IT IS MY GREAT PLEASURE TO INTRODUCE TODAY'S WEDNESDAY AFTERNOON LECTURER, DR. YIXIAN ZHENG, THE CHAIR OF THE DEPARTMENT OF EMBRYOLOGY AT CARNEGIE INSTITUTION FOR SCIENCE IN BALTIMORE, INTERIM CO-PRESIDENT OF THE CARNEGIE INSTITUTION. SHE'S ALSO AN ADJUNCT PROFESSOR OF BIOLOGY AT JOHNS HOPKINS. DR. ZHENG EARNED A BACHELOR OF SCIENCE FROM SZECHUAN UNIVERSITY, BOTH OF HER PARENTS TAUGHT THERE. SHE'S THE DAUGHTER OF TWO ACADEMICS. BUT GREW UP DURING THE CULTURAL REVOLUTION IN CHINA. AND SO HAD SOME INTERESTING EXPERIENCES. I WAS JUST LISTENING TO A VIDEO SHE MADE HOW SHE BECAME A SCIENTIST, IT IS FASCINATING. AFTER HER UNDERGRADUATE EDUCATION SHE MOVED TO UNITED STATES TO STUDY WITH BERL OAKLEY AT THE OHIO STATE UNIVERSITY, WHERE SHE GOT HER Ph.D. IN MOLECULAR GENETICS. AND THEN SHE DID A POSTDOC AT UCSF WITH TIM MITCHISON AND BRUCE ALBERTS WHERE I SUSPECT SHE GOT INTERESTED IN MICROTUBULES. SHE WAS RECRUITED TO THE CARNEGIE INSTITUTION IN 1996, SHE WORKS ON A VARIETY OF ISSUES RELATED TO CELLULAR CYTOSKELETON AND MITOSIS, NUCLEAR FUNCTION AND MICROTUBULES, LAMIN, AGING, MORPHOGENESIS, CELL DIVISION AND DISEASE. PROBABLY PARTLY BECAUSE OF HER UPBRINGING IN CHINA WHERE SHE GREW UP WANTING TO BE A GREAT WRITER, SHE'S PRODUCED A SERIES OF ELEGANT SCIENTIFIC PAPERS DESCRIBING HER JOURNEY THROUGH THE MICROTUBULE ORGANIZING CENTERS INCLUDING HER INITIAL DISCOVERY THAT GAMMA TUBULIN IS WELL CONSERVED IN ALL EUKARYOTES, AND DISCOVERED THE RING COMPLEX IS AN IMPORTANT INITIATOR AND MANY OTHER DISCOVERIES. SHE WAS A PEW SCHOLAR, HOWARD HUGHES INVESTIGATOR FOR OVER A DECADE, WINNER OF ASCV WOMEN IN SCIENCE AWARD, HAS BEEN RECOGNIZED FOR SERVICE AS A COUNCIL MEMBER FOR AMERICAN SOCIETY OF CELL BIOLOGY, CHAIR OF THE INTERNATIONAL AFFAIRS COMMITTEE FOR AMERICAN SOCIETY OF CELL BIOLOGY, SHE'S A NATIONAL ASSOCIATE OF THE NRC COUNCIL FOR NATIONAL ACADEMY OF SCIENCES, AN HONORARY LIFETIME APPOINTMENT FOR EXTRAORDINARY SERVICE. AND SHE'S A SENIOR SCHOLAR IN AGING FOR ELLISON MEDICAL FOUNDATION. TODAY, SHE WILL TALK ABOUT HER RESEARCH ON CELL DIVISION AND GENOME ORGANIZATION IN DEVELOPMENT IN AGING. PLEASE JOIN ME IN WELCOMING DR. ZHENG. [APPLAUSE] >> THANKS, MICHAEL, FOR SUCH A NICE INTRODUCTION, AND THANKS TO MY HOST FOR ARRANGING THIS AMAZING OPPORTUNITY TO COME HERE, TALK TO PEOPLE FROM THE BROADER SCIENTIFIC COMMUNITY AT NIH. SO, I'M JUST GOING TO TRY TO DIVE IN. WHERE IS MY -- OH HERE. I'M GOING TO TRY TO GIVE HIGHLIGHTS ON THE WORK THAT HAS BEEN PUBLISHED, AND SPEND THE LAST TEN MINUTES FOCUSING ON SOME OF OUR MOST RECENT WORK ON GENOME ORGANIZATION. HERE SHOWS A PICTURE OF OUR BUILDING, OUR DEPARTMENT IS PRETTY SMALL. WE HAVE EIGHT FACULTY PLUS TWO STAFF ASSOCIATES, A PRETTY AMAZING DEPARTMENT TO BE IN. SO I'M A CELL BIOLOGIST, I STARTED MY CAREER TRYING TO UNDERSTAND HOW CYTOSKELETAL WORK MOSTLY FOCUSING ON MICROTUBULES, INTERFACE CELLS, AND IT REALLY DEPICTS THE COMPLEXITY OF INTERFACE CELL WITH SO MANY CONNECTIONS EVER NUCLEUS, NUCLEAR LAMINA, CONNECTING THROUGH NUCLEAR ENVELOPE PROTEINS TO VARIOUS FORMS OF CYTOSKELETON AND MEMBRANES ARE HIGHLY CONNECTED TO ONE ANOTHER. SO, THE THING FOR ME WHEN I START MY CAREER IS REALLY TRYING TO UNDERSTAND -- SORRY, I NEED TO GO BACK -- TRYING TO UNDERSTAND HOW, YOU KNOW, YOU REALLY HANDLE WHEN YOU WANT TO GO INTO CELL DIVISION, HOW YOU GO FROM ONE CELL TO TWO CELLS. SO A LOT OF WHAT I'M GOING TO TALK ABOUT ACTUALLY STEMMED FROM OUR INTERESTING CYTOSKELETON, WHAT I WANT TO DO IS FIRST GIVE YOU A HIGHLIGHT ON OUR STUDY OF MITOTIC SPINDLE MATRIX IN CELL DIVISION AND THEN I'M GOING TO SPEND MOST OF MY TIME TALKING ABOUT NUCLEAR LAMINS, BY HIGHLIGHTING SOME OF OUR STUDIES ON HOW NUCLEAR LAMINS ARE INVOLVED IN TISSUE BUILDING AND IN AGING AND SPEND MORE FOCUSED TIME ON HOW LAMINS COULD REGULATE 3D GENOME ORGANIZATION WHICH IN TURN INFLUENCED TRANSCRIPTION GLOBALLY. STARTING FROM THE BEGINNING OF MY CAREER, I WAS VERY INTERESTED IN MICROTUBULES, HOW MICROTUBULE ARE BUILT INTO A BIPOLAR SPINDLE. THE FIELD FOR DECADES HAS BEEN INTERESTED RIGHTFULLY SO FOCUSED ON UNDERSTANDING HOW MICROTUBULE CAPTURED KINETIC CORES AND MAKE TWO CELLS, BUT WHEN STATERRED MY OWN CAREER I FELT LIKE THERE PROBABLY IS REALLY A NEED TO TRY TO UNDERSTAND ABOUT CELL DIVISION THAT'S BEYOND HOW MICROTUBULES CAPTURE CHROMOSOMES BECAUSE CELL DIVISION IS NOT JUST SEGREGATING CHROMOSOMES. YOU NEED TO SEGREGATE SO MANY OTHER THINGS, MASSIVE CHANGES OF NUCLEUS, CELLULAR MEMBRANE INCLUDING NUCLEAR MEMBRANE, NUCLEAR LAMINA DISASSEMBLES AND REASSEMBLES. THE ORGANIZATION WAS MUCH LESS UNDERSTOOD. SO WHEN I START MY LAB, WE'VE DECIDED, OKAY, LET'S JUST SEE IF WE COULD BROADEN BY MITOSIS RESEARCH TO INCLUDE OTHER -- WHAT DID THEY REALLY HAVE INTERFACE FUNCTIONING, HOW CAN YOU SEPARATE AWAY FROM MITOSIS FUNCTION. AND WE'RE LUCKY BECAUSE THE SYSTEM THAT WAS DEVELOPED IN THE '70s BY PEOPLE LIKE MATSUI AND LUCCA, THEY SHOWED YOU CAN TAKE SPERM AND PUT IT IN XENOPUS EGGS, AT THE TIP OF THE SPERM, RECRUIT MATERIALS TO MAKE ACTORS AND WITH TIME CHROMOSOMES MAKE MITOTIC-LIKE CONDENSED COMBSOMES AND MITOTIC SPINDLED ARE ESTABLISHED. YOU CAN MAKE ANTIBODIES AND DEPLETE PROTEINS AND REQUEST WHAT HAPPENS TO THIS PROCESS. AND IF YOU SEE A DEFECT, YOU CAN TRY TO RESCUE IT, AND THEN SEE IF THIS DEFECT IS REALLY CAUSED BY THE PROTEIN. EVEN IF YOUR PROTEIN HAS INTERFACE FUNCTION YOU CAN'T SAY