 Good afternoon everyone. It's my great pleasure to first introduce the provost lecture series and so this is one of the first initiatives the provost office has undertaken since I took the office exact one month ago and since July I have talked to many faculty members about 70 and counting and so based on many conversations and I realized that it's very important to create this the provost lecture series and the purpose is to celebrate milestones in the careers of many or its faculty members in particular we want to acknowledge their research success honor their service to the university and also so we would love to hear their thoughts and learn from their experience when they share their teaching and mentorship of students so in discussion with peter which is very complementary to the president lecture series so we invite external speakers the provost lecture series will be mainly catered towards internal oise faculty members and this also matches very well with the ongoing faculty lunch talks which is more casual and shorter so we also try to avoid the scheduling conflict with those two lecture theories and to so before I start I would like to really thank the people shown on this slide who made the provost lecture series happen so in particular I would like to thank Matsukawa-san from the office of the provost so she worked very hard and to push the process through because we're on a deadline I don't know if you know Ichiro is leaving in eight days and so we're very happy and also with the lift of the COVID prevention starting today so we're able to book the room and make the first lecture on November 1st so in addition also Magai-san from the office of the provost helped us a lot working behind the scenes to to figure out the budget and logistics so in addition I would like to really also pay my special thanks to Kauri-san and she has been amazing in terms of designing the posters and many of you already know her because she designed almost all the fabulous or his posters over different events and also Reg who has been really helpful and who is going to write a Tida story and to promote this series and so this is an example so Kauri-san designed the poster series and also worked very closely with Matsukawa-san and Matsukawa-san found this picture frame from the house close to Naha close to her house and we also try to use take advantage of the OIS facility so Patrick Kennedy who is the section leader in the engineering section so we designed together with using the 3D printer to make the acrylic insert and to make this picture frame work and this each year will be presented to you after your lecture so finally I also want to thank everyone shown on this slide from Peter to Kato-san and many many others and they have been very supportive and we need all the signatures you can imagine to make the provost lecture series official and so the take home message is it takes a village and so we we need to work together closely with the administrators and so they're so actually they're quite busy helping us to promote our research and to make the operation as smooth and possible so finally so I want to introduce professor of Scotland who is going to be the chair of this lecture series and of as a professor at OIS he's also the graduate the dean for graduate school and finally before I forget so after the lecture series so we we will serve some coffee tea and snacks in the patio area but please be mindful so we can prevent the COVID-19 I do not want this becoming the big you know spread event so without further ado off yeah so hello everybody and welcome it's my great pleasure to introduce our main speaker here Ichiro and you all have seen him forever here and I've also seen him forever the question we all put ourselves is of course who is Ichiro right and is a person he likes the minimalistic truth that characterizes a phenomenon and I think this is very peculiar because when you talk to Ichiro for a while he doesn't like to bloom over in things that are completely unnecessary so he's very condensing it's very interesting so if you haven't spoken to him about phenomena that he knows a lot about you missed out and he's not a quitter so he keeps investigating until he is satisfied so he's very stubborn right and of course this is usually for us researchers that that's true actually and so wonder because he's such a investigative character no wonder he likes golf and although Ichiro has always had a burning desire to understand signaling and signaling is you know within the cell cultures and in the bodies is how some kind of signal goes from one place to another to trigger something and this is an exceedingly difficult phenomena to characterize correctly and so whenever there is a model out there the first thing you can think of is oh my god it's another model and and then how many years will it take before they have removed that model or refined the model and he has worked really hard on this over many years and has a very very interesting result right now so what are we talking about in his case well he's very interested in in one of these special molecules that changes shape the epidermal growth factor receptor and it has a domain structure which is in the cell and a domain structure which outside and upon binding something there happens something in here and you get an action point and you can see this a little bit like molecular dances and you wonder what drives all these molecular dynamics well it's actually water and the temperature and because you can have mainly actually this structure or anything in between there water molecules as I say they are the main supplier of energy to transform the