>> SO IT'S MY PLEASURE TO INTRODUCE TODAY'S SPEAKER KELLIE TEN HAGEN. SHE WAS AN UNDERGRADUATE AT CORNELL AND HE WILL GOT HER Ph.D. AT STANFORD AND THEN WENT TO ROCHESTER WHERE SHE WAS RESEARCH ASSISTANT PROFESSOR AND GOT RECRUITED TO THE NIH, TO THE DENTAL INSTITUTE AND SHE'S BEEN INTERESTED IN O-GLYCOSYLATION FOR VIRTUALLY ALL OF HER CAREER, WASOT TENURED TRACKING MECHANISM AND RECENTLY TENURED AT THE NI& THIS IS A LOVELY TURN OUT SO PEOPLE ARE REALLY INTERESTED IN GLYCOPROTEINS AND I AM PARTICULARLY INTERESTED IN O-GLYCOSYLATION WHICH HAS BEEN MAGICAL FOR QUITE A LONG TIME AND WE'RE LOOKING FORWARD TO HEARING ABOUT ESSENTIAL ROLES IN O-GLUE MARIOUS--OGLYCOPROTEINS FOR QUITE A WHILE. >> THANK YOU. SO MAYBE PEOPLE ARE INTERESTED IN O-GLYCOSYLATION, MAYBE EDITORS OF HIGH IMPACT JOURNALS WILL BE INTERESTED TOO. SO THAT NOT WITHSTANDING SO WHAT I AM GOING TO DO TODAY, SO I WILL TALK, I WILL GIVE YOU BACKGROUND ABOUT WHAT IT IS WE STUDY AND WHY WE STUDY IT. I'M GOING TO TALK ABOUT HOW WE DEMONSTRATEED THAT WE WILL TALK ABOUT OLINK GLYCOSYLATION STUDIES AND WE JUST COMPLETED LOOKING AT MECHANISTIC ROLE OF GLYCOSYLATION IN SECRETION EFFECTS AND THEN AT THE VERY END I'LL TALK TO BUT DYNAMIC IMAGING SYSTEM THAT WE'VE SET UP WHEREBY WE CAN IMAGE SECRETION OCCURRING IN REALTIME IN LIVING SECRETING ORGANS. SO THE TYPE OF MODIFICATION THAT WE STUDY IS KNOWN AS MUSEIN GLYCOSYLATION AND IT INVOLVES THE TRANSFER OF A GLUE MARIOUS COSMINE SUGAR INTO THE THESE AND THIS OCCURS IN THE SECRETATTORY APPARATUS SO THAT IS A GO THERE, THIS ADDITION IS PUT ON THESE PROTEINS. SHOW IS GOING TO BE A MODIFICATION THAT'S PRESENT TO PROTEINS THAT ARE TEST INCREASE IN BODIED TO BE MEMBRANE BOUND OR SECRETED AND SO THIS IS THE INITIAL STEP IN THIS MODIFICATION. AND AFTER THAT OTHER TRANSFERASES CAN ADD SUGARS AND THEN THEY CAN BECOME BRANCHED AND ELONGATED AND QUITE ELABORATE SO WHY DO WE CARE ABOUT THIS MODIFICATION. A COUPLE OF REASONS. IT'S KNOWN THAT MUTATIONS IN THE ENZYME, 1 OF THE ENZYMES THAT CATALYZES THIS INITIAL SUGAR EDITION IS RESPONSIBLE FOR THE HUMAN DISEASE FAMILIESIAL TUMOR CALCINOSE AND I GUESS I'LL TALK ABOUT THAT LATER IN THE TALK. ALSO ALTERATIONS IN ENZYMES RESPONSIBLE FOR THESE GLYCAN SYSTEM INVOLVED IN TN SYNDROME AND THERE'S BEEN A HISTORICAL ASSOCIATION BETWEEN CHANGES IN THESE GLYCANS AND THE DEVELOPMENT OF PROGRESSION OF VARIOUS TYPES OF CANCERS IN HUMANS. SO THE COMPLICATING ISSUE WITH THIS PROTEIN MODIFICATION IS THAT INITIAL ADDITION OF GELNEC AND CATALYZED BY A WHOLE FAMILY OF ENZYMES. SO THIS IS THE FAMILY IN DROSOPHILA DROSOPHILA AND THESE ARE 2 TYPE 2 PROTEINS AND IN THE CATALYTIC DOMAIN LIES WITH THE