IT ALSO HAS MITOTIC FUNCTION BECAUSE THE SYSTEM NEVER REALLY HAS GONE INTO INTERFACE. THIS HAS TURNED OUT TO BE VERY PRODUCTIVE, USING THIS SYSTEM WE HAVE BEEN ABLE TO ESTABLISH MANY NUCLEAR PROTEINS, A FEW LISTED HERE, RanGTPase, LAMIN-B, AND A CHROMATIN REMODELER. THEY HAVE WELL ESTABLISHED INTERFACE FUNCTION BUT AFFECT MITOTIC SPINDLES IN VARIOUS WAYS. SOME MEMBRANE PROTEINS ARE LISTED HERE, CDC 48, AND FAN, UXP 9X. PROTEINS HAVE INTERFACE FUNCTION, THEY HAVE MITOTIC FUNCTION. THEN THE QUESTION IS WHY YOU HAVE SO MANY MOONLIGHTING PROTEINS, IN THE NUCLEUS AND THE LIGHT IS SHINING ON MY EYES, IN SO MANY NUCLEAR PROTEINS AS WELL AS MEMBRANE PROTEINS REGULATING MITOSIS. I THINK IT REALLY COMES BACK IN A WAY ADDRESSING THE INITIAL QUESTION I POSE, WHEN YOU THINK ABOUT CELL DIVISION IF YOU THINK BEYOND EQUAL CHROMOSOME SEGREGATING A -- SEGREGATION, YOU VASCULARIZE, DISSOLVE AND HAVE EQUALLY DISTRICTED OR BEYOND MICROTUBULES YOU HAVE ANOTHER NETWORK THAT NEEDS TO BE ORGANIZED AROUND THE SPINDLE AND PERMEATING THROUGHOUT THE SPINDLE, THAT'S HOW YOU CAN PROPERLY DIVIDE THEM TO PREPARE THE CELL FOR THE NEXT CELL CYCLE AND VARIOUS THINGS IN THE NEXT CELL CYCLE, THE CELLS MIGHT WANT TO DO. IN FACT, IN MITOSIS FIELD FOR YEARS, FOR DECADES, STARTING IN THE '60s, BOB GOLDMAN AND COLLEAGUES WERE ONE OF THE FIRST TO PROPOSE MAYBE MITOTIC SPINDLE IS NOT JUST MADE OF MICROTUBULES IN TERMS OF STRUCTURE, AND HERE IS IS A SEE URCHIN SPINDLE UNDER EM, SO THESE ARE FIBERS OF MICROTUBULES, BUT BEYOND THIS MICROTUBULE-LIKE FIBER THERE ARE GRANULAR STRUCTURES SURROUNDING THE SPINDLE, IN SOME OTHER EMs, YOU CAN SEE ELECTRON DENSE MATERIALS PERMEATING THROUGH THE SPINDLE. THIS IS WHERE THE SPINDLE MATRIX IDEA CAME FROM BUT IT HAS NEVER BEEN POPULAR. PEOPLE COMING IN AND OUT, NOT REALLY EVEN A FIELD, BUT IT SEEMS REALLY TO MAKE SENSE THAT IF YOU WANT TO BUILD SUCH A COMPLICATED STRUCTURE TO DIVIDE THE CELL YOU PROBABLY NEED THINGS BEYOND THE MICROTUBULE SO PROBABLY NEED TO ORGANIZE CELLS WITH ANOTHER WAY TO KIND OF MAKE THE CELL DIVISION TO HAPPEN EFFECTIVELY. THE KIND OF INITIAL IDEA HAS BEEN, YOU KNOW, MAYBE MITOTIC SPINDLES, THIS MITOTIC SPINDLE MATRIX COULD REGULATE SPINDLE MORPHOLOGY, COULD INTERACT WITH MICROTUBULES TO MAKE THE SPINDLE MUCH MORE EFFECTIVE. SO NOW AFTER WE REALIZED ACTUALLY THE CELLULAR -- OTHER CELLULAR COMPONENTS SUCH AS NUCLEAR REMNANTS AND MEMBRANES COULD BE PLAYING A ROLE IN MITOSIS, PROGRAMS THE SPINDLE MATRIX ORGANIZEs TO CROSS-TALK WITH TUBULES TO ENSURE PROPER CHROMOSOME SEGREGATION AND PERHAPS PROPER CELL FATE DETERMINATION. INDEED WE HAVE BEEN ABLE TO LOOK AT THIS, THAT'S SOMETHING I'M GOING TO TALK -- HIGHLIGHT MORE. OKAY, TO REALLY GET A HANDLE ON WHAT ARE THE SPINDLE MATRIX MATERIALS, WHAT PROTEINS ARE IN THERE, A FORMER POSTDOCTORAL FELLOW DEVISE IT'S A NICE ASSAY WHICH IS THIS MITOTIC KINASE ON THE MAGNETIC BEADS, AND IF HE PUTS THIS MAGNETIC BEADS, ADDING RanGTPase, ABLE TO INDUCE ASTER FORMATION IN THE FIRST THREE MINUTES, AND THEN BY 15 MINUTES YOU GET VERY NICE BIPOLAR SPINDLE ASSEMBLY. YOU CAN SEE AFTER THIS IS JUST THE LOW RESOLUTION VIEW, YOU CAN SEE THE SPINDLE ASSEMBLY, A WIDER VIEW. THIS IS THE CLOSE-UP. THE ADVANTAGE OF THIS IS THEN YOU CAN USE THIS TO ISOLATE THE SPINDLE BY PUTTING THE REACTION ON THE MAGNET, AND THEN IT WOULD COLLECT THE MAGNETIC IMPEDES AND YOU WASH THE SPINDLE AND WHAT YOU HAVE LEFT IS THE SPINDLE MATRIX. WE DID PROTEOME ANALYSIS IN COLLABORATION TO LED US TO FIND 600 PROTEINS, AND THE MAJOR CRITICISM OF THIS WORK REALLY HAS BEEN -- WELL, WE FOUND A LOT OF THINGS, WHILE WE ACTUALLY -- PEOPLE -- BIOLOGISTS LIKE TO SEE THINGS THAT'S PURE, AND IF WE SEE A LOT OF THINGS, VERY OBVIOUS SUSPECT, WELL, YOU JUST ISOLATED THE CELL AND YOU HAVE NOT DONE MUCH, MAYBE YOU ISOLATED SOMETHING THAT'S AGGREGATED. SO, BUT WE JUST DECIDED TO FORGE FORWARD AND SEE WHAT WE GET. ONE THING WE SPEND TIME ON IS LAMIN-B BECAUSE WE THOUGHT THIS IS A NETWORK, AND PROBABLY HAS SOME STRUCTURES AND LAMIN-B IS INTERMEDIATE FILAMENT PROTEIN SO PERHAPS IT COULD PROVIDE SOME STRUCTURE TO IT. THIS IS HOW WE STUDIED EPSIN. TODAY I WILL TALK ABOUT LAMIN-B AND ANOTHER PROTEIN, A ZINC FINGER PROTEIN AND WILL GIVE YOU HIGHLIGHTS ON THE TWO PROTEIN ROLE IN SPINDLE ASSEMBLY. LAMIN-B, OVER THE YEARS THESE ARE THE PUBLISHED WORKS, SUMMARIZING OVER THE YEARS WE'VE SEEN THAT THE SPINDLE MATRIX PROTEIN LAMIN-B, IT SEEMS TO BE INTERACTING TO HELP A NUMBER OF PROTEINS, SEPARATION IN THE BEGINNING OF SPINDLE ASSEMBLY IN PRO PHASE, IF YOU HAVE CELLS THE CENTERS DON'T SEPARATE QUICKLY. INVOLVED IN SPINDLE ORIENTATION, VENTRICULAR ZONE IN THE NEURONAL PROGENIOR CELLS IF YOU HAVE LAMIN MUTATIONS, LAMIN-B MUTATIONS THE SPINDLES END UP FINDING A LOT OF OBLIQUE ORIENTATIONS WHEN ACTUALLY SHOULD FIND EITHER PARALLEL OR PERPENDICULAR. WE HAVE SPINDLE MORPHOLOGY, THAT'S THE BEST EXPLANATION FOR THESE PHENOTYPES. SO THE PROBLEM WE HAVE OVER THE YEARS HAS ALWAYS BEEN THAT, YOU KNOW, THIS LAMIN-B OR THE OTHER SPINDLE MATRIX PROTEIN THAT I WILL TALK ABOUT SOON, WE LOOK AT IT, IT'S REALLY NOT LIKE MICROTUBULES. LAMIN-B IS HAZY, IN THE CYTOSOL AND SPINDLE, THE OTHER SPINDLE MATRIX PROTEIN IS ENRICHED ON THE SPINDLE AND HAZY. REALLY NOT SUCCEEDED TO IDENTIFY ANY DISCERNIBLE FILAMENTOUS STRUCTURE UNDER EM, SO THEN IT'S REALLY LARD TO MOVE FORWARD TO REALLY UNDERSTAND IF THE