molecules into one structure or another and of course we can always put together like I did here so that you see a dance but the funny thing in molecular dynamics is you can go from the left picture to the right picture in just one go and so this is the sort of continuous movement that you would like to see but it doesn't necessarily be the way it happens inside the cells he has been working very hard on this so he has a very long history in biological science field as you will hear from him and I'm not going to talk about his field exactly he will do that he's an excellent scientist he's an excellent teacher he's an excellent supervisor he's an excellent colleague and collaborator and he has been very very helpful to Oist in many different committee works the simple truth is that Ichiro is just Ichiro and you have to know this fellow a bit right he joined five years before I came here in 2005 and it's for sure that retirement is not going to stop his brain and with that I will welcome Ichiro up and talk about his wonderful stuff okay first of all thank you Ulf for wonderful introduction maybe too kind yes I would I also like thank Amy for giving this opportunity to talk today and in fact this is informal talk actually so this afternoon I'm going to talk about my life in science and technology which was full of challenges so that you may be able to extract some useful hint from my talk so that you should interrupt me anytime whenever you have a question okay so this is my first challenge actually 1983 I was traveling from Tokyo to Heathrow airport in London through North Pole after stopping on college in Alaska because just before boarding Soviet jet airline shot down Korean Airlines flight near Hokkaido the northern island so we could not go through from Tokyo to Russia area so that we diverted to this long way so that's a tough travels because it took more than 20 hours with eight month old daughter with this was terrible so this was 1983 I was arrived MRC laboratory molecular biology here before these three grew up you can see clearly double helical ladders not DNA okay and this is current building they enjoy so there we discovered gene amplification of muscle myosin heavy chain myo three that compensate the deficit of another muscle myosin para myosin gene ang15 so you can see these mutant alleles have two or three copies of myo three genes here and then these vertical arrows suggesting the boundary of amplified fragment so this is the first example of gene amplification in germ line cells of multicellular organisms okay so after this I mean that time Sydney Brenner stepped down as director of MRC LNB and they set up a new unit so called molecular genetic system unit in Adenburg hospital which is here and LNB here so very close and then that was only two percent me and Sydney and me so that I had purchased everything including shelves benches and machines before postdocs and still the graduates arrived there we cloned and sequenced ang13 genes which is very important for synaptic transmission so but we use so-called chromosome walking you don't know young people now that's very untidy laborious work using a lot of radioisotopes so you're lucky you don't need to do anymore because you know you can use next generation sequencing to find this kind of mutations easily and then this is a more recent review and then ang13 formed a so-called priming complex with lab three and rim and then calcium entering through channels so calcium bond ang13 induced tremendous conformational change and they interact with snare complex to initiate membrane fusions for exocytosis so this is a then we did very different project to develop bacterial phage surface display okay so that first we construct this lambda phage vector by inserting amber stop codon just after phage tail protein v here stop codon and followed by long stretch of linkers including end peptidase recognition sequence so that you can cleave after purify those foreign proteins and with enzymes so this was again first example of single molecular observation we use that vector for the expression of e coli beta-galaxides so you can clearly see tetrameric structure so this is a I mean first single molecule observation in history so I wonder John Finch in MRCLMB did all negative staining and took even pictures for us so I think John Finch must be the first person to observe single molecule but I doubt it because when I was excited looking at EM pictures he asked me did you expect this result so I thought he thought I mean this is the one type lambda phage vector with bit of protein contamination so may not be and then this is a Hiroko is my wife we collaborate both in private life and in science okay so the vector you can use initially you should make a cdn library like this my human seed I mean from cells or any organisms and then you should coat micro titer plate or bees are agar bees or magnetic bees with the macromolecules like proteins, lipids, sugars, DNA, RNAs you can select those are plants interact with macromolecules then you can wash off all those unbound phage clones and then you can elute with the end dramatization to release the phage chrome which interacts with those macromolecules and then you can amplify those by infecting E. coli hosts okay and then you can repeat a few times this then eventually you can clone whatever's clone if you're interested by affinity by a puny any questions no so far okay so this is a example we cloned cdn a encoding auto angiom and chauvin syndrome autoimmune disease by quoting the patient serum on the surface of micro titer plate and applied human cdn library to that then I purify those clones and also you