a reagents that's called a tube tandem ubiquitin binding entity. So it's an engineered protein that specifically recognizes ubiquitin chains. So you get different tubes, you can have tubes that recognize all chains. In this case, we have a tube that is specific for lies in 63 linked ubiquitin chain. So this tube, then she can, with that, purify the ubiquitinated proteins that are present in outer segments. And here is just to show you that indeed these tubes, when you take these outer segment homogeneits, the tubes can purify ubiquitinated proteins. And they're seen as a smear because these ubiquitin chains are of such varied lengths between different proteins that you do not see discrete species, but rather a smear. But the important thing is that the smear is present at a much higher level in the bbs knockout than in the wild type animal, which fits very nicely with our immunohistochemistry. So the next step was then to do proteomics on these, these proteins, these ubiquitin proteins that we had discovered that something we did in collaboration with the lab of marine calluxia at Harvard Medical School. And what we found is that there were about and depending where we set the threshold, but we fairly stringent threshold, we find 36 proteins that are enriched in bbs knockout photoreceptor outer segments. The vast majority of them are membrane proteins. And most of them are proteins that are known to reside in the inner segment in some part of the inner segment. The largest category are proteins that are proteins, either in synaptic vesicles or synaptic terminal. We also get transporters ion transporter amino acid transporters that are known to exist within the membrane, the plasma membrane of the inner segment. And we also get some adhesion molecules. But here, again, taking the example of just one such protein we identify syntax in three. And there, the model is that syntax in three accidentally enters the outer segment gets recognized by the ubiquitination machinery which adds a lysine 63 linked ubiquitin chains onto this foreign protein. And so, this ubiquitinated protein can be recognized by the baby's home and the ith machinery to go back into the inner segment. Indeed, we find that syntax in three. The asterisk is just to denote a non specific band, but syntax in three, which migrates here, you can see that it is enriched in these tube eluids is this ubiquitinated proteins specifically in the bbs outer segment. And you can see higher molecular weight species, which may correspond to ubiquitinated forms of syntax in three. And this fits very nicely with what something so I discovered, which is that syntax in three in bbs knockout animals accumulates in the outer segment. So there you have it. The conclusion we have is these these lysine 63 ubiquitin chains in photoreceptor, they mark the unwanted non-soluery protein that are to be removed and redirected back to the inner segment. So, I'm going to leave you with one take home message for today, which is that the baby's home is part of a machinery for quality control of the proteome of Syria. And that's either in Syria of kidney cells of neuron photoreceptor, any cells that has a silly and this quality control can take different form. It can be an on demand control of the celery proteome when the signaling receptor no longer should be in Syria when it is instructed to exit Syria because it is getting activated. The baby's home there will do his job to remove this ubiquitinated signaling receptor. But it also does a job of a just housekeeping of vacuum cleaning the cilium removing proteins that don't belong to the cilium that are also getting ubiquitinated and that are getting constitutively cleared from Syria. So, the conclusions for you today that I hope I convey is that the cilium is really a point of convergence for a range of hereditary disorder with several characteristic features. The baby's home really and its function of quality control. It forms a unifying mechanism for bbs and the failure to control the quality of the celery proteome can easily to specific signaling defects. Or it can lead to more general architectural defects in photoreceptors that lead to a disorganization of the other segment and ultimately to the deaths of photoreceptors. And finally, the part that's the most current part of our work is that the baby's home sorts ubiquitinated protein out of Syria and and uses the slicing 63 ubiquitin chains to recognize its specific cargo. And with that, I will thank the group, I acknowledge the people who did the work today, so I feel shim day, Shredas. None of the work would have been possible without our founders, in particular, our PB and NIH who found who funded the work that I presented today. And with that, I will be ready for your questions. Thank you. Okay, I'm sorry. I can't seem to hear you. Let me see. What's going on. Can you hear me now, Dr. Naturi. Now I can. Yes. So, so for not giving a lot of talks to clinicians you did a wonderful job telling that story allowing us to follow along. You know, it's really interested in the concept of this large flux of rhodopsin bringing along unwanted, you know, proteins. Are there other physiologic models for that that you're aware of where again you have this large flux and now you need this quality control mechanism to to take things back I've not heard of that. Yeah, that's a great question. I mean what what other instance could one see. Yeah, I mean I'm thinking I mean, you know, so it's a classic example of tremendous trafficking flux is the plasma cells so an activated billing for site that really becomes remodeled to a secret IGGs. But yeah, in this case I can't think of anything that the cell does to to counteract this this tremendous tremendous flux so yeah that's a good question actually I haven't thought about that in in other instances and photoreceptors. Thank you. I have a question. Yes, both gone. You said the baby some moves by IFT in primary Celia. How about photos of order segment it is not clear to me how it moves around. Is it IFT or is it some other mechanism. Um, yeah that's a good question so. I mean obviously all this work we've done to image the movement in life cells is all done in cell culture. I don't know of such works that's been done in photoreceptors I believe would be technically exceedingly challenging. So, I, I don't know I'm actually yeah maybe you know what happens to the be so mean dining mutant retina. We know that would upset moves to the outer segment, and we have a paper published that doesn't use IFT, but that's controversial to some people believe, like David Williams that it clearly is IFT, and we say it happens by diffusion. That's a big question and the outer segment doesn't really have a long accident. It doesn't go to the very tip like in primary Celia. We believe to believe this transported by IFT, but what option is not so the question really is, nobody knows how he was, he was always moving about. It could be dining and two as some molecular motor and retrograde transport, but it's not really clear. So you do you have any answer on this. No, I really I mean, in photoreceptors, I do not I mean that's, I think that's a very important question but yeah ones that we haven't really looked at the game that that's something we've only looked at in cell culture. So our research concludes that photoreceptor outer segments are primary Celia but they're different from primary Celia and they're different mechanisms. So that's our research and we talk about this at 11 o'clock. It sounds good I'm looking forward to it. I don't see any other questions, and we are at time do just want to again thank you for anyone that is interested there is the new research seminar. Please just email Megan Johnson or Julie. We can get you that link if you're able and interested for any of the clinicians. Again, thank you we also wish you were here. It's a beautiful blue sky day and no doubt hope hope we can host you at some point so thank you again. Thank you very much.