STRUCTURAL FEATURE OF THIS, IS THERE A STRUCTURE OR JUST RANDOM AGGREGATES. THAT'S BEEN THE BIGGEST PROBLEM WE HAD FACED. SO BUT WE WERE PRETTY LUCKY IN THE SENSE OF HAVING IDENTIFIED THE PROTEIN CALLED BUGZ, A SPINDLE MATE RICK PROTEIN WE IDENTIFIED AT BUGZ. BACKGROUND PUBLISHED IN THIS PAPER, WE FOUND THIS PROTEIN -- WELL, HERE IS BASICALLY A PLOT OF THIS PROTEIN, THE N-TERMINUS, THIS LITTLE BIT, THIS IS DISORDER DISPOSITION, SO ANYTHING BELOW .5 IS CONSIDERED ORDERED, ANYTHING ABOVE THAT IS DISORDERED. AS YOU CAN SEE, THIS REGION IS HIGHLY ORDERED. IN FACT, THIS CORRESPONDS TO PREDICTED TWO ZINC FINGERS. THIS REGION BINDS TO TUBULIN, MICROTUBULES, THE C-TERMINUS IS ESSENTIALLY LARGELY DISORDERED, IT HAS AN INTERESTING MOTIF WHICH IS THE MOTIF THAT BINDS TO PROTEIN SUCH AS BUG 3, GLEBS. WE'VE DEFINED BUGZ IN MY TOTE IS HAS A ROLE IN REGULATING CHROMOSOME ALIGNMENT, SO THIS IS BASICALLY ONE FUNCTION OF MY TOTE IS. THE REAL INTERESTING THING IS THAT THERE IS THIS REALLY LARGE DISORDERED REGION, AND THEN WE ARE ABLE TO MAKE LARGE QUANTITY OF SOLUBLE PROTEINS THAT OFFERS MEANS TO DO BIOCHEMISTRY, AND WHY THAT COULD BE SO INTERESTING. THAT'S BECAUSE IN ADDITION TO THIS SOLID POLYMER SUCH AS ACTIN YOU'VE GOT OTHER POLYMERS, MANY OF YOU HAVE HEARD HYDRA GEL AND LIQUID DROPLETS, THESE POLYMERS HAVE WATER IN IT, HYDRA GEL HAS WATER IN IT, THIS LIQUID DROPLETS HAVE EVEN MORE WATER AND EVEN MORE DYNAMIC STRUCTURES. AND THESE FEATURES, NOW VIBRANT FIELD NOW, THESE ARE CALLED MEMBRANEOUS ORGANELLES THAT COULD HELP ORGANIZED CELLS. IT MAKES SENSE TO US IF YOU THINK ABOUT SPINDLE MATRIX, WHEN YOU GO INTO MITOSIS MANY MEMBRANE BOUND SYSTEMS BECOME VENTRICULARRIZED, PERHAPS THERE ARE FEATURES OF MEMBRANEOUS ORGANELLES AND THEN MOST OF THESE SYSTEMS ACTUALLY ARE FORMED, MOST OF THESE LIKE HYDRA GEL, LIQUID DROPLETS ARE FORMED BY INTERACTION OF PROTEINS THAT EITHER FORMS HAVE THIS REPEATS THAT CAN INTERACT, OR THEY HAVE LOW COMPLEXITY. AND THEY ARE DISORDERED PROTEINS. AND THE WAY THEY ACTUALLY INTERACT, IT'S STILL NOT VERY WELL UNDERSTOOD IN THIS POLYMERIZATION, BUT IT'S KNOWN THAT AROMATIC RESIDUE SUCH AS TYROSINE OR HYDROPHOBIC RESIDUE OF POLAR OR CHARGED RESIDUES DRIVE THE FORMATION OF THIS SEPARATED STATE SO THIS IS CALLED PHASE TRANSITION OR PHASE SEPARATION. NOW I WANT TO SHOW YOU WHAT BUGZ LOOKS LIKE. BUGZ, HERE IS THE TWO ZINC FINGERS, IF YOU LOOK AT BUGZ IT HAS TYROSINE, ABOUT 13 OF THEM, XENOPUS, IN ADDITION IT HAS A LOT OF PROLEINS. EVERY PROTEINS SEEM TO BE HAPPENING NEXT TO HIGHLY HYDROPHOBIC RESIDUE THROUGHOUT THE MOLECULE SO THE MOLECULE IS FAIRLY HYDROPHOBIC YET SOLUBLE AND WE THINK THE PROLEINS PREVENT THIS. THIS GIVES A SENSE MAYBE THIS PROTEIN WOULD FORM A STRUCTURE, NOT LIKE A SOLID POLYMER THAT WE WERE TRYING TO LOOK FOR, MAYBE IT'S ACTUALLY FORMING SOME KIND OF PHASE SEPARATED STATE THAT COULD FUNCTION IN THE SPINDLE MATRIX, INDEED WE TESTED MANY CONDITIONS BECAUSE WE HAVE CHARGED MOLECULES, ONE THING I DIDN'T SHOW YOU, GO BACK QUICKLY TO SHOW YOU, IS THAT IN ADDITION, YOU CAN SEE THIS HIGHLIGHT, THE PROLEIN RICH REGION IS HIGHLIGHTED IN GREEN. OTHER REGIONS ARE POLAR, CONCENTRATED WITH POLAR MOLECULES AND THIS IS PRETTY CHARGED REGION SO THAT'S WHAT THIS PROTEIN LOOKED LIKE, TWO REGIONS OF PROLEIN AND HYDROPHOBIC, CHARGED OR CHARGED OR HYDROPHILIC, WE REALIZED IT UNDERGOES PHASE SEPARATION IN CONCENTRATION AND TEMPERATURE DEPENDENT MANNER KIND OF LIKE ELASTIN PROTEIN. SO BASICALLY IF YOU INCREASE THE TEMPERATURE, INCREASE PROTEIN CONCENTRATION IT WILL FORM BEAUTIFUL DROPLETS, BETWEEN YSP AND BUGZ, HIGHLY REVERSIBLE, NOT COMPLETELY POLYMERIZING BUT REPEATABLE AND REVERSIBLE, A VERY NICE PHASE SEPARATION PROTEIN. WHEN THIS WAS FOUND ACTUALLY THIS WAS THE FIRST PROTEIN THAT YOU HAVE, A PROTEIN, NATIVE PROTEIN, FULL LENGTH THAT UNDERGOES PHASE SEPARATION BESIDES ELASTIN, ALTHOUGH A LOT OF STUDIES ON ELASTIN IS USING FRAGMENTS, AND THIS PROTEIN BY ITSELF UNDERGOES PHASE SEPARATION. WE DID QUITE A BIT OF MUTAGENESIS AND TRIED TO MEASURE MUTAGENESIS PHASE SEPARATION BECAUSE PEOPLE FOUND IN SOME OTHER PHASE SEPARATED PROTEINS YOU CAN MUTATE THE PHENOALANENE AND TYROSINE, SO WE ALSO GOT RID OF THE N-TERMINUS TO LOOK AT THE IMPACT ON PHASE SEPARATION AND THE TAKEHOME MESSAGE REALLY IS THAT BUGZ PHASE SEPARATION MUTANTS ARE DEFECTIVE IN PROMOTING SPINDLE AND SPINDLE MATRIX ASSEMBLY. SO THAT'S KINDS OF NICE BECAUSE THAT AT LEAST SAYS, WELL, THERE IS A STRUCTURE. THE SPINDLE MATRIX COULD BE IN PART THE STRUCTURE INTEGRITY COULD BE IN PART MEDIATED BY PHASE SEPARATION. OKAY. SO, I'M JUST GOING TO TRY TO END THIS BY KIND OF PROVIDING A FEW -- ONE WAY THAT WE BELIEVE BUGZ COULD PROMOTE SPINDLE ASSEMBLY IS THAT AS I SAID, THE N-TERMINUS BINDS TO TUBULIN. WHEN YOU FORM PHASE SEPARATED SPACE YOU CAN SEE A NICE CONCENTRATION OF TUBULIN. AND WHEN YOU GET RID OF THE N-TERMINUS, SO YOU DON'T BIND TUBULIN, IT DOESN'T CONCENTRATE TUBULIN. AND IF YOU USE THIS TWO MUTANT TO TRY TO DO MICROTUBULE ENUCLEATION, TUBULIN BINDING IS -- THE MICRO TUBULIN BINDING SITE IS MICROTUBULE BINDING SITE, IMPORTANT FOR BUNDLINGS, MAKING MICTROTUBULE BUNDLES. YOU HAVE MICROTUBULE POLYMER AND BUGZ LIKE LIQUID ALONG THE MICROTUBULES, PROBABLY FLEXIBLE AND PROBABLY IMPORTANT TO GLIDE MICROTUBULES APART, IT'S NOT A SOLID TO MAKE MICROTUBULE BUNDLE IN THE VERY BIG SOLID SPACE. OKAY. THIS IS BASICALLY