can use by immobilizing sugar on the surface of bees agar bees then you can clone sugar binding proteins they also you can coat DNA on the surface of magnetic bees maybe you can clone DNA binding proteins okay then we made a transatlantic journey from UK to USA San Diego in southern California this is a San Diego bay on top of this hill there is a scripts research institute at that time when 1990 when we moved there they changed the institute name from scripts clinic and research foundation to the scripts research institute now they changed again so-called I mean scripts research because they have branching for leader none kind of thing so this scripts research is surrounded by famous tori pines golf course this is a 15th signature hall in north coast I guess and then that time they established in graduate school as well as the founding new department of cell biology where I was a founding member there so that time there are scripts research institute late 80s or early 90s many people are interested in uh transmembrane signaling by cell surface receptors and they propose so many different models for example this is a dogmatic uh ligand induced dimerization model that tells cell surface receptor adopter monomeric structure is this a monomer and then ligand binding I mean in this case we show only transmembrane domains by removing this extracellular domain intracellular domain for clarity okay and then ligand binding to the extracellular domain induce the dimerization where cytoplasm domain brought together in close proximity and then or to transport force for it for downstream signaling okay this is a dogma there's no model at that time I will explain it and then this piston and scissors model for proposed for bacterial aspartate receptor for tar tar is involving bacteria chemotaxis by detecting attractant aspartate so bring bacteria towards the higher concentration aspartate and then this scissors model is proposed for electropoietin receptor which is essential for proliferation of electrolyte progenitor cells okay so uh when I was first year of graduate student I had a chance to meet one hour and then when I asked what is most important for doing excellent fantastic research and then he mentioned just one word intuition so that intuition keep my mind for throughout my research life and later I learned max sprung also said same thing you know the pioneer scientist must have a vivid intuitive imagination and then also as a first year graduate student I learned this anything fun to be true of equal I must also be true of elephant in terms of size okay in terms of complexity could be human that is proved or supported by this recent genome sequencing because we humans share 37% of gene with bacteria so I started research because my here my intuition came you know God or goddess had only six days to create everything right including cell surface receptors so so there there must be some basic fundamental principle governing the transmembrane signaling of all cell surface receptors that's my intuition okay and then I searched the literature and it found many cell surface receptor may be activated by common mechanisms particularly for these receptors shown in red for example uh varying to glutamate point mutation in transmembrane domain which spontaneously activate all these EGFRB to FGFR IGF insulin receptor FGF activate only one mutation can activate all these receptors the other evidence is uh you can swap extracellular domain or intracellular domain of these receptors and then you know it works for example if you make a human EGF receptor extracellular domain and the human insulin receptor is cytoplasmic domain that chimera is activated by only EGF not insulin and then EGF binding phosphorylate cytoplasmic domain of insulin and vice versa and this chimeric receptor consisting of extracellular domain EGFR and intracellular domain of EPO erythropoietin receptor so EGF activate erythropoietin cytoplasmic domain and vice versa the more interestingly than Koshuran his uh seminar work demonstrates that if you make chimera with bacterial aspartate receptor extracellular domain and human insulin receptor inside the aspartate can induce phosphorylation of insulin receptor right so all these uh data or results suggest some common mechanism throughout these different this distinct receptors okay so this is also encouragement look at this Richard Feynman said learn from science that you must doubt expert okay science is the belief in the ignorance of expert and Syndium is also same similar thing science must be talented amateur not expert so if you think you are expert in the field or professional in the field you should leave that field okay they said so we first analyze our bacteria our aspartate receptor tar which has homodimeric structure on the cell surface and then physically interacting key A histidine kinase with the help of key w adapter when repellent nickel in this case binds to the receptor which activated key A to increase the phosphorylated key y through key A that phosphorylated key y interacts with fluorida and flagella motor basement to induce a clockwise rotation that break apart the flagella bundle so that they stop there they cannot swim and change the direction in contrast the aspartate attractant binding can inhibit key A phosphorylation so that key phosphorylate key y concentration downs now flagella loaded counterclockwise that makes flagella stable bundle so they can swim very long time in this way the bacteria can avoid repellent nickel as a toxic metal ions and then they attracted to nutrient like aspartate through so-called random but bias random work okay so the tar has homodimeric