>>WELCOME TO THE WEDNESDAY AFTERNOON LECTURE SERIES. I'M DR. SERPE, I LEAD THE SECTION OF CELLULAR COMMUNICATION IN CHILD HEALTH AND HUMAN DEVELOPMENT INSTITUTE. I'M VERY EXCITED TO INTRODUCE TODAY'S SPEAKER, DR. SUSAN PARKHURST, DISCUSSING THE TOPIC WE CAN ALL RELATE TO. WOUND REPAIR SHE HAS INSPIRED GENERATIONS OF WOMEN SCIENTISTS AND TODAY SUSAN IS GUEST OF WSA, WOMEN SCIENTIST ADVISORS COMMITTEE. I TOO HAVE BEEN A FAN OF SUSAN FOR A LONG TIME, HER RESEARCH BROUGHT KEY EVENTS TO MANY TOPICS IN IN MODERN BIOLOGY INCLUDING MECHANISTICS OF TRANS-- CELLULAR SCHEDULING AND BIOLOGY OF THE CYTOSKELETON. FUNDAMENTAL SCIENCE THAT MAKES A DIFFERENCE BECAUSE AS SUSAN PUT IT YOU CANNOT MAKE MEDICAL ADVANCES IF YOU DON'T KNOW THE BASES. SUSAN IS PROFESSOR IN THE BASIC SCIENCE DIVISION AT THE FRED HUTCH CANCER RESEARCH CENTER IN SEATTLE AND AFFILIATE PROFESSOR WITH DEPARTMENT OF BIOLOGY, UNIVERSITY OF WASHINGTON. SHE IS BOTH UNDERGRAD AND Ph.D. DEGREES IN BIOLOGY AT JOHNS HOPKINS UNIVERSITY IN BALTIMORE, SHE HAD POST-DOCTORAL FELL PLEA SHIP AT COLLEGE CANCER RESEARCH IN LONDON WITH DR. HOROWITS FOLLOWED BY RESEARCH FELLOWSHIP WITH DR. HOWARD LIPSHIDS, SHE JOINED FRED HUTCH IN 1992 AND ROSE THE RAKES TO BECOME FULL PROFESSOR IN 2000. AT THE HEART OF HER CURRENT RESEARCH, IS THE STUDY OF THE CYTOSKELETON, A STRUCTURE THAT HELPS CELLS MAINTAIN THEIR SHAPE AND PROVIDE MECHANICAL SUPPORT FOR WIDE RANGE OF CELLULAR FUNCTION. FROM CELL DIVISION TO CELL MIGRATION AND METASTASIS. SUSAN IS ESPECIALLY FAMOUS FOR HER CONTRIBUTION TO WOUND HEALING BECAUSE SHE HAD DEFINED THE FIELD BY USING DROSOPHILA FIRST MODEL TO MOLECULAR AND CELLULAR MECHANISM IN WOUND REPAIR PROCESS AND IMPLICATION FOR DISEASE SUCH AS CANCER WHEN THE PROCESS GOES AWRY. HER WORK VISUALIZED CYTOSKELETAL DEVELOPMENT AS WELL AS DURING WOUND REPAIR DEFINING SIGNALING CENTERS SUCH AS RTP ACES THAT MODULATE SKELETAL DYNAMICS AND EFFECTERS DOWNSTREAM THAT BUNDLE ACTIN, MICROTUBULES AND CROSS LINK THEM TOGETHER. ALONG THE WAY, SUSAN HAS PICKED UP NUMEROUS HONORS AND THIS INCLUDES THE MAIN SCHOLAR IN BIOMEDICAL SCIENCES FORMAL SOCIETY SCHOLAR. SHE IS AMAZING AND WIDELY CREATIVE SCIENTIST BUT ALSO A GENEROUS AND THOUGHTFUL CITIZEN BEYOND HER INSTITUTION SERVING CONTINUOUSLY NIH STUDY SECTION AND ADVISORY BOARDS IN THE GENETIC SOCIETY. THE TITLE OF HER TALK IS WOUND REPAIR, DEALING WITH LIFE'S TRAUMAS. SUSAN, WELCOME TO THE NIH. THANK YOU SO MUCH FOR BEING WITH US TODAY. >> I WANT TO START BY SAYING THANK YOU TO ELLEN TO WOMEN'S ADVISORY COMMITTEE FOR THIS INVITATION I'M EXCITED TO SHARE WORK ON WOUND REPAIR WITH YOU TODAY. IT TURNS OUT WOUNDS CAN HAPPEN IN RESPONSE TO MANY DIFFERENT THINGS, FOR EXAMPLE DAILY WEAR AND TEAR, IN TERMS OF TRAUMA, THROUGH CHEMICALS OR OTHER ENVIRONMENTAL STRESSES OR CLINICAL INTERVENTIONS. SO AN ORGANISM NEEDS TO HAVE A WAY TO RESPOND VERY QUICKLY. NOT ONLY DOES THIS HAPPEN AT A SINGLE CELL LEVEL BUT IT HAPPENS AT A MULTI-CELLULAR LEVEL AND ORGANIZE FIZZ MALL LEVEL. THERE ARE DIFFERENCES IN THESE DIFFERENT TYPES OF WOUND REPAIR TO MECHANISMS YOU USE. YOU CAN USE MODEL ORGANISMS TO STUDY DIFFERENT EVENTS AND THEY ARE REMARKABLY SIMILAR TO WHAT HAPPENS IN HUMANS. MY LAB HAS DONE A NUMBER OF THESE BUT WHAT I'LL DO TODAY IS FOCUS ON THE STAGE, WHAT HAPPENS TO SINGLE CELLS. SO TURNS OUT EVERY CELL IN YOUR BOUGH KY IS SUBJECTED TO DAILY WEAR AND TEAR TO ENVIRONMENTAL STRESS, PHYSIOLOGICAL STRESS, AND THEY HAVE TO BE ABLE TO FIX ANY KIND OF MEMBRANE BREECHES. IF YOU WALK DOWN A HILL YOU ARE RIPPING OPEN MUSCLE CELLS AND YOU HAVE TO BE ABLE TO REPAIR THOSE CELL CORTEXES BECAUSE YOU CAN'T REPLACE A EVERY MUSCLE CELL SO IN ORDER TO DO THAT YOU NEED TO HAVE A PROCESS THAT HAPPENS QUICKLY. AND THERE HAVE BEEN A NUMBER OF DIFFERENT ORGANISMS AND SYSTEMS DEVELOPED TO LOOK AT SINGLE CELL REPAIR, AND ALL OF THEM SHARE A LOT OF FEATURES. WHAT I'M SHOWING YOU HERE ARE THE PHYSIOLOGICAL FEATURES THAT WE KNOW HAPPEN NO MATTER WHAT SYSTEM YOU ARE LOOKING AT. SO IF YOU LOOK HERE WE HAVE A MEMBRANE AND CORTICAL CYTOSKELETON, WHEN YOU BREACH THIS CORTEX CALCIUM IS THOUGHT TO FLOW IN, IT CHANGES THE MICROTUBULES AND ACTIN CYTOSKELETON TO DIRECT TOWARDS THE WOUND AND BRING VESICALES TO THE SURFACE AND THOSE VESICALES WITH FUSE WITH EACH OTHER AND WITH THE PLASMA MEMBRANE AND IN DOING SO THEY PLUG THE WOUND. THEN YOU HAVE THE FORMATION OF AN ACTIN MYSIN HERE THAT IS GOING TO PULL THE WOUND CLOSED THE CORTICAL CYTOSKELETON WITH THE OVERLYING MEMBRANE: ONE CLOSED IT HAS TO REMODEL SO YOU HAVE TO MEMBRANE AND CORTICAL CYTOSKELETON TO GO BACK TO NORMAL COMPOSITION AND ORGANIZATION TO CARRY OUT ITS NORMAL FUNCTIONS. SO WHAT WE KNOW THE GENERAL CELL BIOLOGICAL ASPECT OF WHAT HAPPENS IN WOUND REPAIR, WHAT WE DON'T KNOW ARE THE MOLECULAR UNDERPINNINGS. SO WE DON'T HAVE A REALLY GOOD MOLECULAR PATHWAY IN ANY OF THESE ORGANISMS FOR WHAT HAPPENS WITH WHAT IS A START SIGNAL HOW DO YOU KNOW WHAT TO MOLECULES ARE REQUIRED THROUGHOUT DIFFERENT STEPS AND HOW YOU KNOW FOR EXAMPLE THAT YOU FINISHED SO YOU DON'T OVERREPAIR. WE USE DROSOPHILA AS A MODELS ORGANISM, TURNS OUT THERE IS A FLUKE OF DROSOPHILA DEVELOPMENT IN EARLY STAGES WHERE YOU HAVE NUCLEAR DIVISIONS BUT WITH NO CELL DIVISIONS. SO IN THE CENTER OF THE EGG YOU HAVE THE NUCLEI DIVIDE, NOT UNTIL LATER THEY MOVE TO THE SURFACE AND BECOME CELLS. SO DURING THIS EARLY STAGE, THEY ARE ESSENTIALLY A BIG CELL, A MULTI-NUCLEAR CELL BUT LARGE CELL AND LOT OF THE CELLS IN THE BODY THAT ARE UNDERGOING REPAIR SUCH AS MUSCLE CELLS AND GUT CELLS ARE MULTI-KNEW CREATE SO THESE TURN OUT TO BE A GOOD SYSTEM FOR THAT. SO WHAT WE HAVE TRIED TO DO USING THIS AS A MODEL IS TO TAKE TWO BASIC KIND OF APPROACHES ONE IS A GLOBAL APPROACH AND TO USE THE ADVANTAGE THAT DROSOPHILA IS A GENETIC ORGANISM AND YOU CAN DO GENETIC SCREENS THAT ALLOWS YOU TO GENERATE COLLECTIONS OF MUTATIONS THAT AFFECT WOUND REPAIR AND BE ABLE TO LOOK AT COLLECTION AS A WHOLE TO GET GENERAL PRINCIPLES WHAT IS GOING TON FIGURE WHAT HAPPENS FIRST SECOND AND THIRD AND TO START TO BUILD A PATHWAY YOU CAN DO THIS USING GENETIC SCREENS BECAUSE DROSOPHILA IS AMENABLE TO IMAGING YOU CAN USE VISIBLE SCREENS. YOU CAN ALSO LOOK AT SPECIFIC POINTS IN THE PATHWAY. WE WANT TO KNOW WHAT IS START SUGGESTIONNAL DESIGN EXPERIMENTS