THE TAKEHOME MESSAGE, I'M JUMPING A LOT BECAUSE I WANT TO GO TO OTHER THINGS. SO BASICALLY THE TAKEHOME MESSAGE IS THAT BUGZ UNDERGOES PHASE SEPARATION AND THIS BLUE REGION IS MICROTUBULE BINDING SITE AND RED REGION IS STRUCTURED. RED REGION, THE HYDROPHOBIC RESIDUES, ALSO AROMATIC RESIDUES ARE IMPORTANT FOR PHASE SEPARATION, THE REASON WE THINK HYDROPHOBIC RESIDUE IS IMPORTANT WHEN YOU INCREASE THE TEMPERATURE, THAT KIND OF STRETCHES THE MOLECULE. BASICALLY PREVENTS INTRAMOLECULAR INTERACTION, IN FACT WE'VE MAPPED THE REGIONS OF THE MOLECULE, IT PROMOTES INTERMOLECULAR BY INTERACTION, THAT'S WHAT'S DRIVING PHASE SEPARATION. WHEN YOU HAVE MICROTUBULES, HELPS PROMOTING PHASE SEPARATION, YOU CAN SEE MICROTUBULE DROPLETS ALONG -- BUGZ DROPLETS ALONG THE MICROTUBULES. BECAUSE BUGZ COULD CONCENTRATE TUBULIN AND BUNDLE MICROTUBULES, IT CAN PROMOTE SPINDLE ASSEMBLY, ALSO CAN CONCENTRATE KINASE AND HELP ACTIVATE IN MITOSIS, THAT CAN ALSO CONTRIBUTE TO SPINDLE ASSEMBLY. THAT'S THE HIGHLIGHT OF MITOSIS STUDY THAT WE'RE DOING IN THE LAB. NOW I'M GOING TO SWITCH GEARS TO TALK ABOUT NUCLEAR LAMINS AND I'M GOING TO FIRST HIGHLIGHT THE ROLE OF NUCLEAR LAMB INSURE IN TISSUE BUILDING. SO AS I SAID, REALLY, IT'S OUR STUDY OF MITOSIS, LAMIN& INTERACTS WITH MICROTUBULES, PAYING ATTENTION TO INTERFACE, I WAS NOT IN INTERESTED IN INTERFACE IN THE BEGINNING OF MY CAREER. WHAT CAUGHT MY ATTENTION, OH, THIS SYSTEM IS QUITE AMAZING IN INTERFACE, LAMINS FORM THIS POLYMER RIGHT UNDERNEATH NUCLEAR MEMBRANE AND INTERACTS WITH CHROMOSOMES ON ONE HAND AND THROUGH THE NUCLEAR ENVELOPE PROTEINS CONNECTING CHROMATIN WITH CYTOSKELETON, SIMILAR IN CONCEPT HOW THE CELLS ARE -- YOU USE SIMILAR CONCEPT TO SOFT INTERFACE AND MITOTIC PROBLEMS, IN THIS CASE IT'S MAKING SURE YOU CAN ORGANIZE SPINDLE TO SEPARATE THE CHROMATIN, SO I SEE THAT COULD BE VERY INTERESTING, EVEN WE STARTED TO LOOK AT LAMIN BECAUSE THIS CONNECTION IS QUITE INTERESTING. SO HERE I ALREADY HAVE IT WRITTEN DOWN AS AS MAJOR COMPONENTS, MULTIPLE TWO B TYPE, THE PROBLEM WHEN MY LAB FIRST STARTED TO PAY ATTENTION TO LAMIN, MANY PEOPLE REALLY BELIEVE LAMIN IS ABSOLUTELY ESSENTIAL FOR ANIMAL CELLS, IF YOU DON'T HAVE LAMB YOU WON'T BE ABLE TO HAVE A CELL OR NUCLEUS. THAT'S OUR CONTRIBUTION, LAMIN PLAYS A ROLE IN MITOTIC SPACE, STRENGTHENING IS IMPORTANT FOR CELL PROLIFERATION, FOR BASIC CELL STIFLED. SURVIVAL. THE LITERATURE IS NOT CLEAR LAMIN PLAYS SUCH A ROLE, PEOPLE DID RNAi OR GENETIC WORK. WHEN YOU LOOK AT C. ELEGANS THERE'S ONLY SINGLE LAMIN GENE, YOU STILL HAVE MATERNAL SUPPLY. SO BASICALLY THE LITERATURE TELLS US IT'S NOT REALLY CERTAIN LAMIN IS ABSOLUTELY ESSENTIAL FOR CELL FUNCTION. AND THE WAY, WHEN WE APPRECIATED THAT LAMIN'S ROLE HERE REALLY IS CONNECTING CHROMATIN TO CYTOSKELETON MAYBE LAMINA IS INVOLVED IN MORPHOGENESIS BECAUSE CYTOSKELETONS ARE INVOLVED IN MORPHOGENESIS. A TALENTED AND BRAVE POSTDOCTORAL FELLOW CAME TO MY LAB AND DECIDED, OKAY, I'M JUST GOING TO SEE IF I CAN CREATE ANY CELL THAT CAN BE LAMINAL. HE WAS BRAVE BECAUSE MANY SEMINAR SPEAKERS CAME THROUGH MY DEPARTMENT WHEN I TOLD THEM THAT'S WHAT WE WERE DOING, SAYING, WELL, WHAT ARE YOU GOING TO LEARN IF YOU DON'T GET ANY CELLS? YOU WOULD JUST LEARN, OKAY, LAMINS ARE ESSENTIAL. BUT, YOU KNOW, WE JUST FORGED FORWARD. IN HIS TIME HE JUST DID STANDARD MOUSE KNOCKOUTS CREATED DOUBLE KNOCKOUTS, USED TWO ROUND OF DELETION TO GET RID OF LAMIN-A, WAS ABLE TO GENERATE LAMIN MKS CELLS, A DEFINITIVE ANSWER LAMIN IS NOT ESSENTIAL FOR BASIC CELL FUNCTION, ONCE YOU HAVE THE MOUSE MODEL YOU CAN LOOK AT LAMIN IN DIFFERENT ORGANS AND THE MAJOR TAKEHOME, EVERY ORGAN THERE ARE DEFECTS, THE BRAIN IS MOST DRAMATICALLY AFFECTED. BUT IT'S VERY HARD TO TRY TO GET AT THE MECHANISMS USING MOUSE. ANOTHER POSTDOCTORAL FELLOW CAME TO MY LAB FIVE OR SIX YEARS AGO, I'M GOING TO USE DROSOPHILA BECAUSE IT'S MUCH FASTER, THIS IS THE PHENOTYPE, THERE'S ONE LAMIN, B-TYPE LAMIN IN DROSOPHILA, WHEN YOU DON'T HAVE THAT THE TESTES IS VERY SMALL COMPARED TO WILD TYPE. I WOULD JUST QUICKLY HIGHLIGHT WHAT HE FOUND. SO BASICALLY THIS IS THE TIP OF THE TESTES, AT THE TIP YOU HAVE THREE TYPES OF CELLS, HUB CELLS THAT SUPPORTS THE GERMLINE, STEM CELLS AND ALSO CYST CELLS THAT SUPPORT GERMLINE STEM CELLS. THEY DIVIDE TO FORM GERM CELLS AND FURTHER FORMING THE SPERM, THE CYST STEM CELLS STOP DIVIDING AND THEY START TO EXPAND TREMENDOUSLY TO ENCLOSE EXPANDING GERMLINE CELLS. SO THIS EXPANSION BASICALLY IS VERY IMPORTANT MORPHOGENESIS, IF YOU DON'T HAVE THAT TESTES WORK WORK. HE FOUND LAMINS PLAY A SPECIFIC ROLE IN THIS STEM CELL AND CYST CELLS. IF YOU GET RID OF IT, SO THIS IS ACTUALLY AN ILLUSTRATION OF USING GFP KNOCK-IN, WILD TYPE AND MUTANT CELL, AS YOU CAN SEE WILDTYPE CELL EXPANDS TO INCLUDE GERMLINE CELLS, WHEREAS THE MUTANT BASICALLY COULD NOT EXPAND THE MORPHOGENESIS FAILS AND CYST CELLS DON'T ENCLOSE THE GERMLINE CELLS AND THEN SPERMATOGENESIS FAILS. THAT'S REALLY A VERY DRAMATIC DEMONSTRATION HOW LAMINS ARE INVOLVED IN MORPHOGENESIS. ACTUALLY, THE MECHANISM ILLUSTRATED, IF YOU DON'T HAVE B TYPE LAMINS, THE NUCLEAR PROTEIN 153 GETS AGGREGATED AGAINST THE NUCLEAR PERIPHERY AND WHEN THAT PERHAPS THE NUCLEUS ARE NOT ABLE TO RETAIN PHOSPHORYLATED ERK, YOU NEED PROPERLY LOCALIZATION OF 153 TO RETAIN PHOSPHO-ERK IN NUCLEUS, VERY IMPORTANT FOR CYST CELLS TO UNDERGO MORPHOGENESIS. THAT'S REALLY THE