GOLGI LUMEN THEY'RE ALL CAPABLE OF ADDING THE INITIAL GALNABBING, AND THERE'S A FAMILY, LARGER NUMBER OF THESE FAMILY MEMBERS IN HUMANS. SO WOO HAVE 20 IN HUMANS, 19 IN MICE. THIS MAKES FOR VERY COMPLICATED EXPERIMENTS BECAUSE THERE'S A LOT OF FUNCTIONAL REDUNDANCY BUILT INTO THIS FAMILY. SO BECAUSE OF THAT, WE'VE DONE THE MAJORITY OF OUR STUDIES IN DROSOPHILA WHICH HAS FEWER FAMILY MEMBERS AND OBVIOUSLY MANY MORE GENETIC TOOLS. AND WHAT WE'VE TRIED TO DO IS GET A HANDLE ON WHAT'S OCCURRING WITH REGARD TO OGLYCANS IN GROWTH FACTOR SOVILLA DEVELOP AND WANT WHAT WE LEARN IN DROSOPHILA ALLOWS US TO ASK MORE SPECIFIC QUESTIONS IN THE MORE COMPLICATED MAMMALIAN SYSTEMS. SO I'LL GIVE EXAMPLES OF HOW WE TRY TO DISSECT THIS. SO IN DROSOPHILA, OBVIOUSLY THERE ARE A WEALTH OF CONVENTIONAL MUTATIONS THAT HAVE BEEN GENERATED OVER THE DECADE BISE THE WHOLE DROSOPHILA COMMUNITY AND SO WE'VE TAKEN ADVANTAGE OF THESE CONVENTIONAL MUTATIONS, TRANSPOSE ON MEWITITIONS IN FAMILY MEMBERS OF INTEREST, WE'VE ALSO TAKEN ADVANTAGE OF IN VIVO RNA INTERFERENCE YOU CAN PERFORM IN DROSOPHILA SO FOR THOSE NOT FAMILIAR, CAN YOU MAKE TRANSGENIC FLIES THAT HAVE AN INDUIBLE PROMOTER HOOKED UP TO INVERTED REPEATS OF YOUR GENE OF INTEREST, CAN YOU CROSS THESE FLIES TO FLIES THAT EXPRESS THE GAL 4 TRANSCRIPTIONAL ACTIVATOR AND THEN IN THE PROGENY YOU INDUCE DOUBLE STRANDED RNA FOR YOUR GENE OF INTEREST AND THIS IS A GREAT SYSTEM TO PERFORM IN VIVO, RNA, RNAi IN A TISSUE AND STAGE SPECIFIC FASHION SO CAN YOU KNOCK OUT YOUR GENE OF INTEREST WHEREVER YOU WANT TO. OKAY, SO, JUST A SUMMARY OF THIS, THIS WAS WORK THAT THE ENTIRE LAB PARTICIPATED ON BUT IT WAS SPEAR HEADED BY DEWY TRAN AND THE GROUP. SO WHEN WE DO THESE EXPERIMENTS AND WE USE A GAL-4 DRIVER THAT EXPRESSES GAL 4 IN ALL TISSUES OF THE FLY, SO WE'RE DOING A KNOCK OUT OF THESE GENES DURING ALL CELLS DURING ALL DEVELOPMENTAL STAGES. WHAT WE FIND IS THAT 5 OF THESE FAMILY MEMBERS ARE ESEBT--ESSENTIAL FOR VIABILITY. SO THIS DEMONSTRATES THAT AT LEAST OF THESE 5 FAMILY MEMBERS ARE ESSENTIAL AND WE VERIFY THIS WITH CONVENTIONAL MEANs AS WELL WHEN WE KNOCK THESE OUT USING TISSUE SPECIFIC MEMBERS, A NUMBER OF THESE MEMBERS ARE REQUIRE INDEED SPECIFIC TISSUES, SO WE HAVE A NUMBER OF THESE THAT ARE REQUIRED IN THE DEVELOPING DIGESTIVE SYSTEM, WE HAVE A NUMBER THAT ARE REQUIRED IN THE DEVELOPING RESPIRATORY SYSTEM AND THEN WE CAN ALSO USE MORPH LOGICAL CHANGES THAT WE SEE IN TISSUES THAT ARE NOT ESSENTIAL FOR VIABILITY, SO FOR INSTANCE THE EYE AND THE WING OF THE FLY, FLIES DON'T NEED THOSE TO ACTUALLY SURVIVE BUT UNIQUE MORPH LOGICAL CHANGES IN THOSE TISSUES GIVES YOU CLUES AS TO WHAT THESE GENES