structure I said monomer has two transmembrane domain t m1 and tm2 tm1 tm2 and then tm1 it is just stabilized diametric structure so that they do not move during sigling okay so that we replace this transmembrane entire transmembrane domain with random peptide and then searched screened genetically the functional artificial transmembrane domains so this is a summary the transmembrane domain surface consisting of three faces consisting of blue yellow and red blue surface consisting of small hydrophilic amino acid and then red is a huge hydrophobic amino acid and the yellow region is somehow between these two in terms of a hydrophobicity so all those and then we analyze the function of these surfaces these three one by inserting system residues by cross linking of this surface to nearby tm1 which has another assistance okay and then the result is consistent with the rotation model proposed here when upon repellent binding now the attractant may be better as as as stabilize this structure which is most stable structure because tm1 is somehow hydrophobic within the hydrophobic lipid bilayer right so that a has small amino acid with hydrophilic residues so that this is most stable within lipid bilayer and then repellent binding form somehow transgender take this form with b area this this surface facing towards tm1 so that's second stable structures so transmembrane domain rotate about 50 degrees so next we move to human ejf receptors at that time as you can see these textbooks there is a ligand induced dimerization dogma is is around and widely accepted because in the textbook where the receptor tyrosine kinase exists in a dimeric form monomeric form as I explained a ligand binding to the extracellular domain induced dimers where cytoplasmic domain brought together in cross proximity for trans-water oscillation which triggers downstream signaling so this is a dogmatic model already so we examine really easy far is a monomer or dimer so we express easy far on the cell surface and then applied so-called membrane impermeable cell for smpb which cross link the receptor if it's dimeric it's cross link very efficient look at this almost 70 percent of total easy far exists dimer and or more than 80 percent of total cell surface easy far cross linked 80 percent and then the ejf receptor family consisting of four members easy far or rb1 rb2 rb3 rb4 and then we use so-called bimolecular fluorescence complementation assay to see whether it's dimer or monomer in which you can fuse n-terminal or c-terminal half of the fluorescence protein in this case yfp yellow fluorescence protein to receptor easy far so if easy far exists in the form of dimer the fluorescence protein is reconstituted automatically to produce fluorescence right look at this without in the absence of ligand all receptor produce fluorescence indicating the receptor dimer in contrast if you express full-length easy far and easy far lacking cytoplasmic domain they do not produce any fluorescence indicating cytoplasmic domain play a major role to form homodimeric structure okay and furthermore if you add easy f which is ligand of other ejf receptor that cannot enhance any fluorescence indicating 100 percent of ejf receptor dimeric so this is a summary so all those four members of the easy far family takes dimeric homodimeric 1 1 2 2 3 3 4 4 and all combination of heterodimer 1 2 1 3 1 4 2 3 2 4 3 4 so they have in the absence of a ligand have a dim homodimeric or heterodimeric structure and rb3 homodimers and rb3 and rb4 heterodimers exist in the nucleus mainly my cytoplasmic as well because probably rb3 has no kinase activity so that they may have some function in nucleus we don't know what they are doing so then we try to understand how transmembrane domain of easy far moves during signaling by constructing a system replaced mutant this is a linear structure of easy far from n terminals to site extracellular domain and cytoplasmic domain and separated by transmembrane domain in cross harsh region and then we inserted nine consecutive aranine residues because aranine has high propensity to form alpha helix so that this region has expand alpha helix called structure of transmembrane domain towards the outside of cell and then we replace these nine aranines with system one by one to make these c1 to c9 system replace easy far mutant so this is the summary of the result in the absence of a ligand c2 has most efficiently form cross linking and then toward the the efficiency is decreased towards c3 right and then in the presence of ligand obviously you have to prevent endocytosis to observe this cross linking otherwise the cross-link receptor endocytosis is degraded you cannot see this but if you in the presence of endocytosis inhibitor all the system make very efficiently the system cross linking particularly c7 in black is the most efficient to produce cross linking so that suggests upon ligand binding transmembrane domain of ejf rotate about 40 140 degree from c2 to c7 okay so now all those results i mean we quickly go through went through but all those result result is consistent with the this rotation model or twist model the transmembrane domain rotate for both ejf our turn so let's just go back about the completion of this ejf work i heard from sydney brenner he said japanese government plan to set up oise in okinawa and then he asked me the suitability of founding president candidate and then i said no not good you should be president and then he accepted so this is my largest contribution to oise oops then i so so far we used three approaches to detect diametric structure chemical cross linking and and by fc and system cross linking which