Provost Lecture Series: Tomomi Kiyomitsu

Okay. Uh, hi. Sai everyone. Thank you very much for coming today. First, uh, KCO, thank you very much for your excellent introduction. I think this is the best introduction in my research career. Yeah, thank you. And also I'd like to thank uh Amy and also provost office uh members uh for giving me this great opportunity. Uh today I want to uh introduce myself and also my unit research uh to O community for a future development. As you know our body consists of a large number of cells and uh based on this paper adult human body consists of about 40 trillion of cells which are roughly 5,000 fold larger than world population in [music] 2026. It's so huge except uh very uh large number also amazingly we were born as one cell. Please imagine one cell increase its number and cell type to make functional body during early development. This is totally amazing and beyond my imagination. cell can uh increase its number by cell division. Uh this is um cartoon showing a typical uh animal interface cell. The nucleus contain DNA. DNA is very important because this contains a biological information. We receive this information from father and mother and this is very precious and unique [snorts] and determine your identity. Then during interface DNA is replicated precisely. Then during mitosis this DNA is condensed and form this condensed chromosome. After nuclear ember breakdown microtub polymer interact with chromosomes and align and segregate to daughter cell to maintain a genotic information. This structure is called a micotic spindle and this only transiently formed during uh codiction process to segregate [music] chromosomes. And this is the current model of an aromatic spindle. Spindle consists of a lot of short micro tubes. And the micro tube consists of alphabeta tubing dimers. And the tubing dimemer polymer at the plus end micro tubes and shows growth and shrink. And minus end show relatively less dynamics and minus end or micro tube accumulate around [music] this uh micro tube organizing center uh center. So then grow micro tubes and form the uh a micro tubes. This is called aster micro tubes. Then placent interact with cortex or plas membrane. Also placent micro tube interact with chromosome at kinetto core. This connect chromosomes [music] with spindle micro tubes and these uh micro tubes are bundled by microchube binding protein to stabilize [music] this kinetto core micro tubes. And this uh spindle is formed in all ukariote and all developmental stage including this one cell stage. After fertilization the pronucleus migrate and form the first spindle and our long journey starts. Okay let's look at the actual example. This is a a medata 2 cell embryo showing a chromosome and micro tubes like this chromosomes are condensed and align center spindle and beautifully segregated to daughter cell. It's very dynamic and beautiful process. Spindle >> uh ensures equal uh chromosome segregation and abnormal spindle function cause chromosome mis segregation which leads to the gain or loss of chromosomes. This is called anoy and also sometimes uh chromosomes are damaged or clipped by cyto kinetic machinery and abnormally distributed to daughter cell. Now this is called micronuclei and this uh promote uh massive chromosome rearrangement or recently it is reported this is transported other to other uh cell and this is a list of uh showing the uh frequency of anuplo and also chromosome uh misgration rate as As you can see here, yes, cancer cell shows very high frequency of ochromosome misgation. This is uh in my during my m myis and this is a mayotic division which generate sperm or oite as you might know. Yeah, human oite has relatively large number of uh mistration rate. It depends on aging. Now as a researcher extensively study this then now I'm curious uh here this number human zygote also have relatively high number of uh chromosome uh misregation rate. Now several reports suggest uh zygotic division is elapone in m or human. Initially I expected early division should be accurate because if this is abnormal downstream uh this effect is uh transmitted to downstream uh daughter cells. However uh this is different. Okay. So for uh roughly 20 years uh I I'm interested in this mechanism and I have studied a spindle assembly and chromosome segregation. Also we are studying uh spindle positioning because spindle positioning can uh determine the distribution of non genetic information such as data cell size and the location or polarity of cell. Cell division can be classified into two types symmetric or asymmetric division. Symmetric division generate two identical data for clonal expansion whereas asymmetric division gen two different type of data cell to increase cell type diversity. The balance is very important. [music] So now we are uh interested in the mechanism of spin assembly and spin positioning in my unit. Okay, I slightly change my topic. I want to briefly uh share my previous career development. Maybe I hope this would be useful for uh PhD student and postto as KO uh mentioned uh my academic career stud at uh K University in professor Mits lab in his lab he used fishist to study the chromosome segregation he identified a lot of mutant temperature sensed mutant showing Chromosome al phenotype abnormal segregation and also chromosome misregation phenotype. As you know uh fishist has only three chromosomes. That's why we can see uh chromosome uh unequal