TO LOOK DURING THE EARLIEST STAGE AND ASK WHAT IS REQUIRED THEN SO WE CAN LOOK FOR DIFFERENT EVENTS LIKE START SIGNAL STOP SIGNAL, WE CAN ALSO ASK WHAT THE ROLE OF ANY GIVEN FAMILY OF PROTEINS IS. SO DIFFERENT CYTOSKELETAL PROTEINS OR MEMBRANE PROTEINS AND ASK HOW THEY WORK IN WOUND REPAIR. WE DO BOTH OF THESE, I WILL GIVE EXAMPLES OF BOTH. BEFORE I DO THAT I WANT TO EXPLAIN MORE ABOUT OUR SYSTEM AND WHAT YOU CAN EXPECT TO SEE. SO THIS WOULD BE A DROSOPHILA EMBRYO. SURROUNDED BY IMPERMEATE PATELLA MEMBRANE. THERE'S A SPACE FOR FILLED MOSTLY WITH EXTRA CELLULAR MATRIX. WE TAKE A COVER SLIP AND GLUE THE EMBRYO DOWN SO THE EMBRYO IS FREE TO ROTATE INSIDE HERE AND WE IMAGE FROM THE BOTTOM. WHAT WE CAN SEE IS HERE, A STRAIGHT ON VIEW OF THE CELL SURFACE. IF WE WERE TAKING SLICES GOING INTO THE CELL, WE CUT IT HERE AND LOOK FROM THE INTERNAL VIEW IS THAT YOU ARE SEEING HERE OR HERE. SO YOU CAN SEE WHAT'S HAPPENING AT THE SURFACE BUT ALSO WHAT'S HAPPENING BELOW THE SURFACE AS WELL. SO WELCOME DO TIME LAPSE IMAGES AND LOOK FOR ANY PARTICULAR MOLECULE SHOWING ACTIN REPORTER FOR WHAT HAPPENS DURING THE REPAIR PROCESS. SO WHAT WE DID THIS IN MOVIES IT IS DIFFICULT TO SHOW THESE AND COMPARE IN ANYWAY SO WE DO IT IN A TALK OR PAPER IS MAKE A CHAI MOW GRAPH LIKE THIS. SO WHAT WE ARE DOING IS TAKING EACH INDIVIDUAL IMAGE HERE, SHOW A STRIP AND LINE UP OVER TIME. LIKE THIS. BY DOING SO WE CAN SEE CLOSURE OF THE WOUND. WE CAN SEE FOR EXAMPLE ACTIN IS ACCUMULATING HIGHLY AT THE END OF THE WOUND, WE CALL THIS THE ACTIN RING. IT ACCUMULATES AT A HIGHER LEVEL THAN NORMAL BUT NOT AS HIGH AS THE RING WE CALL THIS ACTIN HALO. WHEN WE LOOK IN THIS GRAPH WE CAN SEE FIRST THING THAT HAPPENS IS WOUND EXPANDS THEN IT WILL GENERATE ACTIN RING TO HELP CLOSE ONCE CLOSED IT HAS TO DISASSEMBLE THE RING AND SPENDS TIME REMODELING THE MEMBRANE AND UNDERLYING CORTICAL CYTOSKELETON. WE HAVEN TWO APPROACHES AND DONE TWO SCREENS. ONE IS WE CALL A MICROARRAY SCREEN, THE PURPOSE OF THIS IS TO TRY TO IDENTIFY GENES THAT WERE UP AND DOWN REGULATED IN RESPONSE TO WOUNDING AND OUR HOPE IS TO BE ABLE TO IDENTIFY START SIGNAL. WE CAN A FLY TRAP SCREEN. THIS IS ONE WE ARE TAGGING PROTEINS WITH GFP AND WHAT WE ARE LOOKING IS TO SEE PROTEINS RECRUITED TO A WOUND OR THAT MIGHT BE DEPLETED UPON WOUNDING. I WILL START WITH THE MICROARRAY SCREEN WHAT WE DID HERE, I WILL AGE MYSELF A LITTLE BIT. THERE THE REASON IT IS A MICROARRAY SCREEN IS BECAUSE WE STARTED IT A WHILE AGO. IF WE WERE DOING IT TODAY PROBABLY AN RNA SEQ SCREEN BUT IT WORKED WELL THROUGH RNA SEQ SCREEN SO IF WE TAKE THE EMBRYO AND WOUND IT, SO IN THIS CASE WE ARE WOUNDING IT EIGHT SEPARATE TIMES AND WE HAVE TO DO THAT SO WE CAN GET A ROBUST ENOUGH RESPONSE FOR WOUND REPAIR SO THAT WHEN WE LOOK AT THE DIFFERENCES IN TRANSCRIPTION WE CAN ACTUALLY TELL THEM. SO WE TAKE EMBRYOS NOT WOUNDED, EMBRYOS THAT ARE, WE ICE LATE RNA, MAKE CDNA AND PUT THEM TO MICROARRAY AND THE MICROARRAYS WE WERE USING IN THIS PARTICULAR CASE HAD 12,000 CDNA FROM DROSOPHILA ON IT. AND THAT COVERS 60% OF THE FLY GENES. WE ALSO DID TWO TIME POINTS SO WE WANTED TO KNOW IF TRANSCRIPTION WAS INVOLVED IN A START SIGNAL, AND THE REASON FOR THIS IS SOME OF THE EXPERIMENTS IN TISSUE CULTURE CELLS HAS SUGGESTED THAT WHEN CALCIUM FLOWS INTO THE CELL, THAT IT SETS OFF A TRANSCRIPTIONAL CASCADE STARTING WITH AP 1. AND SORT OF QUANDARY WE HAD IS THAT IN THE DROSOPHILA EARLY EMBRYO, ALMOST ALL OF THE EVENTS ARE RUN OFF MATERNALLY CONTRIBUTED RNAs AND PROTEINS. YOU DON'T REALLY GET ZYGOTIC TRANSCRIPTION UNTIL AN HOUR AFTER THE TIME THAT WE ARE USING THESE EMBRYOS. SO THERE'S VERY LITTLE TRANSCRIPTION AT THIS TIME. THERE'S NOT ZERO, THERE IS A LITTLE BIT BUT NOT VERY MUCH. SO WE EITHER HAD TO HAVE A SITUATION WHERE IN DROSOPHILA THE START SIGNAL WOULDN'T BE TRANSCRIPTIONAL OR WE CAN INDUCE A TRANSCRIPTIONAL SIGNAL IN AN EMBRYO THAT NORMALLY ISN'T HIGHLY TRANSCRIPTIONAL. SO WE WERE INTERESTED IN KNOWING WHICH ONE THAT WAS. AS WELL AS WHAT TRANSCRIPTION WAS DOING IN GENERAL FOR WOUND REPAIR. SO THAT'S THE REASON FOR DOING TWO DIFFERENT TIME POINTS. THE EARLY TIME POINT THE ONE FIVE MINUTES WHICH IS ABOUT AS FAST AS WE CAN WOUND THEM GET THEM OFF THE SLIDE AND ANALYZE THEM. WE SHOULD TELL US ANYTHING THAT'S HAPPENING IMMEDIATELY SO IF THERE IS A START SIGNAL WE SHOULD BE ABLE TO DETECT IT. 30 MINUTES SO THE MOVIES I SHOWED YOU THEY ARE REPAIRING TAKES 30 MINUTES IN OUR CASE SO WE WANTED SOMETHING AT THE END. THAT WILL TELL US EVERYTHING THAT WAS REQUIRED FROM A TRANSCRIPTIONAL POINT OF VIEW THROUGHOUT THE WHOLE PROCESS. THEN WE HAVE A VERY LARGE NUMBER OF RNA AD LINES GENERATED BY THE FLY COMMUNITY WE CAN USE TO VALIDATE THESE. SO WHAT HAPPENED? THESE ARE VOLCANO PLOTS SHOWING YOU THE EARLY TIME POINT AND LATE TIME POINT. THE BLACK DOTS THAT ARE HERE ARE ALL OF THE GENES ON THE CHIP THAT ARE NOT CHANGING AND THE RED AND THE GREEN ARE ONES THAT ARE DOWN-REGULATED OR UPREGULATED. WHAT YOU PROBABLY HAVE NOTICED RIGHT AWAY IS IN THIS ONE THERE ARE NO RED AND GREEN SO IN THE EARLY TIME POINT THERE WERE NO TRANSCRIPTIONAL RESPONSES. EITHER UP OR DOWN. BY 30 MINUTE THERE IS IS A NUMBER OF GENES THAT ARE EITHER UP OR DOWN REGULATED. SO THAT SUGGESTED TO US CONSISTENT WITH THE FACT MANY THE EARLY EMBRYO THERE IS NOT A LOT OF TRANSCRIPTION THAT IN FACT THE EARLY RESPONSE IS PROBABLY NOT TRANSCRIPTIONAL. BUT TO CONFIRM THAT A LITTLE BIT FURTHER, WHAT WE DID WAS TO USE INHIBITORS OF TRANSCRIPTION AND LOOKED TO SEE WHAT HAPPENS TO WOUND REPAIR. THIS IS THE CHIMO GRAPH I SHOWED YOU WILD TYPE EMBRYO ACTIN REPORTER. E YOU GET REPAIRED 15 MINUTES AND REMODELING TAKES ALL THE WAY UNTIL 30. IF YOU LOOK AT EMBRYO INJECTED WITHOUT THE MANITIN OVERALL WOUNDS REPAIR IS PRETTY SIMILAR, THERE IS A LITTLE BIT OF DIFFERENCE AT THE BEGINNING WHERE YOU DON'T GET AN ACTIN RING AS ROBUST AS YOU SEE HERE. TAKES LONGER TO CLOSE AND START TO REMODEL. SO IT HAS PHENOTYPES BUT MOST OF THEM ARE ACTUALLY LATER THAN THE -- WHAT YOU MIGHT EXPECT FOR THE INITIATION OF TRANSCRIPTION. WE AT THE SAME TIME DID ONES FOR TRANSLATION BECAUSE A LOT OF EARLY EMBRYO IN THE FLY HAS PROTEINS AN RNAs THAT NEED TO BE TRANSLATES SOD WE USE PURE MYSIN OR CYCLOHEXIMIDE, WE DO SEE DIFFERENCE THE VERY EARLIEST STAGE THEN WOUND REPAIR IS