is all unidirectional reactions so that we don't know how much ejf receptor exists as a diamond or monomer at steady state right so we searched again literature and then i found fccs so-called fluorescence closed correlation spectroscopy maybe the choice to measure the ratio between ratio of monomers and diamonds and then i found frosting warland who is in singapore so i made another trans pacific journey to singapore so again this is before establishing this biopolis so we rented the space and started the work and then waiting for completion of biopolis here and then this is a slide made by a graduate student showing the principle of this fccs so if the ejf receptor move as a monomer in this tiny point to femtoliter confocal volume you don't see any good cross correlation instead if ejfr move as a dimer you see very good cross correlation so this is a summary of the data after collecting many data forcing mathematically calculate the percentage of dimer okay and then using monomeric gfp and monomeric mrfp in cytotoric as a negative control they as a they monomers and then we made a tripartite construct which has covalently fused mrfp ejfr gfp as a positive they should move together right and then if you analyze this ejfr ejfr homodimer rb2 rb2 homodimer or rb2 ejfr homodimer hedl dimer they are significantly higher than this negative construct control and then i mean statistically indistinguishable from positive control suggesting that that these homodimers and hedl dimers behave as dimers so after spending three years in singapore i completed the journey from tokyo japan to ois i mean okinawa japan so this is a funding members oist again we worked on renting space on the other side of the island and then after i leave only kenji is the main founding member there look like very young okay this is the original design of oist campus entrance so don't forget to request cabinet office to build this bridge and elevators okay okay at oist so we made another challenge to dogma so high impedance neurons with short processes like those of syrians uh the nematode syrians people i mean thought for the last four decades they do not produce any active propagation they send electric signal by passive propagation but in collaboration with jiff wikens and his colleagues we discovered the acl and acl that major gas statory sensor neuron which produce all one and uh depolarization so that's again you know don't believe any dogma it's sometimes wrong and also we did a collaboration with wolf skoglan and his colleagues to analyze uh detergent solubilized dimeric ejf receptor okay this is unlike the previously anticipated people believing if you solubilize ejf receptor by detergent they behave like they are monomeric okay that's why they propose ligand induced dimerical model but if we use ddm i mean uh kind of detergent they cannot break dimers so that you can purify dimerical ejf receptor and then wolf determined this various conformers of ejf receptor all right this is a cryoet good good point you know it's very sensitive to determine various conformers okay if you use single particle cryo eem you can determine very precise structures but they are very stable structure like a crystal structure okay so they they compensate each other the cryoet is not very high resolution but they can detect various different structures so that you know combination of the two may be the best for the structural study so this is a summary in the absence of ligand the ejf receptor dimer has very flexible extracellular domain and relatively stable intracellular domains right then in in the presence of a ligand now extracellular domain becomes relatively stable and then intracellular domain is become flexible so this is a model we propose for ejf r activation so ejf r receptor exists dimers homodimers through interaction of inter intracellular domain and transmembrane domains in this inactive symmetric structure is is a stable before activation right and then extracellular domain has kind kind of this flexible structures and then ligand bind to the open untethered molecules which induce this kind of rotation by interacting the other side of the protomers that as a rigid body that's likely to induce rotation of transmembrane domain which dissociate the symmetric kindness domain into asymmetric inactive kindness domains so this is a model we propose this is consistent with cryoem structure in every other data and then now many other people also analyzed other receptors not only receptor tyrosine kindnesses cytokine receptor receptor gonorrhea cyclase receptor histidine kindness and sensor transducer transmembrane protein patterny gonorrhea receptor receptor tyrosine phosphorus though these are all exist as dimer and they the transmembrane domain is rotate upon ligand binding and then this yellow rotation angle is determined so now some app or group all those receptors you can say the cell surface respect receptor adopt dimeric structure with flexible extracellular domain and stable intracellular domain which induce this stable or heterodimeric structures then upon ligand binding induce rotation of transmembrane domain which dissociate intracellular domain for downstream signaling so this is a relatively recent review they mentioned ligand induced rotation in addition to still ligand induced dimerization that's because max frank said an important scientist a scientific innovation rarely makes its way by gradually winning over and converting its opponent instead what does happen is that the opponent gradually die out so we have to wait for they die so this is a rubber to remember who are brave enough to challenge dogmas and these are