segregation just by looking at the mass of uh chromosomes DNA. So this is nice example to use uh the model organisms. Then he identified a lot of residential genes many of which are conserved in human. Then uh my senior colleagues Gossimasan studied identify human mist and obsess identified the human m binding partners using mass spectrometry and I analyze one of human ming protein called blinking or now this is very famous as KN1. Now we found uh this uh protein is important for uh kinto micro attachment and also mitoic checkpoint. Then I joined yan cheeseman's lab. Here I shift my topic from kinetic core to the cell cordex to understand the mechanism of spindle positioning especially spindle centering to divide symmetrically uh spindle is positioned in center of the cell. The question is how spindle can find its center. Here we found uh two key uh in internal uh gradient derived from chromosome and the centers and these gradient control cortical dining molecule din is a microchip based binding model then uh I I'm happy to show our recent uh result about dining later Then I joined uh professor God's lab as an nonPI assistant professor. However, uh he allowed me to uh uh establish my uh small independent uh group in his lab and also Ian allowed me to continue my postto work. So I really appreciate these two supervisors. Then in Nago University I want to introduce crisper based genome editing. Now we can knock in these tag at uh specific target gene region to induce uh to visualize or deplete or manipulate target protein. I want to yeah achieve this. Then I collaborate with professor Mas Kanimaki uh professor at National Institute of Genetics. He developed excellent tool called aid oxing inducible diagram. So we invited him last week and uh uh he developed uh this technologies. Then uh we use two factors. One is M tag the other is OT1. Both are plant protein. We use this plant protein in human or animal cell. This protein forms CFC EC ED like in human cell and in the presence of plant hormone oxin this recognize this tag and promote polyination which leads to proteasomedate protein degradation. Point is protein degradation halflife is roughly 10 or 20 minutes. Very rapidly we can degrade target protein. As you know DNA is transcribed to mRNA then mRNA is translated to protein and in many cases protein has its own function. Previously I used RNA I to to analyze to degrade mRNA to analyze its function. However RNAI target mRNA but not protein. >> Therefore if protein previously exist we cannot degrade this. That's why uh I think this uh technique is uh provide very nice clean uh result and also very useful especially in uh early embryo division uh research because embryo has a lot of maternal protein. We need to degrade protein to understand its function. Okay. First uh we use this technologies in tissue culture human cell and we visualize dining and we found uh we we successfully degrade dining and we found that is important for chromosome alignment. Then also we introduced uh manipulation >> here I I use light inducible technologies. Uh in short uh we can uh target this protein called numa here spec. Uh then we found this is sufficient to recruit the dining molecule and also to control spin position in live cell. Now we establish this technology. So I want to introduce this technology in uh live uh developmental context for example in uh embriionic cell. However you know human embryo is very very difficult to use for ethical and al technical reason that's why again mo organism model organisms are very important. First candidate is mass but as you may know uh mass sperm uh does not provide functional central it's slightly different frog excellent especially for biochemistry but not suitable for live imaging that's why I was very curious to use fish I found I thought oh fish cleage is slightly different from frog or human but maybe this is the problem of the cytochinesis but not spindle assembled or chromosome segregation but rather this provide a very nice opportunity to find uh chromosome uh spindle spindle on the same uh focal plane that's why this provide a very nice opportunity to capture all spindle in early embraic division and fortunately professor minaka uh N University and he studied uh medical uh sex determination. Then uh also he uh already established a GP advert expressing metat transgenic fish to visualize uh micro tubes. Then he kindly allowed me to use this uh string. Then I found uh yeah I analyzed visualize this uh and there I was very surprised to see oh the inner structure is very different [snorts] from tissue and this orientation nice figure so I want other I think another question why that's why in this video episode explanation of first yeah of course uh it might be good to use human n but this is still corporate that's why that's symbol from my PhD war and also the history I wanted all is very very use and also this is best pandemic Yeah, that's uh keep distance from um competition also I I love driving me pattern that's why I want to use live that's why baby this must have also uh personal reflex and the visual advantage is very for them. For example, maybe you know to me is a model called one meta deta is Japanese I should like also uh uh there are lot of nice resource we should now we in the app not in US that's why we get we should take a condition you might be in There is a difference. This is similar, very similar but some difference. For example, yeah