DISRUPTED SO IT SUGGESTS THAT AT LEAST IN THE DROSOPHILA SYSTEM THE EARLY SIGNALS ARE MORE TRANSLATIONALLY ESTABLISHED THAN TRANSCRIPTIONALLY. THAT WAS A SURPRISE BECAUSE TRANSLATION TAKES TIME AND IS YOU ARE PROBABLY APPRECIATING THIS WHOLE PROCESS AT 30 MINUTES IS VERY, VERY FAST. IT SEEMS LIKE EMBRYO IS ABLE TO DO THAT WELL. IF WE LOOK AT THE LATER TIME POINT JUST TO ASK WHAT EFFECT DOES TRANSCRIPTION HAVE IN THE END, WE KNOW THAT THERE ARE DOWN REGULATED UPREGULATED GENES WHAT WE DID WAS TO MAP THESE ON TO CHROMOSOMES DROSOPHILA. SO DROSOPHILA HAS AN X TWO ARMS AT THE SECOND, TWO ARMS AT THE THIRD AND A TINY FOURTH CHROMOSOME. AND WHAT YOU ARE SEEING HERE, THE BLACK IS CHROMATIN AND HETERO CHROMATIN, ALL THE GRAY DOTS ARE THE CDNA REPRESENTED ON THE CDNA OR MICROARRAY SO THEY ARE EVENLY DISTRIBUTED AMONG CHROMOSOMES AND RED AND GREEN ARE THE UP AND DOWN REGULATED GENES. JUST BY LOOKING AT IT, WE DIDN'T SEE ANY REGIONS WHERE WOUND REPAIR GENES ARE CLUSTERING. SO IT DIDN'T LOOK LIKE THERE WERE ANY KIND OF REGIONS THAT WERE LIKE THAT. WE ALSO TOOK ALL THESE UP AND DOWN REGULATED GENES AND MAPPED TO TRANSCRIPTIONAL ACTIVATION DOMAINS THAT HAVE BEEN ASSIGNED TO DIFFERENT REGIONS OF THE CHROMOSOME AND COULD FIND NO TAGS THAT WERE SPECIFICALLY ENRICHED FOR WOUND REPAIR GENES. SO DOESN'T LOOK LIKE THERE IS A REGION THAT NEEDS TO BE ESTABLISHED AND ALSO TAKING ALL OF THESE GENES UP AND DOWN REGULATED AND LOOKING FOR ANY MOTIF THAT IS IN THE -- THAT WOULD DEFINE AS WOUND REPAIR GENE, DIDN'T YIELD ANY MOTIFS. SO WE DON'T SEE THAT WE ARE HAVING A WOUND REPAIR PROGRAM PER SE, BUT WHAT YOU HAVE ARE LOT OF GENES THAT ARE REQUIRED TO GET UP OR DOWN REGULATED. ONE INTERESTING THING WE DID FIND HOWEVER IS THAT THE SIZES OF GENES VARIED. SO THE AVERAGE SIZE OF THE GENE IN A DROSOPHILA EMBRYO AT THIS STAGE IS ABOUT 2 KB. IF YOU REMEMBER, THIS IS BECAUSE THE VERY, VERY EARLY DIVISIONS ARE JUST THE NUCLEAR ONES AND THOSE ARE HAPPENING EVERY TEN MINUTES. SO THE CELL CYCLE IS VERY, VERY RAPID. IN THAT TIME YOU DON'T REALLY HAVE TIME TO TRANSCRIBE VERY LARGE GENES SO MOST OF THE GENES THAT ARE TRANSCRIBED IN A LIMITED TRANSCRIPTION THAT HAPPENS ARE VERY SMALL AND USUALLY DON'T HAVE INTRONS. SO ROUGHLY TWO AND A HALF KB. WHAT WE FOUND IS THAT THE ONES DOWN-REGULATED WERE IN THE SAME RANGE 2 KB, 2.5 KB SO THE DOWN-REGULATED GENES APPEAR TO BE RECOGNIZING MOSTLY GENES THAT ARE PRESENT IN THE EMBRYO THAT GET DEPLETED SO USED FOR WOUND REPAIR AND DEPLETED AND NOT REMADE. WE DID HOWEVER FIND THAT THE UPREGULATED GENES COULD BE QUITE LARGE. WHEN WE LOOK AT SOME OF THESE WE FIND THESE ARE GENES THAT ARE NOT NORMALLY EXPRESSED IN EARLY EM BEE-BEE OWE IS SO IT SAYS THE EMBRYO IS ABLE TO TURN ON DORMANT GENES NEEDED TO ACTIVATE IN ORDER TO START THE REPAIR PROCESS. IF WE LOOK AT THE DIFFERENT GENES WE GOT, SO WE TOOK THE TOP 15 UPREGULATED AND TOP 15 DOWN REGULATED AND JUST ASKED DO THEY REALLY AFFECT WOUND REPAIR? SO HERE IS A CONTROL WITH AN ACTIN REPORTER SHOWING IT CLOSE AND WHAT YOU CAN SEE IS THAT IN THESE DIFFERENT MUTANTS YOU HAVE PHENOTYPES. ONES THAT DISASSEMBLE THE ACTIN RING EARLY, PREMATURELY DISASSEMBLE IT OR THEY AGGREGATE ACTIN IN THE MIDDLE OF THE WOUND. THEY CAN OVEREXPAND THE WOUND. OR THEY CAN FAIL TO DISASSEMBLE THE ACTIN RING AND KEEP IT TOGETHER FOREVER OR STAY OPEN FOREVER. SO WE CAN SEE THAT ALL THESE DIFFERENT GENES WERE AFFECTING WOUND REPAIR SO WE KNOW TRANSCRIPTION IS PLAYING AN IMPORTANT ROLE BUT JUST NOT IN THE INITIAL STAGES OF WOUND REPAIR. WE CAN MEASURE ALL THESE SO WE CAN TAKE THE DIFFERENT PHENOTYPES THAT WE HAVE, AND WE CAN DO ANALYSIS OF THESE, SO THIS SHOWS YOU A CONTROL HERE. IF WE LOOK AT ALL OF THE DIFFERENT GENES OR ALL 15 TOP UPREGULATED OR THE 15 DOWN-REGULATED WE CAN FIND GENES FOR EXAMPLE HERE WE ARE LOOKING AT THE CONTRACTION RATES OF SLOPE OF THE LINE AS IT GOES DOWN AND WE CAN FIND GENES THAT LIKE THESE OR THESE THAT WILL HEAL FASTER OR SLOWER SO BY MEASURING THE EXPANSION RATE, THE CONTRACTION RATE WHETHER THEY DISASSEMBLE THEIR ACTIN RING WE CAN MAKE A COMBINATORIAL MODEL OF THE DIFFERENT GENE PRODUCTS WE HAVE AND WHAT KIND OF PROCESSES THEY AFFECT. THAT LETS US TAKE THIS LARGE GROUP OF GENES WE HAVE IDENTIFIED AND START TO PLACE THEM INTO A MOLECULAR PATHWAY. AND UNDERSTAND WHAT ARE THE DIFFERENT EVENTS AND WHAT TYPES OF GENES YOU NEED IN ORDER TO CARRY OUT THOSE PROCESSES. WHEN WE WERE DOING THIS WE FOUND AN INTERESTING SET OF GENES. THAT HAD TO DO WITH INSULIN SIGNALING. WHAT I'M SHOWING YOU HERE IS A VERY, VERY SIMPLIFIED MODEL OF INSULIN SIGNALING WHERE YOU HAVE INSULIN BINDING TO RECEPTOR AND THEN SETTING OFF A CASCADE OF DIFFERENT SIGNALING EVENTS THAT WILL EVENTUALLY LEAD TO EFFECTOR PROTEINS BEING EXPRESSED. WE IDENTIFIED ONES FROM THE TOP OF THE PATHWAY TO THE BOTTOM OF THE PATHWAY. AND IT MADE US WONDER IF CONONCAL INSULIN SIGNALING WAS INVOLVED IN CELL WOUND REPAIR. THAT WAS A LITTLE BIT SURPRISING TO US BECAUSE YOU CAN IMAGINE THAT YOU COULD HAVE INSULIN SIGNALING WHEN YOU HAVE MULTI-CELLULAR REPAIR OR TISSUE REPAIR SIGNALING FROM ONE CELL TO ANOTHER BUT IN THIS CASE WHEN LOOKING AT A SINGLE CELL AND YOU HAVE INSTANT SIGNALING IT MEANS YOU HAVE TO BASICALLY HAVE A CELL SECRETE INSULIN AND TAKE IT UP AGAIN, THE SAME CELLS YOU ARE HAVING AUTOCRINE SIGNALING. AND THE QUESTION WOULD BE WHY TO DO THAT IF YOU HAVE TO HAVE A RAPID EVENT, WOULDN'T IT BE EASIER JUST TO SIGNAL WITHIN THE CELL AND NOT HAVE TO SIGNAL OUT AND THEN SIGNAL BACK IN? SO WE DID LOOK TO SEE IF THE CANONICAL PATHWAY WAS BEING INVOLVED HERE SO PIP 3 GFP IS ONE REPORTER PEOPLE USE TO LOOK AT INSULIN SIGNALING PATHWAY, AND WHAT WE CAN SEE IS I'M SHOWING YOU ACTIN IS RECRUITED TO WOUND, THIS IS DOUBLE LABELED WITH PIP 3 GFP, PIP 3 GFP IS RECRUITED TO THE WOUND. SO SUGGESTING THAT AT LEAST THIS CANONICAL PATHWAY IS INTACT AND BEING USED. IF IT IS REALLY THIS PATHWAY, IF WE WERE TO KNOCK OUT ONE OF THESE UPSTREAM FACTORS, THAT THIS WOULD NO LONGER BE RECRUITED. THAT TURNS OUT TO BE THE CASE. SO IF WE KNOCK OUT CHICO HERE, NOW WE DON'T TURN ON THE REST OF THE INSULIN SICKNALLING PATHWAY AND YOU CAN SEE WHILE PIP 3 IS HERE IT DOESN'T ACCUMULATE AT A WOUND. SO AGAIN SUGGESTED THAT IT REALLY WAS THE CANONICAL INSULIN SIGNALING PATHWAY OCCURRING HERE AND