ARE ACTUALLY DOING DEVELOPMENTALLY. SO AN EXAMPLE OF THIS ARE PHENOTYPES WE CAN SEE IN THE WING AND I'LL JUST BRIEFLY GO THROUGH 1 OF THESE STUDIES. SO THE WING HAS BEEN USED AS A MODEL SYSTEM TO STUDY CELL ADHESION AND THE FACTORS THAT ARE RESPONSIBLE FOR CELL ADHESION DEVELOPMENTALLY, SO THE WING IS COMPRISED OF 2 EPITHELIAL CELL LAYERS WHICH ARE SHOWN HERE, THAT ADHERE TO 1 ANOTHER VIA INTEGRIN MEDIATED CONTRACTS TO THE SECRETED EXTRA CELLULAR MATRIX SO WHEN THESE CELL LAYERS ADHERE TO 1 ANOTHER, HAVE YOU THIS BEAUTIFUL NICE FLAT ADULT WING. WHEN HE'S ADHESIVE CONTACTS ARE DISRUPTED, THESE 2 CELL LAYERS SEPARATE ASK THIS REGION FILLS WITH FLUID AND WHAT YOU GET ARE THESE WINGED BLISTERS WHICH IS AN OBVIOUS PHENOTYPE AND USED TO SCREEN BY A NUMBER OF LABS TO IDENTIFY GENES, PROTEINS THAT ARE INVOLVE INDEED CELL ADHESION EVENTS AND NOT JUST IN DROSOPHILA BUT IN MAMMALS. SO FOR INSTANCE, THE VARIOUS INTEGRINS AND THEIR INTERACTING PROTEINS AND PARTNERS BOTH EXTRA CELLULARLY AND INTRACELLULARLY. OKAY. SO THIS IS WORK DONE BY LEE PING ZANG IN MY GROUP ASK WHAT LEE PING NOTICE SIDE THAT WHEN 1 MEMBER OF THE TRANSFERASE STUDY WAS KNOCKED OUT, THE FLIES ARE VIABLE SO IT'S NOT ESSENTIAL. HOWEVER, THEY GAVE THIS VERY CHARACTERISTIC BLISTERS PHENOTYPE ON ALL OF THEIR WINGS, SO JUST TO SUMMARIZE WHAT LEE PING FOUND OUT THROUGH USING A COMBINATION OF GENETIC AND BIOCHEMICAL ANALYSIS, SHE FOUND THAT PGOOD NIGHT 3 IS REQUIRED AND IT'S IN MEDIATED CELL ADHESION AND MORE OVER SHE FOUND THAT IT GLYCOSYLATEDS AN EXTRA CELLULAR PROTEIN KNOWN AS TIGGRIN, AND IT IS IMPORTANT BECAUSE IT IS KNOWN FROM PRIOR STUDIES TO BE AN INTEGRIN BINDING PROTEIN. SO OUR NEXT QUESTION THEN WAS HOW WAS PGAUNT 3 AFFECTS TIGGRIN, SO LEE PING DID A CAREFUL ANALYSIS LOOKING AT SYNTHESIZATION LOOKING AT DEVELOPMENT THROUGHOUT THE WICK AND SHE FOUND THAT TIGGRIN IN A WING AS IT DEVELOPS IS NORMALLY SECRETED TO THIS ADHESIVE SURFACE BETWEEN THE 2 CELL LAYERS SO IT'S PART OF THE EXTRA CELLULAR MATRIX BUT IN OUR MUTANTS IT NEVER GETS SECRETED TO THIS INTERFACE. WHEN HE DOES A CROSS SECTION OF THESE CELLS AND LABELS WITH CADHERON TO OUTLINE THE CELL WHICH IS YOU CAN SEE IN THE MUTANT IS THAT IT'S TRAPPED INSIDE THE CELLS. OKAY SO WE HAVE A SITUATION HERE TO LOOK AT DO DOFFILLA TO LOOK AT THESE GENES, SO IT AFFECTS THE DEVELOPMENT BY INFLUENCING THE COMPOSITION OF THE EXTRA CELLULAR MATRIX. SO OUR NEXT QUESTION WAS THEN NOW KNOWING THIS, DOES O-GLYCOSYLATION INFLUENCE EXTRA CELLULAR MATRIX COMPOSITION IN MAMMALS. SO TO ADDRESS THIS QUESTION, WE TURN TO THE MAMMALIAN CELLULAR GRAND FOR A NUMBER OF REASONS. THE PRIMARY REASONS, IS THESE TRANSER ACES