MECHANISM. THAT'S BASICALLY SHOWING LAMIN COULD USE ONE MECHANISM SUCH AS ENSURING NUCLEAR CORE COMPONENT PROPER LOCALIZATION, THEREFORE PROPER PHOSPHORYLATION TO HELP ORGANOGENESIS. I'M GOING TO GO QUICKLY THROUGH AGING, SO BASICALLY THE CONCEPT REALLY IS IF LAMIN IS IMPORTANT FOR MAKING BASICALLY MORPHOGENESIS, ORGANOGENESIS, THE MAJOR PROBLEM IS ON AGING IS ORGANS AND TISSUES CANNOT MAINTAIN THE SHAPE SO PERHAPS LAMIN COULD BE ONE OF THE CULPRITS IN AGING. AND INDEED THERE IS LITERATURE ON STUDIES, FINDINGS, THERE'S SENESCENCE ASSOCIATED PHENOTYPE, MEANING WHEN CELLS GETTING TO SENESCENCE STATE, SECRETE INFLAMMATORY CYTOKINES AND CHEMOKINES. IT'S UNCLEAR IF THIS IS HAPPENING IN VIVO, THIS IS HOW THE GENETICIST DECIDED I'M GOING TO USE FLY TO TACKLE THIS BECAUSE IT'S TOO HARD TO STUDY THIS IN HUMANS OR MOUSE BECAUSE IT HAS TOO MANY -- TWO IMMUNE SYSTEMS, ADAPTIVE AND INNATE. IN DROSOPHILA YOU ONLY HAVE INNATE, FAIRLY SIMPLE IMMUNE SYSTEM, THE MAJOR ONE, HUMORAL OR FAT BODIES, HUMORAL REGULATES HUMORAL RESPONSE OF IMMUNE SYSTEM AND JUST RIGHT UNDERNEATH THE CUTICLE SHOWN IN BLUE. ANOTHER LOCAL IMMUNE SYSTEM IS THE DROSOPHILA GUT, SO HE LOOKED THROUGH THE TISSUES OF YOUNG AND OLD FLIES, ONE VERY STRIKING CHANGE RIGHT ALMOST RIGHT OFF THE BAT, THE FAT BODY CELLS SHOWN HERE EXHIBITED VERY CLEAR LAMIN-B 1 REDUCTION UPON AGING BUT REALLY ONLY LAMIN-B. LAMIN-A DIDN'T SEEM TO CHANGE. IF HE LOOKED AT THE GUT, GUT DOESN'T SEEM TO CHANGE LAMIN TO JUST SUMMARIZE HIS WORK, THE FINDING REALLY MADE, IT TURNS OUT UPON AGING THE FAT BODY CELLS REALLY HAVE A VERY SIGNIFICANT LOSS OF LAMIN-B, YEAH, I THINK IT'S ABOUT AT LEAST 50%. AND THIS REDUCTION LEADS TO SYSTEMIC UPREGULATION OF SYSTEMIC INFLAMMATORY FACTORS THAT GETS SECRETED, THAT'S REALLY LED TO THE LOCAL REPRESSION OF THE GUT IMMUNE RESPONSE. WE ALL KNOW THAT IN OUR GUT WE HAVE TO MODULATE OUR MICROBIOME. SO YOU HAVE TO HAVE AN IMMUNE SYSTEM THERE TO MAKE SURE YOUR MICROBIOME WON'T GO OUT OF CONTROL. NOW, IF YOU HAVE THIS SYSTEM, IF YOU HAVE TOO MUCH INFLAMMATORY FACTORS HERE, THAT REPRESSES LOCAL IMMUNE SYSTEM, THAT WOULD CAUSE THE OVERGROWTH OF THE MICROBIOME, THAT COULD CAUSE DAMAGE, AND THEN THE GUT BASICALLY THE GUT STEM CELLS TRY TO REPAIR THAT DAMAGE, SO THE REPAIR IS JUST DEFECTIVE BECAUSE YOU ARE DEALING WITH OLD GUT. YOU END UP WITH BASICALLY MISDIFFERENTIATION, INCREASED PROLIFERATION OF MISDIFFERENTIATION OF THE GUT STEM CELLS, THAT LED TO THIS GUT HYPERPLASIA. THIS IS ONE WAY THAT LAMIN-B REDUCTION IN THIS CASE POST-MITOTIC CELL, IN THIS CASE CONTINUOUSLY BEING REPLACED SO THEY DON'T SEE LAMIN-B 1 LOSS BUT THIS LOSS IN THE FAT BODY COULD INFLUENCE OTHER SYSTEMS. SO NOW MY LAB -- HY YUNG TOOK THIS PROJECT TO HIS LAB IN CHINA, I HAVE ONE LITTLE PROJECT LEFT IN MAMMALS TO HAVE AN EXAMPLE. WE HAVE AN EXAMPLE, ALSO IN PART DRIVEN BY LAMIN-B 1 REDUCTION IN EPITHELIAL CELLS, I'M NOT GOING TO TALK ABOUT THAT BECAUSE I WANT TO REALLY TALK ABOUT GENOME ORGANIZATION AND TRANSCRIPTION, HOW LAMIN COULD PLAY A ROLE IN THAT. SO, NOW, WHY DO WE REALLY WANT TO LOOK AT GENOME ORGANIZATION, AND LAMIN'S ROLE? THE MAIN REASON REALLY IS KIND OF WILD, IT'S KIND OF A WILD IDEA, BUT IT STILL REMAINS THE IDEA I'M MOST EXCITED, THAT IS IF YOU THINK ABOUT DO WE REALLY UNDERSTAND MORPHOGENESIS, HOW DOES MORPHOGENESIS OCCUR? WE HAVE TRANSCRIPTIONAL AND SIGNALING CASCADE AND MECHANICAL TRANSDUCTION, ALL CASCADE IDEA. I'M A CELL BIOLOGIST, THAT DOESN'T MAKE SENSE. I WANT TO UNDERSTAND MORPHOLOGICAL CODE. I FELT THIS CONNECTION BETWEEN LAMIN, CYTOSKELETON AND LAMIN, CHROMATIN, WHERE YOU COULD SEE IT. IF YOU EXERT FORCE TO CHANGE NUCLEAR LAMINA YOU MIGHT CHANGE CHROMATIN ORGANIZATION, THAT MIGHT HELP YOU TO REALLY UNDERSTAND IF YOU WANT TO BECOME ELONGATED EPITHELIAL CELL, WANT TO HAVE A DIFFERENT KIND OF 3D CHROMATIN ORGANIZATION, BY INFLUENCING LAMIN, THEN YOU ESTABLISH THAT TRANSCRIPTOME, RIGHT? SO THIS IS BASICALLY A DRAIN, THAT'S WHAT I'M -- A DREAM. THIS DREAM IS NOT UNFUNDED. LAMIN INTERACTS WITH LATS, PEOPLE HAVE FOUND THAT THIS LAT REGIONS DISASSOCIATE, MOVE AWAY FROM NUCLEAR LAMINA REGION TO BECOME INTERNALIZED, AND THAT INTERNALIZATION SHOWS SPECIFICITY, HOPEFUL AND ENCOURAGING. THE QUESTION THEN IS COULD THE DISASSOCIATION OF LADS TRANSCRIPTOME AND OTHER THING I DIDN'T ADD HERE BECAUSE I THINK IT'S PROBABLY, YOU KNOW, EVEN CRAZIER, COULD THE INTERACTION BETWEEN THE CYTOSKELETON AND NUCLEAR ENVELOPE CHANGE THE LAMINA MeSH WORK WHICH LEADS TO DISASSOCIATION OF LADS, THAT COULD HOPEFULLY HELP YOU DECIDE WHICH LADS GOES INSIDE AND DETERMINE THE TISSUE-BUILDING, WE ALREADY SHOWED LAMIN IS IMPORTANT FOR TISSUE BUILDING. IT'S VERY COMPLICATED. YOU HAVE TO FIRST UNDERSTAND IF YOU DON'T HAVE LAMIN, WHAT IS THE CHANGE ON THE GENOME ORGANIZATION IN 3D? SO ONE THING I WANT TO SAY, IN THE FIELD IN LAMIN FIELD BECAUSE NUCLEAR PERIPHERY IS CONSIDERED REPRESSIVE ENVIRONMENT, SO FOR YEARS PEOPLE SAY, NUCLEAR PERIPHERY IS JUST -- LAMIN PLAYS REPRESSIVE ROLE, WHEN WE REMOVE LAMIN FROM ES CELLS AND OTHER CELLS WE DON'T SEE THAT. SO YOU CAN LOOK AT GENE EXPRESSION CHANGE, THIS IS WILD TYPE VERSUS LAMIN TRIPLE KNOCKOUT ES CELLS, AND YOU CAN SEE THIS IS FULL CHANGE OF GENE EXPRESSION, YOU CAN SEE ASSOCIATION WITH LAMIN-B 1, USING THE TECHNIQUE, AND YOU SEE NO CORRELATION. I CAN HAVE UPREGULATED OR DOWNREGULATED GENES, WITH GENES IN THE LAD OR