NOT SOME EXTRA PATHWAY OR MOONLIGHTING. SO WHAT WE DID IS LOOK FOR REAGENTS THAT EXISTED FOR THESE DIFFERENT FACTORS AND ASKED WHAT THE PHENOTYPE WAS. ALL THE ONES THAT ARE CIRCLED IN RED ARE ONES WE HAVE RNA EYE LINES FOR AND KNOCK DOWN AND ASK IF THEY HAVE EFFECT ON WOUND REPAIR. THESE ARE THE DIFFERENT CHIMO GRAPHS SHOWING THE PHENOTYPES. THEY ARE REMARKABLY SIMILAR FOR ALL THE DIFFERENT FACTORS. WHERE YOU SEE OVEREXPANSION, INTERNAL ACTIN ACCUMULATION, ET CETERA. SO SUGGESTED TO US INDEED IT IS REALLY THE CANONICAL INSULIN SIGNALING PATHWAY BEING INVOLVED HERE AND YOU HAVE TO HAVE SOME TYPE OF AUTOCRINE SIGNALING. THE FACT THAT IT WAS AFFECTING THIS REPORTER WHICH IS ACTIN REPORTER, SUGGESTEDDED TO US THAT IT MUST BE AFFECTING ACTIN IN SOME WAY. WHEN WE LOOK AT THE DOWNSTREAM EFFECTORS THAT ARE KNOWN FOR INSULIN SIGNALING THERE ARE A LEAST A COUPLE THAT AFFECT ACTIN. WE LOOKED AT TWO OF THESE. THOSE ARE SHOWN HERE SO THERE'S -- THIS IS A PROTEIN THAT COMBINED TO ACTIN FILAMENTS, A SUBSTRATE FOR AKT. WE LOOK AT CHICKADEE THE DROSOPHILA ORTHO LOG AND KNOWN TO BIND O TO ACTIN MONOMERS I'M SHOWING YOU AN EMBRYO WOUNDED SO YOU CAN SEE THE WOUND FROM ACTIN AND IF YOU LOOK AT BURDEN YOU CAN SEE ACCUMULATING AT THE WOUND AND PROFILIN TO THE CENTER. AND IF YOU KNOCK DOWN THE BATH WAY WITH CHICO AS WE DID BEFORE THIS ACCUMULATION IS LOST SO BOTH FACTORS ARE ACTING DOWNSTREAM OF THE INSULIN SIGNALING PATHWAY. ONE THING THAT WAS CURIOUS TO US THOUGH IS THAT THE PATTERN OF THESE ARE NOT THE SAME. SO THE WAY THAT BURDEN IS RECRUITED TO A WOUND HOW PROFILIN IS NOT THE SAME. THAT IS REFLECTED IN THEIR MUTANT PHENOTYPES. SO THIS SHOWS YOU KNOCK DOWN OF GIRDIN, THE XY VIEWS AND CHIMO GRAPH, FOR PROFILIN. IN THIS CASE WE CAN'T REMOVE THE GENE BUT WE CAN REDUCE SIGNIFICANTLY. REMOVE IT WE DON'T RECOVER EMBRYOS TO LOOK AT. YOU CAN SEE DIFFERENCES IF YOU START WITH A GRAPH YOU CAN SEE THIS ONE OVEREXPANDS A LITTLE BIT COMPARED TO THIS ONE AND IS DELAYED NUCLEUSING, WHEREAS THIS ONE ISN'T. WE CAN SEE DIFFERENT ACCUMULATIONS OF ACTIN AND PHENOTYPES ASSOCIATED WITH STAYING OPEN AND DISASSEMBLING THE ACTIN RING. SO THEY ARE NOT DOING EXACTLY THE SAME THING. WHAT THAT HAS LED US TO IS A MODEL FOR HOW THIS MIGHT WORK IN SINGLE CELL REPAIR. SO WHAT I'M SHOWING YOU HERE, THIS WOULD BE THE EMBRYO, THIS WOULD BE THAT MEMBRANE THAT SURROUNDS THE EMBRYO AND THIS IS THE EXTRA CELLULAR SPACE. WHAT HAPPENS WHEN WOUNDING IS WE ARE BREACHING THE EMBRYO CORTEX AND YOU GET AN INFLUX OF CALCIUM, THIS CAUSES MANY THINGS TO HAPPEN BUT ONE OF THE THINGS THAT IT INDUCES EXOTIETOSIS. ONE OF THE THINGS THAT ICE EXOCYTOSE IS INSULIN PEPTIDE, THE INSULIN IN FLIES THIS CAN THEN BIND TO THE INSULIN RECEPTOR, AND WHAT IT'S GOING TO DO IS BINDS TO DIFFERENT AFFINITIES FROM WITHIN THE RECEPTOR AS IT'S DIFFERENT DISTANCES FROM THE WOUND. AND IN DOING SO WILL SET OFF A TRANSCRIPTIONAL OR SORRY A CASCADE WHICH WILL TURN ON ONE OF THESE DOWNSTREAM FACTORS AND THOUGH DIFFERENT DEPENDING ON DISTANCE OF UNDERSTAND LYNN RECEPTOR FROM THE WOUND AND AFFINITY OF INSULIN FOR THAT SIGNALING PROCESS. THAT TURNS OUT TO BE REALLY IMPORTANT. AS I SHOWED YOU, RIGHT AROUND THE WOUND YOU HAVE ACTIN RING AND THEN YOU HAVE A LOWER LEVEL OF ACTIN BUT STILL ACCUMULATION IN ACTIN HALO, THOSE ARE DIFFERENT ARCHITECTURES OF ACTIN, THEY REQUIRE DIFFERENT WAYS WHICH THE ACTIN ASSEMBLIES ARE PUT TOGETHER. AND THESE DIFFERENT FACTORS ARE KNOWN TO ASSEMBLE ACTIN IN DIFFERENT WAYS. ONE OF THE ADVANTAGES THEN TO SIGNALING OUT AND SIGNALING BACK IN EVEN IF IT TAKES LONGER IS TO BE ABLE TO TURN ON PRECISE GENES THAT WOULD GIVE YOU DIFFERENT ASSEMBLIES AND DIFFERENT REGIONS FROM THE WOUND AND ALLOW YOU TO CLOSE THE WOUND. SO WE ARE DOING MORE WORK ON THIS TO TRY TO UNDERSTAND EXACTLY HOW THOSE ARE WORKING TOGETHER. SO IF I JUST SUMMARIZE WHAT THE MICROARRAY SCREEN TOLD US, IT TOLD US THE INITIATION OF THE REPAIR RESPONSE AT LEAST IN DROSOPHILA REQUIRES TRANSLATION. RATHER THAN TRANSCRIPTION, THOUGH YOU DO NEED TRANSCRIPTION FOR THE LATER EVENTS OF WOUND REPAIR. IT ALSO GAVE US 253 GENES INVOLVED IN CELL REPAIR BETWEEN THE UP AND DOWN REGULATED ONES. AND WE HAVE ONLY LOOKED AT A HANDFUL OF THOSE, SO FAR, 15 UP AND DOWN REGULATED ONES BUT EVEN FROM THAT SMALL SUBSET WE HAVE ALREADY IDENTIFIED INSULIN SIGNALING BEING REQUIRED TO REGULATE ACTIN DYNAMICS IN THIS PROCESS SO WE NOW HAVE NUMBER OF OTHERS TO LOOK AT THAT WILL HELP US BUILD THIS MOLECULAR PATHWAY WE ARE TRYING TO DO. SO I WILL BRIEFLY TELL YOU THE OTHER SCREEN WE DID. THIS IS A GFP TAGGED SCREEN. WHAT WE ARE DOING IS TO TAG OR TAKING PROTEINS THAT ALREADY TAGGED BY THE COMMUNITY, AND ASKING IF THEY ARE RECRUITED OR DEPLETED UPON WOUNDING. WHAT I'M SHOWING YOU HERE ARE SOME EXAMPLES SO WE HAD 1400 LINES GENERATED IN THE DROSOPHILA COMMUNITY WHERE GFP TAGGED PARTICULAR PROTEIN. FROM THIS 1400 WE IDENTIFIED 141 LINES RECRUITED TO WOUNDS. SO WHAT I'M SHOWING YOU HERE, THIS IS THE ACTIN REPORTER SHOWS YOU WHAT THE WOUNDS LOOK LIKE SO THE WOUNDS ARE SIMILAR. AND THE GREEN IS WHAT WE CALL THE FLY TRAP, SO THIS IS THE GFP THAT IS FUSED TO THE PROTEIN OF WHATEVER 1400 PROTEINS HERE. YOU CAN SEE THAT FOR EXAMPLE THIS ONE AND THIS ONE ARE IN THE PLUG REGION. SO IF YOU LOOK AT THE ACTIN RING THEY FILL THE SPACE IN BETWEEN WHERE THE MEMBRANE PLUG WOULD BE. IN THIS CASE IT IS DEPLETED FROM THE AREA WHERE THE RING IS. YOU CAN SEE ONES LIKE THIS WHERE IT FORMS A RING BUT THIS IS ACTUALLY COMPLETELY INSIDE THE ACTIN RING. YOU CAN HAVE ONES THAT FORM A RING OVERLAPPING WITH THE ACTIN RING, THIS FORMS A RING IN HALO AND THIS IS JUST HALO. WE GET A NUMBER OF DIFFERENT PATTERNS AND WE CAN LOOK AT THESE TO SEE LIKE WHAT TYPE OF STRUCTURES THESE THINGS ARE AFFECTING. WE ALSO GET IN ADDITION TO SPATIAL ASPECT A TEMPORAL ONE SO HERE I SHOW YOU TWO EXAMPLES WHERE YOU HAVE EMBRYO WHEN WOUNDED WILL ACCUMULATE IS GFP ASSOCIATED PROTEIN EARLY AND BE GONE LATER OR DOESN'T REALLY COME UP TO THE LATER STAGES. SO BY LOOKING AT THESE GENE AND WHERE THEY AFFECT THE DIFFERENT PARTS OF THE WOUNDING PATTERN THEY AFFECT AND THE TIMING WE CAN ALSO USE THIS TO ESTABLISH A PATHWAY, WHAT IS FIRST AND SECOND AND WHAT KIND OF FEATURE THEY ARE HAVING. SO FROM THE FLY TRAP SCREEN THEN WE HAVE IDENTIFIED 141 PROTEINS