institutional agencies to support this project and what's next is to rejoin my family in san diego this is a look at these two more far behind okay and one more my daughter taking this picture so that's whole family in san diego so last not but not least i'd like to thank my wife hiroko for her contribution well obviously without uh which his uh i could not be here today okay thank you for your attention and i'm ready to take questions do we have any questions thank you very much for an extremely impressive uh and inspiring talk i i learned a lot so there is a very interesting data showing like the 70 percent of ejfr dimerized without ligand in vitro in vitro data so three millimolar is that the concentration is it really physiological range or not well that that's good question though here we we changed all sort of different concentration and find best concentration to to detect dimers but i don't know whether it's i mean it's this is the in vitro experiment right and then uh that kind of close linking efficiency is not very great particularly if you do it on ice and kind of things so that's why uh people didn't detect any dimers before we did mostly they use different cross linkers so we find i try to find many best best cross linkers to detect this so that's difference so you know i believe it's not 70 percent or 80 percent i i think 100 percent is dimer because other data indicate so it's is that is that's answer your question well i was wondering whether three millimolar is like a too high that will induce artificial dimerization without ligand in the cell if you use too high cross linker consented too high you don't see any receptor i see look at this it's all covered up all the proteins you don't see thank you questions thank you thank you for this great talk in bacterial chemo receptor arrays the tar receptor exists in in in the sort of almost a crystalline hexagonal lattice right oh yeah yeah that's right is is that consistent with the rotation and is it uh is it actually physiologically that the tar receptor exists you know the tar has a so-called hemp domain in the inside site plasmic domains that has a cog wheel structures so that's consistent with transmembrane domain rotations the rotation induced rotation of a cog wheel of hemp domains that induce winding unwinding of long alpha helical bundles in this height of present tail that open up methylation residues and rearrange those kindness domain well place positions so that i believe is also the rotation is consistent with this regression of activity all questions are there only ah can you okay yeah thank you very much so i have been with you for a long time but it was a very good opportunity for me to learn how your research starting from the early days in the UK carry on until the your time in the west and then in uh attacking dog dogma i think you had a lot of difficulty for example uh you use the like fluorescent protein to monitor the state of a monomeric versus dimeric but uh wonder any like a criticism that those attachment of the fluorescent component would affect the modernization dimerization states yeah well full of critics you know i had you know if you modified whatever could happen something funny or wrong or bad so we we did more than i would say 10 different approaches well i omitted everything and then all those 10 different techniques converge to to the 100 diamond so that i i believe and then not only ourselves but many others you know many other receptors also use it i mean take took homo dimeric structure head of dimeric structure and then rotate transmembrane domain so that uh that kind of uh data accumulating years and over the years so i think i believe the model is correct but you know i mean still now many people don't i mean believe in that ligand induced dimerization model i don't know how you know but you know that psychologically they don't want to accept the new models or what you know that's mark sprunk i mean indicate they have to die and for the like a turn the ejf and all those after this dimeric rotation model appears very correct but the other other mechanism of a transmembrane signaling mechanism used by other types of receptors or this rotation is the only or most common ways of a extracellular i wouldn't say every receptor behavior like this so maybe piston like movement or scissors or what maybe c source may may be but it's not very beautiful isn't it if different receptor has different mode so you know maybe there is some exceptions but i i also think maybe your question is more general because there are other other molecular constructs in the membranes we call receptors right and and they behave a little bit different but i would say for in your field acetylcholine receptor which multiple transmembrane domain receptors yeah again those transmembrane domain rotate to open and close the that channels because this is the most economical movement of the transmembrane domain in comparison to this translated you know movement or or up and down or or this kind of you know so maybe this is the case also for like a dopamine or serotonin receptors as well i i guess so i don't know right thank you more questions or are we zooming in on the end here well it was a very very nice lecture for us and i would like to all of us give you a hand again i can give you yeah sure so next so ichiro thank you again for the wonderful talk and you earned your gift so off is going to present we have one more your unit members would like to share thank you i'm so honored that i have been there research unit administrator for his unit and mariam unit and this is from all the members of mariam unit thank you very much