NOT IN THE LAD. IF YOU DON'T HAVE LAMIN IN THE CELL, YOU DON'T REALLY AFFECT GENES IN THE LADS, YOU AFFECT GLOBAL GENE EXPRESSION. THAT'S THE FIRST THING WE LEARNED BY DOING THIS CLEAN KNOCKOUT. IS THAT BASICALLY SOMETHING YOU DON'T UNDERSTAND, YOU WILL NEVER UNDERSTAND, JUST RANDOM. WE FELT MAYBE IT'S NOT TOTALLY RANDOM, IMPORTANT TO LOOK AT 3D GENOME WORK WHEN YOU DON'T HAVE LAMIN. IN ES CELLS THE CHANGE IS NOT DRAMATIC BUT CLEAR. THIS IS A CHROMATIN, REGION OF CHROMOSOME 10, WILDTYPE AND LAMINAL CELLS, PLOTTING TO SHOW THE TOPOLOGICALLY ASSOCIATED CHROMATIN DOMAIN TADS DO NOT CHANGE IF YOU DON'T HAVE LAMIN, YOU DON'T AFACT TADS, WHICH MAKES SENSE, LAMIN IS AT THE NUCLEAR PERIPHERY, SHOULDN'T AFACT TADS. IT AFFECTS TAD-TAD INTERACTION, EACH OF THE BLACK LINES OUTLINE ONE, THIS IS ONE TAD, RIGHT? SO WHEN YOU START TO COMPARE INTERTAD INTERACTION NOW YOU CAN SEE THE CHANGE. BASICALLY THERE'S ONE TAD, THERE'S AN INCREASED INTERACTION IN TKL CELLS, BETWEEN THIS TAD AND THIS TAD. ON CHROMOSOME 10. IF YOU DON'T HAVE LAMIN YOU'RE CHANGING THE MUCH LARGER SCALE CHROMATIN STRUCTURE. SO NOW TO REALLY UNDERSTAND THIS BETTER, JUST LOOKING AT TADS IS NOT ENOUGH. YOU HAVE TO REALLY DIAL IT MUCH LOWER AT HIGHER RESOLUTION. TO DO THAT A TALENTED PERSON IN MY LAB REALLY SHE DEVELOPED A HIDDEN KNOCKOFF MODEL TO TRY TO SEPARATE THE CHROMATIN DOMAINS INTO DIFFERENT FEATURES, BY USING LAMIN ASSOCIATION LADs, HISTONES, H1, H3, ALSO HISTONE MODIFICATIONS, VARIOUS HISTONE MODIFICATIONS, ABLE TO SEPARATE THE CHROMATIN DOMAIN INTO SIX DIFFERENT FEATURES, SO WE KIND OF HISTONE LAMINA ASSOCIATED DOMAINS SO HE USED A COLOR FROM RED TO PURPLE TO KIND OF JUST NAME THEM, AND THEN THE RED REGION BASED ON HISTONE MODIFICATION IS MOST ACTIVE, NEXT IS YELLOW REGION, ORIGINAL, GREEN REGION IS LESS ACTIVE, WITH THIS MODEL HE WAS ABLE TO SEPARATE LADS, ONE IS B, THE OTHER IS P. I NEED TO NEED YOU TO REMEMBER THIS BLUE AND PURPLE ARE LADS REGIONS AND THEY ARE SEPARATED BY THEIR FEATURE OF THIS BLUE REGION, ACTUALLY IS HAVING HIGHER ENRICHMENT OF H3 K 27 TRIMETHYLATION, THE PURPLE REGION HAS A LOT MORE LAMIN-B ASSOCIATION. SO THIS IS VERY IMPORTANT FOR US TO BE ABLE TO FURTHER STUDY THE RELATIONSHIP AND HOW THE 3D IS CHANGING BECAUSE NOW WE CAN MAP OUR HIGHLAND REGION TO THE TADS, AS YOU CAN SEE THE PURPLE LADS, THE LADS, PURPLE REGION OF THE LADS, OFTENTIMES IS REALLY LONG, WHICH IS REPRESENTING MOSTLY HETEROCHROMATIN REGION OF CONSTITUATIVELY HETEROCHROMATIN REGION, OFTENTIMES COULD COVER MORE THAN ONE TAD, WHEREAS THE BLUE REGION, BLUE LADS REGION, ARE MUCH SHORTER, SOMETIMES COULD BE ONE TAD LONG, BUT SOMETIMES IT'S LESS THAN ONE TAD LONG, ALL OF THE REST OF THE INTERIOR CHROMATIN, THERE ARE MUCH SMALLER AND OFTEN HAVE MULTIPLE -- ONE TAD COULD HAVE MULTIPLE FEATURES OF CHROMATIN. SO THROUGH THIS ANALYSIS AND LOOKING AT IN DEPTH ANALYSIS OF THE DATA, WE PREDICTED THAT WHEN YOU LOSE LAMIN, IT LOOKS LIKE THE PURPLE LADS REGION ARE ACTUALLY EXPANDING, SO THIS IS AGAIN CHROMATIN, CHROMOSOME 1, 4, 13 AND 14. THAT'S BASICALLY FROM THE GLOBAL ANALYSIS IT PREDICTS THAT THE PURPLE LADS REGION ARE EXPANDING. AND BUT YOU REALLY WANT TO SHOW THAT, SO WE PERFORMED A FISH EXPERIMENT, BASICALLY BY DESIGNING OLIGO ARRAYS ON SELECTED REGIONS AND DID OLIGO PAINT, THIS IS A VERY COOLs METHOD ALLOWING YOU TO LOOK AT THE VOLUME AND SURFACE AREA OF THE SPECIFIC REGIONS YOU PAINTED. AND AS YOU CAN SEE, TRIPLE KNOCKOUT ES CELLS HAVE SIGNIFICANTLY EXPANDED PURPLE LADS AS ALL OF THIS -- EACH ONE OF THE FOUR PROBES SHOWED EXPANSION. WHEREAS HIGH C ANALYSIS SUGGESTED THAT THE BLUE LADS REGION LOOKS LIKE IT'S KIND OF -- IT FALLS INWARD. AGAIN, YOU CAN USE OLIGO PAINT TO SHOW PICKING DIFFERENT REGIONS MEASURING FROM THE NUCLEAR PERIPHERY AWAY FROM YOUR FISH PROBE, YOU CAN SEE BLUE REGION BASICALLY SHIFTS INWARD. SO, NOW, THIS IS ONE FINDING. BASICALLY THIS 3D GENOME CHANGE SEEMS TO BE PROBABLY STARTING FROM THE PURPLE REGION AND BLUE REGION CHANGING DIFFERENTLY, PURPLE REGION IS EXPANDING, BLUE REGION IS GOING INWARD, IN THE PAST, PEOPLE HAVE BEEN ALWAYS CONFUSED AND TROUBLED BY THE FACT WHEN YOU DON'T HAVE LAMIN, YOU DON'T SEE A CLEAR CHANGE ON THE LADS. HERE OUR DATA SUGGESTS, WELL, THE REASON YOU DON'T SEE THAT IS BECAUSE YOU ARE LOOKING AT LADS AS A TOTALITY, YOU ARE NOT SEPARATING THEM BECAUSE THEY ARE CHANGING DIFFERENTLY. I WILL GIVE YOU A MODEL TOWARD THE END. THE OTHER THING WE TRIED TO DO, WE WERE ABLE TO DO BECAUSE WE ARE ABLE TO REALLY LOOK AT CHROMATIN REGIONS WITH FEATURES, IS TO REALLY LOOK AT ENHANCER ACTIVITIES, THE ACTIVE ENHANCER, H3, K 27 ACETYLPEAKS, IT'S CORRELATED WITH TRANSCRIPTIONAL ACTIVITY. YOU CAN LOOK AT THE INTERACTION CHANGES BETWEEN THESE CHROMATN REGIONS, AND SEE HOW IT'S CORRELATING WITH CHANGE OF ENHANCERS. AND BASICALLY THE TAKEHOME MESSAGE FOR THIS PART OF THE FIGURE, SO HERE BASICALLY THERE'S NO CORRELATION BETWEEN LAMIN ASSOCIATION AND GENE EXPRESSION ACTIVITY. BUT WE DO SEE SOME CORRELATION BETWEEN ENHANCER, ACTIVE ENHANCER CHANGES FOR ENHANCERS THAT'S UPREGULATED, CHROMATIN REGIONS THAT SHOW A DISASSOCIATION FROM THIS MOST REPRESSIVE LADS REGION, THE PURPLE LADS REGION. WHEREAS FOR ENHANCERS THAT'S DOWNREGULATED WE SHOW INCREASE INTERACTION WITH THE OTHER INACTIVE REGION, THE BLUE LADS REGION. SO THIS REALLY SHOWS THE ONLY WAY YOU CAN UNDERSTAND THE GENE TRANSCRIPTIONAL CHANGE