AND IT IS GIVING US A PRETTY GOOD VIEW OF WHAT'S HAPPENING AT DIFFERENT TIMES IN THE REPAIR PROCESS. SO THIS IS OUR GLOBAL APPROACH USING FLIES AS A GENETIC SYSTEM TO BE ABLE GET COLLECTIONS OF PROTEINS AND LOOK AT THE GENES AS A COLLECTION TO UNDERSTAND THE GLOBAL UNDERPINNINGS OF THE MOLECULAR PATHWAY. ANOTHER THING WE CAN DO WITH THIS MODEL IS LOOK AT SPECIFIC THINGS. WE CAN LOOK AT SPECIFIC EVENTS OR MOLECULES SO IF WE WANT TO LOOK AT START SIGNAL STOP SIGNAL OR REMODELING OF THE CORTEX WE CAN LOOK AT THOSE DIFFERENT EVENTS AND ASK WHAT IS REQUIRED BUT ALSO LOOK AT DIFFERENT FAMILIES OF PROTEINS AND ASK WHAT DO ROGUE GTP ACES DO OR ANEXINS DO OR ANY DIFFERENT SET OF MOLECULES WE MIGHT BE INTEREST IN LOOKING AMOUNT. I WILL GIVE YOU A COUPLE OF EXAMPLES HOW WE ARE ABLE TO DO THAT. AND THE KIND OF THINGS WE ARE LEARNING. I WILL START WITH ROGUE GTPASES. CLASSIC MOLECULES MOAN KNOWN TO AFFECT THE ACTIN CYTOSKELETON, THIS SHOWS YOU ROGUE THEY CYCLE BETWEEN GTP BOUND STATE AND IN GTP BOUND STATE BIND TO EFFECTOR PROTEINS AND CARRY OUT A NUMBER OF EVENTS ALL UNDERLYING THEME IS THEY AFFECT CYTOSKELETAL REGULATION. SO WORK DONE IN XENUPISOOCYTES IN WOUND REPAIR SHOWN THE THREE ROGUE FAMILY GTPASES WHICH IS RHO RAP AND CD 42 FORM CONCENTRATION BRAINS AROUND THE WOUND SO THIS SHOWS YOU ACTIN AND RHO, CDC 42 AND RHO SO RHO IS IN A RING AT THE EDGE OF THE WOUND. YOU CAN SEE A CONCENTRIC RING, CDC 42 ON THE OUTSIDE. SO THEY HADN'T DONE RAC SO THEY DON'T KNOW WHAT IT IS DOING IN THIS PARTICULAR CASE BUT WHAT THEY SHOW IS THESE PROTEINS ARE FORMING THESE DIFFERENT REGIONS AND THEY ARE AFFECTING ACTIN POLYMERIZATION AND DEPOLYMERIZATION. SO IN THIS CASE WHAT THEY ARE LOOKING AT IS THESE REPORTERS ARE SHOWING THEM ARE ACTIVATED RHO AND ACTIVATED CDC 42. WHAT RHO IS DOING IS CAUSING ACTIN TO BE POLYMERIZED SO IF THIS IS AN ACTIN RING, THE RHO IS ON THE INSIDE CAUSING MORE ACTIN TO BE POLYMERIZED. CDC 42 ON THE OUTSIDE DOES THE OPPOSITE, IT ASKS FOR ACTIN TO BE DEPOLYMERIZED OR DISASSEMBLED. SO THE WAY THAT THIS RING WILL GET SMALLER SO THE WAY THAT IT'S GOING TO TRANSLOCATE INWARD IS BY CONTINUOUSLY POLYMERIZING ACTIN ON THE INSIDE AND DEPOLYMERIZING ON THE OUTSIDE. AS OPPOSED TO CONTRACTING IN A CONTRACT TILE FORM THEY KNOW THAT BECAUSE IF THEY INHIBIT MYSINUOUSING A CHEMICAL INHIBITOR Y 27632 OR BELOW STATIN, THIS PROCESS WILL PROCEED SO YOU DON'T NEED MYSIN FOR THIS TO HAPPEN BUT WHAT THEY CALLED THIS IS ACTIN TREADMILLING. SO YOU HAVE ACTIN THAT IS FORMING HERE AND WHEN IT DISASSEMBLES MOVE TO INSIDE AND FORM NEW ACTIN. THAT WILL ALLOW THE CYTOSKELETAL TO CLOSE AND WILL PULL THE MEMBRANE WITH IT. SO WE WANTED TO SEE IF THE DROSOPHILA SYSTEM WAS DOING EXACTLY THE SAME THING OR NOT, SO WE LOOK TO SEE IF RHO RAC AND CDC 42 ARE CALLED TO WOUNDS AND TURNS OUT THEY ALL ARE. IF YOU CO-LOCALIZE WITH ACTIN YOU CAN SEE RHO IS INSIDE THE RING, WHEREAS RAC IS OVERLAPPING THE RING AND CDC 42 OVERLAPS THE RING AS WELL AND GIVES MORE A HALO REGION. WHAT I'M SHOWING YOU HERE IS A GRAPHICAL REPRESENTATION OF WHAT'S HAPPENING HERE SO HERE IS THE WOUND AND HERE IS THE ACTIN RING AND HALO, RHO IS INSIDE THE WOUND. AND YOU HAVE A HIGHER AMOUNT OF CDC 42 AND RAC IN THE WOUND. THIS TAPERS OFF WHEREAS THIS STAYS MORE CONSISTENT INTO THE HALO REGION. THIS WAS INTERESTING AND WHAT WE WANTED TO KNOW IS HOW IT WAS BEING -- WHAT IT WAS DOING IN TERMS OF ACTIN REGULATION IF IT WAS ACTING THE SAME AS IN XENOPUS OR NOT. ONE MAJOR DIFFERENCE WE FOUND IS THAT IF WE WERE TO TREAT DROSOPHILA EMBRYOS WITH MYSIN INHIBITORS SO Y 2763, WOUND REPAIR STOPS DEEDS IN ITS TRACKS SO IN OUR CASE THE ACTIN RING IS CONTRACTILE. THAT WAS VERY INTERESTING IN TERMS OF RHO FAMILY GTPASES SO THEM IN THE XENOPUS SYSTEM ARE USING THE SAME DOWNSTREAM MOLECULES TO CARRY OUT THE SAME KIND OF ROLE, CLOSING A WOUND, BUT THEY ARE DOING IT THROUGH A DIFFERENT MECHANISM THAN DROSOPHILA ARE DOING SO ONE CASE YOU NEED CONTRACTION, IN THE OTHER CASE YOU NEED TREADMILLING. WE USED THIS SYSTEM NOW TO LOOK DOWNSTREAM AT EFFECTORS AND ASK WHAT TYPES OF THINGS ARE DOING AND WE CAN ASSIGN DIFFERENT DOWNSTREAM EFFECTORS TO DIFFERENT PARTS OF THE WOUND REPAIR PATHWAY. OR IN SOME CASES WE KNOW DIFFERENT PARTS OF THE PATHWAY BUT WE DON'T KNOW WHAT THE DOWNSTREAM EFFECTORS ARE YET BUT WE KNOW THEY ARE NOT THESE. WE ARE CONTINUING TO DO THIS. BUT WE ALSO WANTED TO START LOOKING UPSTREAM AND ASK HOW DO WE REGULATE THESE GTPASES. BECAUSE IT IS A LITTLE BIT INTERESTING BECAUSE IT IS THOUGHT THE INITIAL SIGNALS COMING IN ARE UNIFORM. WHAT WE ARE SEEING HERE ARE CONCENTRIC RINGS OF GTPASES. SO WE NOTE DIRECTLY UPSEEM OF GTPASES ARE RHO GAPS SO WE THOUGHT WE WOULD LOOK AT THESE AND SEE IF THEY WERE RESPONSIBLE FOR GIVING YOU THIS BREAK IN ASYMMETRY. SO DROSOPHILA HAS A NUMBER OF RHO GEFs MORE THAN 20 BUT ONLY THREE ARE CALLED TO WOUNDS, THAT'S THE THREE SHOWN HERE. SO RHO GEF 2, 3 AND PEBL. YOU CAN SO SEE THE PATTERNS ARE DIFFERENT, RHOGEF 2 IS A RING, PEBL IS A HALO AND RHOGEF 3 HAS BOTH. IF I PUT WITH ACTIN AS WELL YOU CAN SEE THIS IS INSIDE THE RING OVERLAPPING THE RING AND THIS IS OUTSIDE THE RING. SO IF WE DO OUR GRAPHICAL MODEL THE GEFs WOULD LOOK LIKE THIS COMPARED TO GTPASES I SHOW YOU EARLIER. SO E WANTED TO GET -- THEY ARE PATTERNED SO WE ALREADY SEE A PATTERN IN RHOGEFS WE SEE IN GTPASES AND WANTED TO KNOW THE RELATIONSHIP BETWEEN THE GEFS HERE AND THE GTPASES. ONE WAY WE CAN DO THAT IS TO KNOCK OUT THE GTPASES OR GAPS IN GTPASES ARE RECRUITED. I WILL SHOW AN EXAMPLE OF THAT HERE. THIS IS RHO 1 RECRUITMENT IN WILD TYPE EMBRYO. AND IF YOU REMOVE GEF 2 WHAT YOU FIND IS THAT RHO IS NO LONGER RECRUITED. BUT IF YOU REMOVE THE OTHER GEF PEBL OR RHOGEF 3 YOU SEE RECRUITMENT OF RHO. REMOVEK THESE AFFECTS THE ACTIN CYTOSKELETON BUT THEY ARE BEING RECRUITED. SO THAT MEANS THE RHO GEF 2 IS RESPONSIBLE FOR RECRUITING RHO. WE CAN DO THE SAME THING BY REMOVING THESE SAME GEFS AND LOOK AT REF AND CDC 42. WE FIND THAT RHOGEF 2 LOOKS LIKE NEEDED TO RECRUIT RHO. PEBP NEEDED FOR CDC 32 AND RHOGEF 3 IS NEED FORD RAC. SO SOMETHING THAT LOOKS LIKE THIS SO MORER OR LESS A ONE O TO ONE CORRESPONDENCE IN THIS CASE IN TERMS OF THE RHOGEF AND RHOGTPASE, THAT MADE US REFINE OUR QUESTION BECAUSE WE WANTED TO KNOW HOW THESE WERE PATTERNED RELATIVE TO EACH