fish, metaka pear produce egg every day. You know we collect egg today then tomorrow we can get egg from the same pair. This is very nice for experiment also. Yeah, genome size is smaller than zebra fish. This facilitate generating also premise temperature is relatively wide. This is very attractive to me. Also, uh Medaka has a lot of inbredad lines, a lot of closely related species. That's why I feel a strong uh potential to medaka especially for my research purpose. Of course, zebra fish is very excellent model, but for us, maybe metag would be better. And also again, zebra fish is very popular. That's why I I prefer uh using metacapish. In addition, in Japan, medaka fish are used in elementary school uh in fifth grade uh science class to help students understand how animals pass on life. like this we compare human and fish that's why I thought uh if uh we can get a nice results >> might be good for outreach activities okay then let's move on uh then uh I'm interested we are interested in the mechanism of spindle assembly and the positioning because uh these are important for uh to maintain a genomic inform information and non-genetic information during uh development. However, uh we already know a lot of mechanisms and key factors but they depends on automatic cell models. Also, we know uh early embionic division called cleavage have very unique features. For example, large cell size also transcription is off or suppressed and the cell cycle is very rapid SM at least in fish and frog and also checkpoint is not functional at least in medakata. However, chromosome should be equally segregated. That's why I want to look then this is the first uh quick I I just quickly check uh chromosome and spindle in two cell stage and later stage then we found look chromosomes are almost similar very similar but micro spindle structure and size are very different. So >> this is the same embryo then they use the same tubing protein. However the assemblies are very different to segregate chromosomes. Based on this we hypothesized mechanism of spindle assembly and positioning would be different between cleage and somatic division. To understand that first we tried to uh establish uh transgenic uh metacol line to visualize chromosome. Uh here we inject uh try to estab guide and donor construct coding mch cherry at uh rc1 gene locus. This is chromosome marker. Then we uh finally get uh this uh not clean string. Then after that we crossed uh this uh ej for tubing expressing metacapish strength to visualize both chromosome and micro tube. And this is the first medical fish showing some IQ. And uh as I mentioned before uh in fish uh cell divide as the periphery of the egg then we invert it like this. Then we fix the seven image. Then he show the sculp. Okay. Then if it's the egg in the channel made by agaros then we took the image using [music] uh spinning disco microscope. Okay this is one of the highlight of my talk. Then this is a a male prucleus. This is a female pronucleus. Then after fertilization they fused and form a nice beautiful bipolar spindle and they they undergo repeated cell division process. It's very dynamic and also very beautiful. Now we took uh time-lapse imaging roughly for for example 10 hours. Of course imaging cause damage phototoxicity to the embryo. However, uh this embryo still continue uh normally the development then they can hatch. Therefore, now we believe we can capture the physiological uh dynamics of uh cell division process. >> This provide a lot of new information. Uh for example, uh cell cycle is roughly 30 minutes. uh then uh mic duration from nuclear breakdown to an onset is roughly 10 minutes. It's very fast. In tissue culture human cell it takes roughly 30 minutes but in this case only 10 minutes fish embryo can capture the 24 pair of chromosome within 10 minutes and nicely segregate without a clear uh error. Also maybe you already know this uh nucleus is positioned in the center of the cell then divide again the orientation is very nicely controlled to make symmetrical pattern. So we don't know why but this is very uh exciting and uh we already published this then you can of course download this then please use this under the uh license of CCB4 uh yeah and recently we got this award and I described some explanation so if you're curious uh please check this then we uh carefully analyze as been uh lengths during development. Then we found uh dynamic structure change uh during early embroenesis. Especially I want to emphasize that metac spindle has dense uh microtub network at the spindle midplane around the uh chromosomes. This is specific uh in early uh embryo and this seems to be stabilized. That's why I want to know uh this mechanisms uh in the literatur group suggested uh RCC1 this is we use this as a chromosome marker but this is actually enzyme to produce GTP bound form of RAM then this GT l GTP a dissociate inhibitor in pushing from uh spindle assembly factors. Therefore chronicize that RP actually the skin color found which standardize for n to test that we want to redesign product but by use this system to make a long story so we should >> yeah uh we could uh establish RCC This is your mid global flag homozygous knocking strain. Then we injected mRNA encoding OT1 plus uh MJ H3V which uh we can uh monitor the expression of OT1 by this red fluoresence. At the same time we can monitor the chromosome dynamics. As you can see here in response to the