WHEN YOU DON'T HAVE LAMIN TO UNDERSTAND INTERACTION CHANGE. I CAN ALSO SHOW SPECIFIC BUNDLE HERE, BASICALLY HERE IS TO LOOK AT TWO SPECIFIC LOCI, ONE IS RIAN LOCUS, DOWNREGULATED IN ES CELLS, ANOTHER IS NOD 1 LOCUS. THIS IS A 4C ANALYSIS, THIS IS A HI C ANALYSIS, YOU DON'T HAVE LAMIN, THIS RION LOCUS EXHIBITED INCREASED INTERACTION WITH THE INACTIVE CHROMATIN REGION. THAT'S BLUE. THIS IS THE BLUE LADS. THAT'S INACTIVE. IT HAS A DECREASED INTERACTION WITH THIS HILAND RED REGION, WHICH IS ACTIVE, SO THAT COULD EXPLAIN WHY THIS IS DOWNREGULATED. ON THE OTHER HAND, IF YOU LOOK AT THIS UPREGULATED NOD1 LOCUS YOU CAN SEE PROBABLY ONE THING THAT CONTRIBUTES TO ITS UPREGULATION IS BECAUSE IT'S PICKING UP THE -- IT'S BASICALLY STARTING TO DISASSOCIATE, SO YOU GO UP FROM HERE, LOOK AT DECREASED INTERACTION, BLUE IS DECREASED INTERACTION, WITH THIS PURPLE LADS, INACTIVE REGION, AND SECOND ONE AND THIRD ONE. SO THIS REALLY IS CORRELATED WITH BASICALLY YOU HAVE LADS CHANGE CHANGE AND INTERACTION CHANGE, THAT MAKES CHROMATIN FIND ITSELF IN DIFFERENT NEIGHBORHOOD, DIFFERENT ACTIVITY REGION, THAT CAN EXPLAIN GLOBAL EFFECT ON GENOME-WIDE TRANSCRIPTIONAL CHANGE. OKAY. SO I'M GOING TO GIVE YOU A MODEL AND I WILL SPEND A FEW MINUTES TO ENTERTAIN A COUPLE IDEAS. ONE IDEA ESSENTIALLY. SO BASICALLY THE TAKEHOME MESSAGE IS SO THE TRADITIONAL UNDERSTANDING OF NUCLEAR LAMINA IS ALWAYS NUCLEAR LAMINA IS A TETHER, FOR THE LAMINA ASSOCIATED CHROMATIN. BUT WHAT WE FOUND, IF YOU REMOVE THIS MESH WORK YOU DON'T HAVE WHOLESALE INWARD FALLING OF THE LADS REGION, INSTEAD THIS PURPLE REGION EXPANDS, AND WE BELIEVE THIS EXPANSION BASICALLY MAKES NO SPACE ANYMORE FOR THIS BLUE LADS REGION, WE DRAW IT IN THE MORE CONDENSED MANAGER, MORE ENRICHED FOR K 27 TRIMETHYLATION, WHICH HAS A ROLE. WE THINK THIS REGION FALLS INWARD. WE HAVE DATA TO SHOW THAT. WHEN THIS REGION FALLS INWARD, THEN IT CHANGES CHROMATIN INTERACTIONS. AND IF YOU PUSH AN EXISTING -- THIS GENE, IF YOU PUSH THIS GENE INTO A WAY MORE TOWARD THE REPRESSED PURPLE REGION, IT'S GOING TO BE REPRESSED, IF YOU PUSH THIS AWAY OR CLOSER, IT BECOMES EXPRESSED, THAT'S WHY THE IMPACT IS GENOME WIDE. WHY IS THIS IMPORTANT? THAT'S THE FIRST QUESTION. WHY AM I EXCITED ABOUT THIS? THE REASON IT'S EXCITING REALLY IS AGAIN YOU COME BACK, WE SAID BASICALLY IF YOU DON'T REMOVE ALL THE MESH WORK, HAVE YOU THE DRAMATIC CHANGE OF CHROMATIN ORGANIZATION. NOW YOU CAN IMAGINE WE KNOW IN FACT FROM OTHER STUDIES I DIDN'T HAVE TIME TO TALK ABOUT IS WHEN WE REMOVE ALL LAMINS YOU CAN SEE THE NUCLEAR AGGREGATE TOWARD THE CENTROSOME, LAMINS HAVE A ROLE IN KIND OF GETTING -- MAKING SURE THAT THE UNDERLINING, WHATEVER FORCE IS EXPERTED IN THE NUCLEUS IS MODULATED. YOU CAN THINK PERHAPS CELLS ARE EXERTING FORCE TO MOVE AND EXERTING FORCE ALONG THE NUCLEAR SURFACE, YOU COULD IMAGINE THAT FORCE COULD HELP TO CHANGE THE MESH SIZE OF THE NUCLEAR LAMINA. WHEN THAT MESH SIZE CHANGES, IT'S LIKE YOU'RE LOCALLY REMOVING SOME LAMIN MESH, THAT COULD LOCALLY OPEN UP THIS PURPLE REGION, THAT COULD THEN ALLOW IN THE NEIGHBORING BLUE LADS TO FALL INWARD, AND THEN YOU KNOW THAT CELL MORPHOGENESIS AND DIFFERENT TRAJECTORY DIFFERENTIATION COULD HAVE IMPACT ON DIFFERENT REGION OF THE NUCLEAR SURFACE. THAT COULD ACTUALLY ONE DAY PERHAPS HELP US TO UNDERSTAND HOW THIS KIND OF MORPHOGENESIS WOULD HAVE PERHAPS DIRECT IMPACT IN HOW YOU ESTABLISH 3D GENOME ORGANIZATION. THEREFORE UNIQUE TRANSCRIPTOME THAT'S LINEAGE SPECIFIC. PROBABLY NOT IN MY LIFETIME WE'RE GOING TO UNDERSTAND THIS BUT AT LEAST KIND OF JOURNEY FOR TEN YEARS, SO FAR SO GOOD, SO FAR IT LOOKS LIKE LAMIN REGULATES MORPHOGENESIS AND LAMIN CONNECTS CYTOSKELETON TO CHROMATIN, AND THEN IF YOU DON'T HAVE LAMINS YOU COULD HAVE THIS SIGNIFICANT INWARD FALLING OF CHROMATIN THAT WILL LEAD TO THE CHANGE TRANSCRIPTOME CHANGE, SO PERHAPS THIS COULD BE A NODE THAT WILL ALLOW US TO UNDERSTAND THE MORPHOLOGICAL IMPLICATIONS OF HOW YOU ORGANIZE DIFFERENT TRANSCRIPTOME. THAT'S IT. SO I'M GOING TO QUICKLY ACKNOWLEDGE PEOPLE IN MY LAB, I THINK I'M DOING OKAY WITH TIME. CELL DIVISION STUDY IS A LONG-TERM, I START MY LAB TO TRY TO UNDERSTAND CELL DIVISION. WE DID SOME GAMMA TURC WORK. WE DECIDED NOT TO DO MORE BUT CONCENTRATE ON SPINDLE MATRIX, IT'S BETTER TO WORK ON THIS, I BELIEVE IT'S VERY IMPORTANT. SO MANY OF THESE PEOPLE CONTRIBUTED, NO ONE IS CURRENTLY IN MY LAB NOW. AND ACTUALLY TRYING TO RECRUIT MITOSIS PERSON RIGHT NOW. AND THEN THE GENOME ORGANIZATION WORK AND ALSO AGING ARE MOSTLY DONE BY THIS GROUP OF PEOPLE, JIABIAO WITH FISH WORK, AND XUELIANG HELPING MODEL CHROMATIN AND HAIYANG DID AGING AND DROSOPHILA WORK, AND I'VE HAD A NUMBER OF COLLABORATORS AND SOME OF THEM I'M STILL COLLABORATING, HERE ARE MY FUNDING, I FINISHED MY ELLISON FUNDING, IMPORTANT FOR US TO DO WORK IN AGING. OKAY. THANK YOU VERY MUCH. [APPLAUSE] >> DO WE HAVE TIME FOR SOME QUESTIONS? PLEASE GO TO THE MICROPHONES. YES, HERE. >> THANK YOU. VERY INTERESTING TALK, INTELLECTUALLY EXCITING. >> THANK YOU. >> ALTHOUGH SCIENCE HAS BEEN REMOVED FROM ME, LOT OF IS INTACT. MY QUESTION IS YOU MENTIONED THE THREE STAGES OF LIFE, FROM EMBRYO, SPINDLE SHAPE, ALL THE WAY THROUGH THE SENESCENCE AND THEN YOU TOUCHED ON THE DISEASES. MY QUESTION IS WHETHER THIS PROTEIN STRUCTURE OF THE TUBULE OR LAMIN CHANGES CONFIRMATIONALLY AS FAR AS PERHAPS THE PROLIEN THAT YOU MENTIONED IS GOING