OTHER, HOW YOU GOT CONCENTRIC RINGS, TURNS OUTS THESE ARE RESPONDING TO THE GEFs THAT ARE REGULATING THEM. SO WE KNOW GO UP A STEP AND SAY OKAY IF THIS IS THE PRE-PATTERN THAT SET, HOW DO WE GET THIS TO COME OUT IN A PATTERN? THE OTHER THING I WILL POINT OUT, THIS IS HAPPENING ABOUT 30 OR 45 SECONDS AFTER WE WOUND. WHEREAS THIS IS HAPPENING TEN SECONDS TO 15 SECONDS AFTER WE WOUND. SO WHATEVER IS WORKING UPSTREAM HERE HAS TO BE DOING IT IN LESS THAN 10 SECONDS. SO THIS IS WHERE WE TOOK ADVANTAGE OF THE SCREENS THAT WE DID AND WE WENT BACK TO OUR FLY TRAP SCREEN AND SAID OKAY, OUT OF THE GENES WE IDENTIFIED HOW MANY OF THEM ARE RECRUITED IN LESS THAN TEN SECONDS? THERE ARE COUPLE OF THEM THAT ARE. THIS SHOWS YOU TWO. SO THERE'S ONE THAT'S CALLED THE NEXIN B 9 AT THREE SECONDS AND THIS IS ANOTHER ONE CALLED FLY TRAP 74, RECRUITED IN A WEIRD PATTERN BUT AT THREE SECONDS. SO WE DECIDED TO LOOK AT ANXB 9 THE REASON FOR THIS IS ANXINS ARE FAMILY OF PROTEINS TO STABILIZE ACTIN. THEY RESPOND TO CALCIUM WHICH WE THINK IS INWARD FLOW AS SOON AS YOU RUPTURE A WOUND. OR CORTEX. AND IN OTHER SYSTEMS OF WOUND REPAIR TISSUE CULTURE CELLS, ANXINS ARE IMPLY CATTED IN LATER STEPS BUT IN THE REPAIR PROCESS AT LEAST. SO WHAT WE WOULD HYPOTHESIZE THEN IS THAT IF ANNEXIN B 9 WAS REQUIRED TO REGULATE RHOGEFO THEN RECRUITMENT TO WOUND SHOULD BE DISRUPTED IN ANNEXINB 9 MUTANT SAME AS RHOGTPASES ARE AFFECTED BY GEFs. SO WE LOOKED IN THAT CASE AND SAID IN WILD TYPE RHOGEF 2 IS RECRUITED TO WOUND AND ANNEXIN B 9 MUTANTS IS NOT. THAT SAYS WE NEED ANNEXINB 9 TO GET RHOGEF 2. IF YOU LOOK AT GEF 3 AND PBL THEY ARE RECRUITED TO WOUNDS STILL SO YOU DON'T NEED ANNEXINB 9 FOR THOSE. IF WE LOOK, IT WOULD SAY ANNEXINB 9 IS UPSTREAM OF RHOGEF 2 WHICH IS UPSTREAM OF RHO 1. SO THE NEXT QUESTION WE WANTED TO ADDRESS THEN IS SINCE ANNEXINS ARE KNOWN TO REGULATE ACTIN STABILIZATION IS IT SOME WAY AFFECTING ACTIN STABILIZATION IN THAT MECHANISM OF ACTION IN THIS PATHWAY? WHAT WE DID WAS TO DISRUPT ACTIN BY INJECTING TRUNCULIN B THAT DE POLYMERIZES ACTIN THAT EXISTS. THIS SHOWS YOU THAT SO IF YOU INJECT, YOU CAN SEE ARC TIN IS DISRUPT AND RHOGEF 2 IS NOT RECRUITED TO A WOUNDS ANY MORE. ANXB 9 THOUGH IS. SO IF YOU DO INJECTIONS YOU CAN SEE ACTIN IS DISRUPTED BUT ANXB 9 HAS NO PROBLEM GETTING TO A WOUND SO IT DOESN'T NEED THAT ACTIN IN ORDER TO GET TO A WOUND. THAT SUGGESTS THAT ANXB 9 COULD BE DOING SOMETHING TO ACTIN WHICH THEN IS REQUIRED FOR RHOGEF 2 TO SHOW UP AND ACCUMULATE AT A WOUND. SO THE WAY WE WANTED TO TEST THIS WAS TO SAY IF WE WERE TO TAKE AN ANXNB 9 MUTANT WHERE RHOGEF 2 IS NOT RECRUIT AND STABILIZE ACTIN BY SOME MEANS OTHER THAN ANXB 9 WE MIGHT BE ABLE TO BYPASS THAT REQUIREMENT HERE. AND GO ON WITH THE PATHWAY. SO WE INJECTED FILOITIN IT WILL STABILIZE ACTIN IN WHATEVER FORM IT IS AND WE CAN ASK WHAT HAPPENS SO WE ARE TAKING THIS EXACT MUTANT, ANXB 9 RHOGEF 2 IS NOT ACCUMULATING AND INJECTING PHALLOIDIN. WHAT YOU SEE IS RHOGEF 2 CAN BE RECRUITED. SO IT REALLY SUGGESTS THAT WHAT ANXB 9 IS DOING IS STABILIZING ACTIN. SO THIS JUST SUMMARIZES WHAT I'M TELLING YOU, ANXB 9 STABILIZE ACTIN THAT WILL ALLOW RHOGEF 2 TO BE RECRUITED AND HRO 1 WILL FOLLOW THAT PATTERN. SO THAT'S GREAT IN TERMS OF THIS PATHWAY. IT DOESN'T REALLY TELL US WHAT'S GOING ON OVER HERE. BUT WHAT I DIDN'T REALLY TELL YOU IN THE BEGINNING IS THAT WHILE ANXB 9 WAS PART OF THE FLY TRAP SCREEN, THERE ARE ACTUALLY THREE ANXNs IN FLIES. THIS IS JUST SAYING THERE ARE TWO DIFFERENT ACTIN. THERE ARE THREE ANXs IN FLIES SO ANNEXIN B 10 AND 11. THERE ARE NO GFB CONSTRUCTS OR INAF CONSTRUCTS WHEN WE STARTED THOSE SO WE GENERATED THOSE AND ASK IF THESE WERE HAVING AFFECT ON OTHER TWO RHO GEFS SO THE FIRST THING WE DID IS LOOK AT EXPRESSION. SO THIS IS ANXB 9 I SHOWED IN RESPONSE TO WOUNDING, THIS IS B 10. THIS IS B 11. WHAT YOU PROBABLY ARE ALREADY REALIZING IS WE HAVE THE SAME PATTERN CROPPING UP AGAIN. THESE ARE PATTERNS AND THEY ARE FORMING CONCENTRIC RINGS. SO WE ARE NOT CATCHING A BREAK YET IN TERMS OF WHAT'S GOING FROM THE ASYMMETRIC OR THE SYMMETRIC INITIAL RESPONSE TO ASYMMETRY WE ARE SEEING HERE. BUT THESE ARE PATTERNING IN THAT REALLY EARLY TIME POINT. SO IF WE COME BACK TO THIS THEN WITH KNOW THE GEFS ARE A PATTERN AND THE ANNEXINS ARE PATTERNED SO THE GEFs ARE RESPONDING TO THE PATTERN BEING SET BY THE ANNEXINS. THAT MADE US GO BACK TO WHAT WHICH KNOW ABOUT THE BEGINNING OF THIS PATHWAY WHERE THERE IS A CALCIUM INFLUX AND WE WANTED TO GO BACK AND ASK, IS THAT REALLY A SYMMETRIC SIGNAL OR IS IT ASYMMETRIC FROM THE BEGINNING? SO WE USED A G CAMP REPORTER, THIS IS AN UNWOUNDED AND WOUNDED EMBRYO, THIS SHOWS YOU WHERE THE WOUND IS, YOU CAN SEE THAT THE RESPONSE IS UNIFORM OVER THIS WHOLE AREA. SO THE INITIAL RESPONSE IS UNIFORM AND WITHIN THREE SECONDS YOU ARE SEEING A PATTERN RESPONSE. SO THAT MEANS BESTILL HAVE ANOTHER STEP HERE WE DON'T KNOW WHAT IT IS. BUT LESS SECONDS TO FIGURE IT OUT IN. SO WE ARE GOING BACK TO OUR SCREEN WITH FLY TRAPS AND TRYING TO LOOK AT SOME OF THE OTHER ONES THAT ARE RECRUITED TO WOUND IN LESS THAN TEN SECONDS AND ASK IF WE CAN BIND WHAT IS RESPONDING TO CALCIUM THAT THEN PATTERNS THESE IN A SHORT TIME. ONE OF THE IDEAS WE HAD IS THIS IS SOME WAY AFFECTING LIPIDS. BECAUSE ALL THESE ARE MEMBRANE BOUND SO THEY ARE SORTED TO MEMBRANES AND HAVE PREFERENCES FOR CERTAIN LIPIDS. WE THOUGHT PERHAPS WHAT CALCIUM INFLUX COULD DO IS ALTER THE LIPID COMPOSITION RIGHT AROUND THE WOUND AND THAT WOULD AUTOMATICALLY CAUSE THESE THINGS TO BE SEGREGATED TO PARTICULAR REGIONS. THAT TURNS OUT NOT TO BE THE CASE. THERE ARE SOME REGIONS THAT WE SEE WHERE THERE'S ACCUMULATION OF PARTICULAR LIPIDS BUT IT DOESN'T ACCOUNT FOR THIS TYPE OF ASYMMETRIC PATTERN. WE ARE STILL STUCK WITH WHAT EXACTLY IS THIS QUESTION MARK AND WHAT IS CAUSING IT. ONE THING I WILL SHOW YOU, AS WE ARE DOING THESE STUDIES WE ENDS UP FINDING OUT MANY OTHER THINGS AS WELL. THIS IS JUST TO SHOW YOU WHAT HAPPENS WHEN WE WERE IMAGING, SO THIS EMBRYO. I WILL REMIND YOU WE HAVE AN EMBRYO IN CASED IN A MEMBRANE, WE ARE GOING TO COVER SLIP AND IMAGING FROM BELOW. IF IT IS BEHAVING PROPERLY, THIS IS WHAT YOU SEE WITH AN ACTIN REPORTER. HOWEVER, SOMETIMES WHEN YOU DO THIS, THIS IS WHAT HAPPENS. SO BECAUSE THE EMBRYO IS NOT TETHERED, INSIDE THE