expression of MJ fluoresence. Now JFD fluoresence decrease suggesting aid system work in metagapish embryo. This is a movie in response to expression of the red signal. Green signal is reduced. Then we could see very very abnormal chromosome seation phenotype something like that. Yeah. Then when I first look at this, oh this is very similar to fish is cut a mutant and then in those days professor was still in oy that's why I print out this [music] image and visit his office and show that then he praised me. Yeah when I was a future student I he really praised me but in this situation yeah he praised me. I was very happy. And after that uh yeah this cause embarity if we look carefully this shows several uh slightly different phenotype uh depends on the depletion level it's slightly difficult to see but uh yeah it shows lagging chromosome or micronucle or lagging or this non-isjunction phenotype but in all cases we also found uh abnormality of the spindle uh dense microchip network is not formed when we deplete RCM protein. So now we speculate uh this is one of the cause reason to uh induce this abnormal segregation in embry and now we can degrade and protein in later timing if we use culture we just degrade that's it but now we can we have developmental axis right then we can uh by controlling the timing of uh oxygen addition we and degrade this triggered protein in later stage. Then we can analyze its phenotype and we can compare. So yeah we could degrade RCC1 in later stage and interestingly we couldn't see any uh chromisation phenotype suggesting RCC1 is dispensable in late stage embry uh it cause slightly uh delay and this cause uh additional uh defect after mitosis especially nuclear the envelope deformation is just is very uh abnormal but uh yeah chrome spindle assembly and chromosomeation is basically a normal so this is a summary of this paper uh first uh I would like to thank these external contributors and the point is yeah previously we and others found uh in uh small somatic cell RCC1 is known essential likely uh centrosome dependent nucleation is enough to make functional aspindo but uh in rur uh uh embryo [sighs] chromosome derived rccc1 dependent GTP signal is essential become essential to make a functional uh spindle. This situation is similar to all site. Okay. Then now we established a basic strategy. Uh we can establish knocking strain for gene X and analyze its localization and depression phenotypes in early and late stage embryo. Then uh today I I did not I do not show the data but we already uh succeeded in performing rescue experiment using wild type and mutant after depletion of endogenous one uh to understand the domain function. Now we already established one, two, three, four, five at least five uh diagonal green strain. Then one is now evaluating and now uh uh our other as a member uh are trying to establish new strengths. Let me briefly uh introduce show our recent result about dining. So uh as I mentioned before uh dine is microchub uh dep dependent uh m minus m directed motor d is highly conserved uh in ukarot and dining transport almost all organ toward minor end that's why very important also during mitosis d is important for chromosome spin attachment and also for uh focusing and also spin the positioning in at least uh in uh smallotics. I'm wondering uh where and how dining works [music] and extremely large embryo. This is this shows a meta embryo and human uh HC16 cell with same scale. Now you realize how big it is. Then uh first uh we establish uh dining em uh no queen strain and also dining a global nine strain. So here we visualize dining using Merry together with EJ tubing. Dining is shown in magenda and yeah please look did you see something uh interesting. We could see uh halo like enrichment at at aa periphery in cytolas. Oh the dining uh shows halo like enrichment at the as the periphery in cytoplasm in metaphase. This is totally unexpected. I have never seen such uh cytoplasmic enrichment in smaller cell and also uh in other organisms for example in circans uh as a group visualize endogensteining but they have never uh seen such uh cytoplasmic enrichment likely due to the size of the embryo. Okay. Then also we analyze dinens localization. Dino chain is dining binding pattern. They shows a very similar localization pattern. Now we found this enrichment is well regulated during cell cycle to understand its function. Next we deplete dining interaction using this uh inhibitor called P150 CC1. Then after injection uh this disrupt dining localization at central or spindle or head like this. This cause a phenotype at metaphase but most uh unexpected phenotype was observed in [music] anaphase. This is control in anaphase you can see large aa expansion something like that right. However when we deplete the dining we could see a lot of microtub nucleation inside plasm. This cause very abnormal cleavage like that. Maybe this is easier to understand. In normal situation, your micro tube dominantly uh glow at the a like this. However, when we inhibit dining, we could see a lot of feather like microchip nucleation in sight plasma. I do not explain the detail. If you're curious, please check this file archive. Now, uh yeah, we already submitted this uh manuscript. Okay, this is a summary. So, initially we hypothesized uh mechanism of spindle assembly and positioning are different between early and later stage divisions. Now, we can provide a strong evidence