TO CONFORM IN SOME WAY, OR IS IT LATER DEPEND ON THE OTHER PROTEIN STRUCTURE SUCH AS BRANCH LIKE ALANENE. WE SAW SOMETHING YEARS AGO AT THE UNIVERSITY OF PENNSYLVANIA BUT THAT WAS RELATED TO MYOBLAST FORMATION. >> OKAY. SO ARE YOU SPECIFICALLY ASKING ABOUT THE PHASE SEPARATION OF THE BUGZ PROTEIN AND WHAT AMINO ACID MIGHT -- DOES PROLIEN PLAY A ROLE? >> ARE YOU CHANGING STRUCTURE OF PROTEIN FROM EMBRYONIC AND MITOSIS AT THAT STAGE, A FORMAL GROWTH, THE DISEASED STATE, WHICH IS CHAOTIC. >> RIGHT. YEAH, I GUESS ONE GOOD EXAMPLE TO USE WOULD BE LAMIN, I MEAN, YOU KNOW, DURING DEVELOPMENT YOU DEFINITELY NEED LAMIN TO HELP YOU TO MAINTAIN THE STRUCTURE AND TO SUPPORT MORPHOGENESIS. AS YOU GET OLDER, THE PROTEIN JUST STARTED TO DECAY, WE KNOW BY STUDYING THE FAT BODY IN DROSOPHILA SOME CELLS WHEN THEY GO INTO SENESCENCE OR WHEN THEY STOP DIVIDING FOR A LONG TIME LAMIN-B 1 LEVEL BECOMES REDUCED. AND THAT REDUCTION IN FAT BODY CASE LEADS TO THE REPRESSION OF THE INFLAMMATORY CHEMOKINES AND CYTOKINES, BUT, YOU KNOW, BASICALLY THIS IS ALL DOWNSTREAM CONSEQUENCE. THE QUESTION IS HOW DOES LAMIN-B GET REDUCED. WE ACTUALLY STILL DON'T KNOW. WE DID A LOT OF WORK. WE STILL DON'T REALLY HAVE A VERY GOOD CLUE ABOUT WHY LAMIN-B 1 BECOMES REDUCED UPON AGING. IT COULD BE REGULATED BY UBIQUITINATION. >> SENESCENCE MAYBE. >> YES, SENESCENCE IS THE BIGGEST TRIGGER BUT STILL IT'S NOT A DIRECT -- IT CANNOT EXPLAIN HOW LAMIN GETS REDUCED. >> THANK YOU. >> THANK YOU. >> VERY GREAT TALK. TWO QUESTIONS. ONE IS ABOUT THE BUGZ PROTEIN. IN VITRO ASSAY WHERE YOU SHOWED VERY NICELY VACCINATE WHEN YOU RAISE TEMPERATURE. WHAT IF YOU TAKE INTERFACE EXTRACT, THAT'S THE SIGNAL TO STOP PHASE SEPARATING IN VIVO. >> SO I THINK THAT'S IN VITRO IS AN ARTIFACT WHERE WE JUST -- WE ONLY HAVE ONE -- TUBULIN IS A TEMPERATURE -- IT'S TEMPERATURE DEPENDENT. IN VIVO IT IS TOO. IF YOU COOL DOWN THE CELLS. YOU CAN THINK ABOUT BUGZ PHASE SEPARATION AS TUBULIN, LIKE TUBULIN, BECAUSE IN IN VITRO ASSAY IN THAT SENSE YOU JUST REALLY HAVE ONE PROTEIN, DON'T HAVE REGULATORS. IN VIVO FOR BUGZ IT'S ALSO SIMILAR TO TUBULIN, WE'VE DONE THIS EXPERIMENT TAKING TISSUE CULTURE CELLS, WARM IT UP, COOL IT DOWN ON ICE, YOU CAN SEE BUGZ ASSOCIATION WITH MICROTUBULE IS DECREASED. I KNOW WHAT YOU'RE ASKING IS HOW CAN YOU EXPLAIN THIS TEMPERATURE DEPENDENCE. >> THAT WASN'T MY QUESTION. MY QUESTION SIMPLY IS THERE SOME CHEMICAL PROPERTY WITHIN BUGZ FOLDING DOMAIN SENSITIVE TO SOME CATION THAT'S RELEASED IN MITOSIS AND THAT ISN'T PRESENT IN INTERFACE, CATALYZING PHASE& SEPARATION SPECIFICALLY AT THE RIGHT TIME OF CELL CYCLE. >> ACTUALLY SHE WAS IN MY LAB, THIS IS IS A NUCLEAR PROTEIN INTERFACE. IT DOES HAVE A ROLE IN REGULATING SPLICING. >> OKAY. >> SO WE SUSPECT INTERFACE THAT'S ALREADY UNDERGOING PHASE SEPARATION, MITOSIS IS WITH DIFFERENT PARTNERS. >> SECOND QUESTION, BEAUTIFUL STORY ON THE TAD-TAD INTERACTION. ONE THING NOT CLEAR ARE YOU SAYING TAD-TAD INTERACTIONS ARE SELF ORGANIZING, RIGHT? THAT'S ESSENTIALLY THE IMPLICATION BECAUSE WHEN YOU LOSE THE LAMIN-B, THE TAD-TAD INTERACTIONS CHANGE AND YOU GET THIS ODD INTERACTION BETWEEN DOMAINS THAT AROUND SUPPOSED -- IS THIS THING HERITABLE, DOES THIS CHANGE, COMPARED G1 TO THE NEXT G1. >> THAT WOULD BE REALLY INTERESTING TO DO. BUT WE HAVEN'T -- THIS IS BASICALLY A WHOLE POPULATION. WE HAVE NOT DONE THIS CELL CYCLE STATE YET. >> OKAY. >> THAT WOULD BE A VERY GOOD THING TO DO. >> ASSOCIATION, DISASSOCIATION, PHASE SEPARATION, YOU NEED THE FOLDS TO SEPARATE CHROMOSOMES. DO YOU HAVE SIMULTANEOUS INCREASE OF THE ATP TO SEPARATE IN TERMS OF THE BIOMAGNETIC FORCE YOU MENTIONED IN ONE SENTENCE SOMEWHERE THERE, SO DO WE KNOW ENOUGH, THIS MOLECULAR ASSOCIATION GENERATES ENOUGH, WHAT IS THE SOURCE, DO YOU HAVE A HIGHER LEVEL OF PRODUCTION OF ATP OF MITOCHONDRIA DURING SEPARATION? >> A VERY GOOD QUESTION. I HAVE MY COLLABORATOR IN CHINA, ONE STUDY THAT'S WHAT THEY ARE TRYING TO DO. THEY BELIEVE WHEN CELLS GO INTO MITOSIS THERE'S INCREASE IN CERTAIN TYPE OF ATP PRODUCTION THAT IS MEDIATED BY MITOCHONDRIA, BY ONE OF THE TRANSPORTERS. I DON'T REMEMBER THE DETAIL. I CAN'T EVEN TELL YOU EXACTLY WHAT IT IS. BUT, YES, THERE ARE INDICATIONS CELLS GO INTO MITOSIS, INCREASED PRODUCTION OF ATP, THAT'S HELPING TO SUPPLY ATP FOR MANY MOTORS, RIGHT, TO GET CELLS TO DIVIDE, THERE ARE A LOT OF MOTORS THAT NEED TO BE VERY ACTIVE. MOVING CHROMOSOME TO WORK, HYDROLYZE ATP TO PULL CHROMATIN APART, AND CYTO KINESIS NEEDS ADDITIONAL MOTORS. YES, DEFINITELY I THINK THAT IDEA IS ON THE RIGHT TRACK. YOU NEED TO PRODUCE MORE ATP. >> LOOKING AT THE VIDEO THEY DID NOT LOOK SOMATIC, ONE WAS SMALLER, THE OTHER IS LARGER. SO IF TRUE WHAT I SEE THERE? >> WELL, SO ACTUALLY I'M SORRY, I PROBABLY DIDN'T EXPLAIN WELL ENOUGH. SO YOU ARE LOOKING AT PSEUDOSPINDLE FORMATION. WHERE THERE'S NO CELL, THERE'S NO DNA, ALL WE HAVE IS PUTTING MICROTUBAL ORGANIZING CENTER, AND BEADS, PUTTING, FORMS A PSEUDOCENTROSOME. NUCLEATE, MAKING A PSEUDOSPINDLE. SO YOU ARE NOT LOOKING AT CELLS, THE BEADS ARE THE SAME SIZE, ON ONE HAND I HAVE THIS ENLARGED SPINDLE, ONE SPINDLE THAT'S ELONGATING, ON THE OTHER, ON THE RIGHT, YOUR RIGHT HAND, THE SMALLER SPINDLE BECAUSE YOU'RE SEEING MANY MORE. YES, I'M SORRY. I CONFUSED. >> OH, ALL RIGHT. THANK YOU. GOOD LUCK. >> THANK YOU. >> ALL RIGHT. SO LET'S THANK DR. ZHENG FOR A STIMULATING LECTURE. [APPLAUSE]