MEMBRANE IT CAN MOVE AROUND THAT IS WHAT YOU ARE SEEING HERE, IT MOVES AROUND. AND FROM A WOUND REPAIR ANALYSIS POINT OF VIEW THIS IS REALLY A PAIN. BECAUSE THE THINGS ARE NOT IN THE RIGHT PLACE YOU CAN'T GET THE MOVIES I SHOWED YOU BECAUSE THEY ARE MOVING ALL OVER THE PLACE AND IT IS HARD TO DO ANY MEASUREMENTS. T BUT SOMETIMES YOU GET LUCKY, SO THIS EMBRYO HAPPENED TO BE DOUBLE LABELED SO NOT ONLY DID IT HAVE ACTIN BUT ALSO HAD ANNEXIN B 9 IN GREEN. WHETHERREN WE IMAGE THIS, THIS IS WHAT WE SAW. WHEN WE IMAGE THIS, THIS IS WHAT WE SAW. IF WE DRAW A LINE THROUGH IT AND LOOK UNDERNEATH WHAT IS HAPPENING THE RED IS THE ACTIN CYTOSKELETON, THE GREEN IS THE ANNEXIN. YOU CAN'T SEE MEMBRANE BUT MEMBRANE WOULD BE ABOVE THE RED AND THE WHITE DASH LINE ONE WHERE THE MEMBRANE IS. WHAT YOU CAN SEE ANNEXIN B 9 IS GOING TO PLUG REGION BUT REACHING OUT AND OVER AND TOUCHING THE PLASMA MEMBRANE. AND WE CAN SEE THAT ACTUALLY IN THE SCANNING EMs, SO THIS IS AN EMBRYO. THERE IS A WOUND HERE. AND IF YOU ENLARGE IT YOU CAN SEE THE PLUG AND REGION AROUND IT. AND TO ENLARGE MORE YOU CAN SEE HERE IS A PLUG AND YOU CAN SEE THAT IT IS REACHING OUT AND TOUCHING THE MEMBRANE AROUND IT. SO IN TERMS OF LOOKING AT THE EARLIER STAGES WHEN ASKING HOW DO YOU PLUG A WOUND HOW DO THE BEST SCHOOLS COME TO SURFACE, SOME OF THESE REAGENTS ARE ALLOWING US TO LOOK AT THOSE EVENTS AS WELL. SO FROM THIS APPROACH LOOKING AT SPECIFIC THINGS, THESE ARE SPECIFIC EVENTS THAT ARE HAPPENING, SPECIFIC MOLECULES, WHAT WE FOUND IS THAT IF PATHWAY WE KNOW EXISTS CALCIUM GOES AT LEAST THROUGH ANNEXIN RHOGEF AND RHOGTPASES SO THESE ARE RESPONDING TO THESE RESPONDING TO THESE SO HOPEFULLY WE KNOW WHAT THEY ARE RESPONDING TO SO FROM A SYMMETRIC SIGNAL TO ASYMMETRIC ONE. WE KNOW THAT THE FUNCTIONS OF RHOGTPASES ARE TO AFFECT ACTIN RING FORMATION STABILITY CONTRACTION AND GENERAL ORGANIZATION OF ACTIN. AND ONE OF THE THINGS REALLY NICE ABOUT THIS IS THAT WE KNOW THERE'S A DIFFERENCE IN THE FLY WOUND MODEL FROM THAT DESCRIBED IN XENOPUS. I OLD YOU XENOPUS THEY TREADMILL, IN THE FLY CASE YOU NEED CONTRACTION AND MYSIN PRESENT. THIS IS NICE BECAUSE IF IN THIS CASE YOU HAVE TWO DIFFERENT ORGANISMS USING THE SAME GROUP OF PROTEINS, TO CONTROL THE SAME PROCESS WHICH IS WOUNDS CLOSURE BUT BY VERY DIFFERENT MECHANISMS. WHAT WE WANT TO KNOW IS HOW THEY ARE DOING THAT. BECAUSE BOTH OF THESE SYSTEMS ARE PRESENT IN BOTH ORGANISMS YET ONE CHOOSES ONE AND ONE CHOOSES ANOTHER. THE REASON THAT WOULD BE IMPORTANT IS IN DISEASE STATE WHERE YOU LIKE SAY CHRONIC WOUND ASSOCIATED WITH DIABETIC IF YOU KNOCK OUT ONE PATHWAY, IT CAN'T FUNCTION, IF U YOU GETS THE OTHER PATHWAY TO TURN ON SINCE ALL THE COMPONENTS YOU NEED ARE ALREADY THERE, THEN YOU MIGHT BE ABLE TO GET WOUND REPAIR TO GO FORWARD INSTEAD OF HAVING CHRONIC WOUNDS. I THINK WE ARE GETTING SOME TRACTION IN THAT BECAUSE HERE IS A CASE WHERE WE HAVE BEEN ABLE TO CAUSE TREADMILLING TO HAPPEN IN A WOUND. SO WE MIGHT BE ABLE TO TRIGGER TREADMILLING INSTEAD OF CONTRACTION WHEN THERE'S NO MYSIN OR THAT PATHWAY AVAILABLE TO CLOSE A WOUND. SO GOING BACK TO WHAT WE KNOW ABOUT CELL WOUND REPAIR IN ALL ORGANISMS WE HAVE CELLULAR RESPONSES WE KNOW HAPPEN, STARTING FROM THE BREACH OF THE CORTEX IN THE PLUGGING THE ACTIN MYSIN RING FORMATION CLOSURE AND REMODELING. AND WHAT THE FLY SYSTEM IS LETTING US DO IS HAVE A REALLY NICE TOOLBOX TO STUDY FROM A GLOBAL VIEWPOINT WHAT'S HAPPENING AND HOPEFULLY BE ABLE TO GIVE US THE MOLECULAR MECHANISM THAT UNDERLINE WHAT WE SEE HERE PHYSIOLOGICALLY AS WELL AS TO ASK WHAT THE ROLE OF PARTICULAR FAMILIES OF PROTEINS LIKE RHOGTPASES OR ANNEXINS ARE IN THIS PROCESS SO WE HAVE OUR WORK CUT OUT FOR US AND CONTINUING TO DO THESE KINDS OF THINGS. IN THIS CASE WHEN I SAY WE, I JUST WANTS TO ACKNOWLEDGE ALL OF THE PEOPLE IN THE LAB THAT HAVE BEEN WORKING ON DIFFERENT ASPECTS OF WOUND HEALING, WE HAVE ALSO HAD A LOT OF HELP FROM MORFO METRICS ANALYSIS, IMAGING QUANTIFYING OR IMAGE ANALYSIS AND BIOINFORMATICS. I ALSO NEED TO REALLY ACKNOWLEDGE ALL OF THE DIFFERENT COMMUNITY RESOURCES THAT ARE FUNDED BY NIH THAT MAKE THIS WORK POSSIBLE FOR LABS OF ANY KIND AND OF COURSE OUR FUNDING FROM NIH. I'M HAPPY TO TAKE ANY QUESTIONS. THANK YOU VERY MUCH, SUSAN. THAT WAS A TRULY LOVELY TALK. THANK YOU. I HAVE ALREADY A NUMBER OF QUESTIONS MT. CHAT BUT FIRST LET ME REPEAT THE CME CODE FOR TODAY, 37919. YOUR FIRST QUESTION COMES REGARDING THE FIRST PART OF YOUR TALK. JAMIE WOULD LIKE TO LEARN MORE ABOUT THE INSULIN PATHWAY. AND SHE SAYS WE USUALLY THINK OF INSULIN AND INSULIN LIKE PEPTIDES AS LONG RANGE SYSTEMIC SIGNALING MOLECULES. ARE THERE CONDITIONS WITHIN THE EXTRA CELLULAR SPACE OF THE EMBRYO THAT WOULD KEEP THE ILP HIGHLY LOCAL TO WOUND? >> IT'S KNOWN THAT EXTRA CELLULAR SPACE IS FILLED WITH EXTRA CELLULAR MATRIX, IF YOU INJECT A DYE INTO THAT SPACE, IT TAKES ABOUT TWO MINUTES FOR IT TO COMPLETELY GO AROUND THE EMBRYO AND BECOME EVEN. SO THERE IS A BARRIER, IT DOESN'T GO INSTANTLY. IN TERMS OF DIFFUSION BUT IT IS NOT GOING TO BE HELD BACK VERY LONG. FOR THE WOUNDING, IF YOU ARE WOUNDING AND HAVING A RESPONSE RIGHT AWAY, IF THE PEPTIDE GOES OUT, PROBABLY THE FIRST RECEPTORS THAT IT COMES TO WILL BE THE ONES THAT IT BINDS TO DEPENDING ON AFFINITY SO TAKE LONGER FOR IT TO DIFFUSE FURTHER TO GET FURTHER ONES. SO I THINK THERE IS SOME RESTRICTION BECAUSE OF THE EXTRA CELLULAR MATRIX BUT EVENTUALLY SHOULD GO ALL THE WAY AROUND THE EMBRYO. >> SO STAY WITH INSULIN PATHWAY, DOES REVERSAL OF INSULIN RESISTANCE IMPROVE WOUND REPAIR? >> THERE ARE A LOT OF STUDIES DONE WITH INSULIN WHERE THEY TOOK MAMMALIAN CELLS AND PUT THEM IN INSULIN AND LOOKED TO SEE WHAT HAPPENED. BUT I THINK THE GENERAL THOUGHT WAS THAT YOU WERE SOMEHOW MAKING CELLS FRAGILE AND IT WASN'T JUST -- IT WASN'T INSULIN SIGNALING PER SE BUT THEY WERE FRAGILE SO THE KIND OF ANALYSIS THEY DID WEREN'T REALLY LOOKING AT INSULIN BUT I THINK WE CAN GO BACK AND DO THAT NOW. BECAUSE NOW I THINK AT LEAST IN THE FLY SYSTEM IT IS CLEAR INSULIN SIGNALING REQUIRED NOT JUST A SECONDARY EFFECT. BUT NOBODY HAS DONE THAT YET; WOULD BE INTERESTING TO FINDS OUT. >> ROLAND OWENS ASKING HAVE YOU LOOKED AT WOUND REPAIR IN THE PRESENCE OF BACTERIA? >> NO MY FLYER WAS REALLY CLEAN. NOT INTENTIONALLY. IN THE EARLY EMBRYO YOU DON'T