ARE ABUNDANT THROUGH GLAND DEVELOPMENT AND THE SECONDARY REASON AND ACTUALLY A VERY, VERY IMPORTANT REASON IS THE AMOUNT OF EXPERTISE WE HAVE IN NIDCR WITH REGARD TO SALIVARY GLAND DEVELOPMENT AND THIS IS WORK DONE BY ETN IN MY GROUP. AND SO, LET ME JUST KIND OF WALK YOU THROUGH SALIVARY GLAND DEVELOPMENT. SO THE SALIVARY GLAND BEGINS TO DEVELOP AS A SINGLE EPITHELIAL BUD SHOWN HERE SURROUNDED BY CONDENSED MESSENTERY AN KINE AROUND THE BUD AND AROUND THE BUD S&P RICH OGLYCOPROTEINS AND THEW IS IMPORTANT IN MEDIATED SIGNALING EFFECTS THAT ALLOW THE APPROPRIATE PROLIFERATION OF THIS EPITHELIUM AND THEN ALLOW THE BUD GROWTH AND BRANCHING TO FORM THIS VERY ARBORIZED GRANULAR STRUCTURE THAT OCCURS LATER ON IN DEVELOPMENT AND SO, WHAT TEN DID WAS TO LOOK AT SALIVARY GLAND DEVELOPMENT IN MICE THAT ARE DEFICIENT FOR THE TRANSFERASE THAT IS MOST ABUNDANTLY EXPRESSED AT THIS STAGE IN DEVELOPMENT AND THAT'S KNOWN AS GELNECK 1 AND SO WHAT HE NOTICED WAS THAT IN THE GALNECK T-1 MICE THE SUBMAN DIPULAR GLAND GREW SIGNIFICANTLY LESS THAN THE WILD-TYPE LITTER MATE CONTROLS SO THIS IS SHOWN HERE LOOKING AT GLAND GROWTH OVERTIME IN THE KNOCK OUTS AND YOU CAN COMPARE THIS TO THE GLAND GROWTH THAT YOU SEE IN THE WILD-TYPE AND HETEROGENEOUS ROW ZYGOUS LITTER MATE CONTROLS AND THIS IS QUANTITATED HERE BY COUNTING BUD NUMBER SO THERE'S REDUCED GROWTH IN GLANDS THAT LACKED THIS TRANSFERASE. AND THIS REDUCED GROWTH WAS DUE TO REDUCED EPITHELIAL CELL PROLIFERATION SO WE LABELED CELLS WITH EDU, GROWING BUDS WITH EDU AND YOU CAN SEE DECREASED PROLIFERATION IN THE GAL T-1 KNOCK OUTS AND YOU CAN SEE THIS HERE BY THE LITTER MATE CONTROLS. WE KNOW FROM DOING A SERIES OF PC R AND BIOCHEMICAL EXPERIMENTS THAT IN THESE T-1 KNOCK OUT GLANDS THERE WAS REDUCED FGF 1 EXPRESSION AND EXPRESSION OF THE DOB STREAM TARGET ETD 4 AND THERE WAS REDUCED PHOSPHORYLATION OF AKT AND MAP KINASE. AND SO BECAUSE WE KNOW THESE ARE ABUNDANT IN THE BASE MEMBRANE, WE WANT TO LOOK AT BASE MEMBRANE AND WE DID THAT HERE USING A LECTIN THAT SPECIFICALLY DETECTS OLINK GLUE MARIOUS COPROTEINS AND ECADHERON LABELS THE EPITHELIAL CELLS. SO CAN YOU SEE IN THE WILD-TYPE, YOU CAN SEE THE STAINING ALONG THE BASE OF THE MEMBRANE AND YOU CAN SEE THIS IS DRAMATICALLY REDUCED IN OUR T-1 KNOCK OUT ANIMALS INDICATE THANKSGIVING ARE CHANGES AT THE BASE OF THE MEMBRANE. WHEN WE LOOKED THE MAJOR COMPONENTS OF THE BASE OF THE MEMBRANE, WHICH ARE LAMINA ALPHA 1 AND COLLAGEN 4, YOU CAN SEE A REDUCTION IN BOTH OF THESE SO THIS IS LAMMEN ALPHA 1 IN THE T-1 KNOCK OUT ANIMALS RELATIVE TO THE WILD-TYPE LITTER MATE CONTROLS AND COLLAGEN 4 ALONG THE SPACE OF THE MEMBRANE VERY MUCH REDUCED IN