uh showing this. Okay. And uh in the future uh we want to understand this mechanisms and also we want to know how this is remodeled uh during uh uh empiric divisions. Maybe you might want to know whether these mechanisms are conserved between medaka and other vertebbrates. Of course uh cleage pattern are very very diverse between different organisms. However, uh for example, this is a recent report from uh other lab showing the human embryo in first uh first stage and late stage. If we look this carefully, of course uh microtubal probe are different between these experiment. That's why we should be very uh cautious. But this data suggest uh in early embryo to me uh micro tube is more dense near the chromosome compared to later stage similar to embryo that's why I think uh partially uh mechanism may be conserved we want to ex uh continue our approach and also in the future we also want to uh intro introduce new technologies to manipulate uh spindle formation or spindle positioning using for example laser operation or optogenetic technologies. Also uh personally I want to identify new genes essential for embed assembly and positioning. Previously we receive uh we had a unit review. Then one uh reviewer said our research is good but we only target all the players. I agree. Yeah. as a first step which we should focus on all the player established one but I I personally believe uh there are so unknown uh early embryo specific essential genes in the future I want to identify this using several techniques such as massspec then if we could identify this new gene I want to show that to that reviewer also this the same reviewer gave us very nice word blue ocean. This might be the best word which I received in my research career. As you know blue ocean means very pioneering and very uh yeah less competitive work. The opposite word is red ocean very competitive. Of course there are advantage and disadvantage but I I personally prefer a blue ocean in the future. Yeah, we want to get something uh we want to uh yeah discover something very unexpected and totally exciting uh finding this is acknowledgement. I'd like to thank uh all uh previous and current members As Ko mentioned uh we started our unit during the corona pandemic then after that we gradually enjoyed Okinawan food and nice restaurant something like that currently uh our unit have f five students uh so far I have not yet uh graduated any PhD student that's why this is my key one of the key goals in the next uh five years I I want to graduate uh PhD student they already have a nice data that's why I'm op optimistic also uh yeah this is uh very uh impressive pictures to me they kindly celebrated my birthday sorry then yeah I'm very happy to be able to work uh with these students and other members I also O want to uh thank former member at Nag University and collaborators and uh in Oy uh recently we share the general club with CO France and Midori and recently we co-organized this workshop it was very nice and I uh nasan kindly gen synthesize several chemical and also yeah we'd like to thank especially animal resource section for medical feeding. At the beginning uh I and my wife uh came to the lab every weekends [clears throat] for feeding but recently yeah they kindly helped that it is very uh important to for our research. I'd like to thank uh these organizations for grants. Then when I consider the tenure, of course, I want to uh thank all previous uh supervisor teachers, but especially I'd like to thank uh these uh sensei Shinshi in Okinawa was without their supervision or mentoring or support maybe I could I couldn't get this current position. I really appreciate uh their support. Also I want to express special thanks to Isam. Uh she established a lot of no strain and established live imaging and system in medical. So this is Okinawan's world. Thank you very much. [applause] very much for the fantastic talk. We really enjoyed. So maybe we can take a couple questions. So any questions or comments? >> Thank you. It was very nice story especially establishing new system is a lot of work and then it's impressive. Uh I have two question. The first question is I was surprised medaka toreate a very wide range of temperature >> from 4° to 37ish degrees. So uh the is there any um good temperature for embryogenesis or embryogenesis also tolerate uh whole range of temperature. >> So yeah >> ah yeah good question and uh I can show you this one first. Yeah, maybe this one, right? Okay. Yes. So, this is the permacy temperature for adult fish. >> Then for embryo, the situation is slightly different. If I understand correctly, uh, of course, it depends on different species. Then uh roughly primive temperature is 20 to [music] 37° for embryo. >> Okay. >> But this is very slightly different in different [music] species. That's why but this is interesting because if we compare different species we can narrow down the essential genes required for uh to explain this uh permissive temperature. >> Okay. Thank you. And the second question is, can you um locally target dining? For example, you showed a really nice hall localization of dining. Can you specifically knock down or knock uh Nokia knock down that local locally dining? >> Locally knock down dining. >> Yeah, >> locally knock [music] down dining. uh recently as a group reported uh optogenetically controlled dining inhibitor. Okay. Now we currently we are using a P150 CC1 flag but this can be Yeah.