HAVE IMMUNE RESPONSE YET SO IT WOULDN'T RECOGNIZE THE BACTERIA AS BEING FOREIGN, YOU WOULD HAVE TO WAIT UNTIL LATER STAGE WHEN IT HAS INNATE IMMUNE SYSTEM AND RECOGNIZE THE BACTERIA. AS BEING SOMETHING THAT IS NOT GOOD SO NOT SURE IT WOULD WORK IN OUR SYSTEM FOR THAT REASON BUT IT IS A GOOD QUESTION. >> WE ALSO HAVE A CROSS SPECIES QUESTIONS SUCH AS COMING FROM ANONYMOUS, IS IT KNOWN WHICH MECHANISM XENOPUS LIKE IS USED FOR CELLULAR WOUND HEALING IN MAMMALIAN CELLS? >> THAT APPEARS TO BE CONTEXT DEPENDENT. SO THERE ARE CASES WHERE IT LOOKS LIKE IT IS TREADMILLING AND THERE ARE CASES IT LOOKS LIKE IT IS CONTRACTILE. IT MAY BE THAT IN MATTERS IN WHAT CONTEXT YOU ARE LOOKING IN ALL CASES. WE HAVE SEEN THAT WITH A NUMBER OF DIFFERENT PARAMETERS WE HAVE LOOKED AT IN JUST THE FLY. IT MATTERS FOR EXAMPLE IF YOU WOUND WITH A NEEDLE OR WOUND WITH A LASER YOU GET A DIFFERENT RESPONSE. SO IT COULD BE THAT CONTEXT IS REALLY MAKING A BIG DIFFERENCE FOR WHAT MECHANISM YOU USE. >> THANK YOU. CHUCK IS ASKING FROM THE CHIP GENE CHIP ANALYSIS NOT YET EXAMINED IN DETAILS, DO YOU HAVE ANY OTHER PATHWAYS YOU THINK MAY BE INVOLVED IN WOUND HEALING? >> THERE ARE DEFINITELY ASSOCIATIONS, IF YOU DO BIOINFORMATICS AND SAY WHAT KIND OF HUBS DO YOU HAVE IF YOU LOOK AT THE 253 GENES WE DEFINITELY HAVE SIGNALING HUBS AND CYTOSKELETON HUBS AND -- ACTUALLY MOSTLY SIGNALING HUBS BUT THERE ARE HUBS OF DIFFERENT THINGS OR DIFFERENT TYPES OF MOLECULES THAT COME UP AND WE ASSUME THAT THERE ARE LINKED PROCESSES BASED ON THAT. WE HAVEN'T LOOKED AT ALL OF THOSE YET. INSULIN WAS THE FIRST BUT THAT'S THE KIND OF PLACE WE ARE GOING TO GO NEXT. FOR SURE. >> I HAVE A BIT OF A PUZZLE WHEN I'M THINKING ABOUT WOUND HEALING ESPECIALLY LISTENING TO YOU. YOU ARE DESCRIBING AN ARMY OF EFFECTORS COMING THROUGH TO HELP WITH THE WOUND HEALING AND I CAN SEE HOW THEY WILL LINE UP AT THE BEGINNING OF THE PROCESS BUT HOW DO THEY STOP? WHAT THEY ARE DOING? >> THAT IS A GOOD QUESTION. IN SOME CASES IT COULD BE THE HALF LIFE OF THE PROTEIN, IT GETS CALLED THERE BUT IF IT HAS A HALF LIFE OF FIVE MINUTES MAYBE IT IS GONE AND NOT REPLACED. THAT WOULD BE ONE WAY TO DO IT. YOU CAN HAVE OTHER COMPLEXES COME IN AND TAKE THEM AWAY. WE DEFINITELY HAVE TO HAVE SOME MECHANISM OF GETTING RID OF THE CALCIUM, SO WE CAN SEE THE CALCIUM FLOWS IN BUT IT HAS TO GO AWADE. SO THERE HAS TO BE A MECHANISM OF RETURNING IT TO THE PERIVENTILIN SPACE SO I ASSUME THERE ARE SOME TYPE OF PUMPS THAT CAN DO THAT. WE DON'T KNOW WHAT THOSE ARE. AND WE ARE LOOK FOR THEM. THERE HAVE TO BE A NUMBER OF DIFFERENT THINGS THAT FROM PHYSICAL REMOVAL TO JUST WEARING OUT OR MAYBE BEING SOMEHOW MODIFIED SO THEY ARE NO LONGER EFFECTIVE. >> THANK YOU. MORE QUESTIONS COMING ON THE CHAT HERE. BEN WHITE. IS ASKING YOU MENTION THAT AT LEAST SOME OF THE MOLECULES O OF CELLULAR WOUND REPAIR ARE CONSERVED BETWEEN XENOPUS AND FLIES. IS IT KNOWN WHEN THIS FORM OF WOUND REPAIR EVOLVED? IS IT EUKARYOTIC INNOVATION? >> I'M NOT AWARE OF ANY STUDIES WHERE PEOPLE HAVE LOOKED AT THAT TO KNOW, TO ASK WHAT THE MOST ANCIENT TIME IS. BUT AT LEAST IN ALL THE MODELS THAT I SHOW YOU PEOPLE LOOK AT CELL WOUND REPAIR, THE MOLECULES LOOK LIKE THEY ARE THE SAME, THEY ARE NOT ALWAYS USED THE SAME WAY AS I SHOWED YOU AN EXAMPLE TWO MECHANISMS, SAME MOLECULES ACHIEVING THE SAME GOAL IN THE LONG RUN. SO I DON'T KNOW THE ANSWER TO THAT. I DON'T KNOW THAT ANYBODY HAS DONE A STUDY TO KNOW HOW FAR BACK IT GOES. >> MORE QUESTIONS COMING. ONE IS A VERY DEER DEAR TO ME, BECK KY FROM A STAFF SCIENTIST IN MY LABORATORY. INTERESTED IN GLUTAMATE RECEPTORS. HE ASKS ARE THERE GLUTAMATE RECEPTORS FUNCTION ESPECIALLY CAUSE IMMUNE FLUX PHENOM YOU ARE DESCRIBING AT THE ONSET OF WOUND HEALING THAT ARE IMPORTANT IN HERE? >> I KNOW AT LEAST ONE CAME UP IN THE SCREEN. WE HAVE NOT LOOKED AT THAT. WE HAVE NOT LOOKED AT THAT YET. TO SEE WHAT IT DOES OR WHAT KIND OF EFFECT IT HAS. THERE ARE LOT OF GENES THAT CAME UP, BUT I KNOW AT LEAST ONE GLUTAMATE RECEPTOR IS PRESENT IN THE TOP HITS. >> I SEE. I COULDN'T HELP NOTICING EVERY TIME YOU ARE DESCRIBING A WOUND WITH THIS AMAZING COLLECTION OF GPP YOU HAVE SOMETIMES DOTS IN THE MIDDLE OF THE ONE ESPECIALLY THAT FLY TRAP 74 GRID PATTERN. THAT LOOKED TO ME LIKE A LOT OF VESICLES DEPLOYING EXOCYTOSING AT THE SAME TIME. I MIGHT BE COMPLETELY OFF AND GET WRONG IDEA IN BETWEEN ONE YOU HAVE THAT WILD VESICLE RELEASE PHENOMENA WITH PROVIDING THE MEMBRANES AND WHEN YOU SEE THIS PROTEINS COMING UP BUT IS THERE ANY CONNECTION? >> ONE IS A REAL INTERESTING THING AND ONE IS AN ARTIFACT. SO THE FLY TRAP 74 WAS A REAL PATTERN. IT COMES UP IN THOSE DOTS, NOT ON THE SURFACE, IT IS SLIGHTLY LOWER BECAUSE I SHOWED YOU A MAX PROJECTION. SOMETIMES WHEN WOUNDING WITH A LASER, BECAUSE IT WOUNDS IN A CIRCLE YOU GET -- IF YOU HIT THE MEMBRANE IT BURNS THE MEMBRANE AND YOU GET A SLIGHT GRID PATTERN. IF YOU ARE GFP GENE IS NOT INCREDIBLY BRIGHT, ONE OF THE ARTIFACTS OF IMAGING IS YOU CAN SEE THE GRID PATTERN OF THE MEMBRANE AS A VERY LIGHT PATTERN ON TOP OF THE ONE YOU ARE LOOKING AT. WHEN YOU DO A MAX PROJECTION. AND I TRIED TO REMOVE THE SLICE THAT HAS THE MEMBRANE WHEN I DO THAT BUT I CLEARLY MISSED ONE BECAUSE -- SO SOMETIMES WHEN YOU SEE THAT REALLY FAINT PATTERN IT JUST FROM THE LASER HITTING THE MEMBRANE AND NOT QUITE BEING AS LOW AS IT NEEDED TO BE. BUT THE FLY TRAP I SHOWED YOU IT WAS VERY STRONG PATTERN THAT IS A GPFP PATTERN, IT IS CONSISTENT EVERY EMBRYO WILL SHOW THAT, AND IT COMES UP IN A GRID PATTERN AND COALESCE INTO ANOTHER PATTERN. >> THOSE ARE VERY INTERESTING FINDINGS TO MOVE FORWARD IN THE FUTURE. DEFINITELY CHARMED BY THAT KIND OF A BEAUTY WHEN YOU ARE CLOSING THOSE WOUNDS. >> EXACTLY. WE GOT ALL KINDS OF PATTERNS, MOST -- I SHOWED YOU ACTIN RINGS AND HALOS WE DID GET SOME LIKE THE FLY TRAPS WHERE IT IS A VERY SPECIFIC SUBSET OF DOTS OR REGIONS THAT ARE COMING UP. SO FAR WE DON'T KNOW EVERYTHING THEY CORRESPOND TO. >> IF THERE ARE NO MORE QUESTIONS THEN I WOULD LIKE TO THANK YOU ONCE AGAIN I KNOW MY VOICE DOES NOT REPRESENT THE HUNDREDS THAT ARE ON THIS CALL THAT ENJOYED YOUR STORIES. THANK YOU VERY MUCH FOR BEING WITH US TODAY, SUSAN. AND FOR TEACHING US A BUNCH ABOUT WOUND HEALING. THANK YOU. >> THANK YOU VERY MUCH FOR THE OPPORTUNITY. TO TALK ABOUT OUR WORK.