THESE T-1 KNOCK OUT ANIMALS. OKAY. AND SO WHEN WE QUANTITATED THE STAINING OF EACH 1 OF THESE, WHAT YOU CAN ACTUALLY SEE IS THAT ALONG WITH A REDUCTION OF BOTH LAM THEN CAN COLLAGEN IN THE BASE, WE AWE AN INCREASE CELLULARLY. SO LAM INTERFERON WAS ACCUMULATING AND A DECREASE IN THE BASE OF THE MEMBRANE INDICATING THESE PROTEINS IN THE ABSENCE OF T-1 ARE NOT BEING PROPERTILY SECRETED TO THE BASE OF THE MEMBRANE, SO IF THESE THINGS ARE GETTING ALL HUNG UP ON THE CELL, THE NEXT THING WE WANTED TO KNOW IS HAS THERE BEEN AN INDUCTION OF ER-STRESS. AND THAT'S BEEN SHOWN HERE, SO IN THE ABSENCE OF T-1 WE DO SEE AN INDUCTION OF E. R. STRESS BY XPB-1 SPLICING AND ALL BRANCHES OF THE EXPRESS PATHWAY ARE ALSO ACTIVATED SO WE SEE INCREASED LEAVE ANDROGEN OF ATF 6 AND INCREASED TRANSLATION OF ATF 4. SO, OUR CHANGES IN THE BASE MEMORY AND COMPOSITION RESPONSIBLE FOR THE GROWTH DEFECTS THAT WE SEE. SO WHAT WE DID IS WE EXOGENOUSLY ADDED BACK COMPONENTS OF THE BASE MEMBRANE TO THE GLANDS TO SEE IT QUEY COULD RESTORE GROWTH AND WE WERE ABLE TO DO THAT SO THESE ARE GLANDS AGAIN FROM T-1 KNOCK OUT ANIMALS, EITHER GROWN IN THE A--BITS SENSE OR PRESENCE OF LAM NIN 111 AND YOU CAN SEE THAT WE CAN GET RESTORATION OF GROWTH. THIS IS QUANTITATED HERE BY BOTH BUD NUMBER AND EPITHELIAL AREA AND ADDITIONALLY WE WERE ABLE TO RESTORE FGF 1 AND ETB 4 EXPRESSION WHEN WE ADD BACK BASEMENT MEMBRANE COMPONENTS TO THESE. SO OUR MODEL FOR WHAT IS OCCURRING IN THIS SITUATION IS THAT O-GLYCOSYLATION BY VIRTUE OF THE ACTIVITY OF GEL T-1 IS RESPONSIBLE FOR THE SECRETION OF BASEMENT MEMBRANE COMPONENTS WHICH THEN ACTIVATE VARIOUS SIGNALING TRANSDUCTION PACT WAYS AND--PATHWAYS AND REGULATE PROLIFERATION TO ALLOW GLAND GROWTH, BUT IN OUR T-1 KNOCK OUT A--BITS MALS HA WE FIND IS THAT THE BASEMENT MEMBRANE AND DISRUPTED BECAUSE THEY'RE NOT APPROPRIATELY SECRETED TO THE BASE MEMBRANE. WE SEE A REDUCTION IN FGF SIGNALING, REDUCE EPITHELIAL CELL PROLIFERATION AND AN INDUCTION IN ER STRESS. SO TO SUMMARIZE THESE 2 STUDIES THAT I JUST TOLD YOU ABOUT, SO WE HAVE 2 INSTANCES WHERE YOU KNOCK OUT A TRANSFERASE AND YOU'RE AFFECTING SECRETION, OKAY SO IN THE DROSOPHILA SYSTEM YOU'RE AFFECTING SECRETION OF AN EXTRA CELLULAR MATRIX COMPONENT THAT RESULTS IN ABERRATIONS IN CELL ADHESION DURING DEVELOPMENT, OKAY? AND IN THE MAMMALIAN SYSTEM, YOU'RE AFFECTING SECRETION OF THE BASE OF MEMBRANE COMPONENTS WHICH IS RESULT NOTHING ABERRATIONS AND CELL PROLIFERATION DURING ORGAN GENESIS, SO GETTING BACK TO OUR AGE OLD QUESTION HERE, HOW DOES O-GLYCOSYLATION INFLUENCE SECRETION MECHANISTICALLY AND TO ADDRESS THIS WE TURN BACK TO THEIR DROSOPHILA NOW TO ANOTHER ORGAN IN DROSOPHILA THAT HAS VERY INTERESTING PROPERTIES. SO, THIS IS THE DROSOPHILA VENT RICKULOUS, SO THIS IS THE FUNCTIONAL EQUIVALENT OF THE STOMACH, OKAY? AND THIS IS A VERY INTERESTING ORGAN BECAUSE IT HAS THESE VERY SPECIALIZED P. R. CELLS HERE THAT ARE HIGHLY SECRETOR NEUROECTODERMAL NATURE, THESE PR CELLS ARE RESPONSIBLE FOR SECRETING THE VAST MAJORITY OF THE PARATROPIC MEMBRANE THAT WILL LINE THE WHOLE GIVING--YOU JESTIVE TRACT OF THE DROSOPHILA LARVA AND IT'S THE FUNCTIONAL EQUIVALENTS OF OUR MEMBRANE SO IT ACTS AS A LUBRICANT FOR FOOD PASSAGE, PROTECTIVE BARRIER FROM THE IBT--INTEGRATE CULTS AND IT'S VERY, VERY IMPORTANT. SO THESE CELLS DO A VERY IMPORTANT JOB, AND ADDITIONALLY WHAT WE FOUND IS THAT THEY ALSO HAD SOME VERY UNIQUE PROPERTIES. SO UNLIKE MOST CELLS IN DROSOPHILA WHERE THE SECRET ARYEH APPARATUS AND ALL DISPURSED AND NOT REALLY LOCALIZED THEY HAD A VERY HIGHLY APICKLY SECRET ORY AND THIS IS THE MARKER FOR THE GOLGI AND YOU CAN SEE IT STAINING INTENSELY ALONG THE APICAL SURFACE OF THESE PR CELLS. ADDITIONALLY WHAT THEY HAVE WITHIN THEM ARE SECRETORY VESICLES SO THIS IS DROSOPHILA MUSSEN AND CAN YOU SEE IT LIGHTS UP THESE VESICLES IN THE PR CELLS. SO SOME VERY UNIQUE CHARACTERSISTICS ABOUT THESE SECRETORY CELLS THAT WILL ALLOW US TO DETECT ANY KIND OF CHANGES IN THE POLARITY OF THE SECRETORY APPARATUS AND IN THE GRANNUALS AND IN THE SECRETION OF THESE MEMBRANE COMPONENTS. AND SO THIS IS WORK DONE BY LEE PING ZANG AND THE GROUP THAT'S IN PRESS RIGHT NOW. I THINK IT'S COMING OUT NEXT WEEK. SO WHAT LEE PING FOUND BY LOOKING AT THE VENT RICKULOUS IS THAT THERE IS 1 MEMBER OF THE PDM FAMILY THAT'S EXPRESSED VERY, VERY SPECIFICALLY IN THESE HIGHLY SPECIALIZED PR CELLS, SO THIS IS PGANNA 4 AND SHE BEGAN INTER GAUNTING ITS ROLE IN PC CELL FUNCTION AND SECRETORY APPARATUS STRUCTURE AND SHE LOOKED AT CONVENTIONAL 4 AS WELL ARNAi THAT KNOCKS IT DOWN SPECIFICALLY IN THESE PR CELLS. AND SO WHAT SHE FOUND WAS IT WAS REALLY QUITE INTERESTING, IN A WILD-TYPE SITUATION, HAVE YOU--IF YOU STAINED 4 COMPONENTS OF THE MEMBRANE, CAN YOU SEE HAD HERE, RIGHT ALONG ALONG THE REGION AS IT'S SECRETED AND WHEN WE KNOCK IT DOWN AND INTERESTINGLY AGAIN IN THE WILD-TYPE, YOU CAN SEE GOLGI STAINING ALONG THE APICAL SURFACE OF THESE PR CELLS IN THE INNER APICAL SURFACE AND WHEN HE KNOCKS DOWN 4, THE CELLS GET LARGE AND THE GOLGI DISBURSES THROUGHOUT THE CELLS. SO WE'VE LOST IT IN THE ABSENCE OF THE TRANSFERASE. WHEN YOU KNOCK IT DOWN THE CELLS GET LARGE AND THE GRANNUALS DISAPPEAR, SO WE AFFECTED SECRETORY SYNTHESIS AS LSO HOW IS GLYCOSYLATION INFLUENCING SECRETION, THE FORMATION OF THE SECRETORY APPARATUS, OF VESSELS AND SECRETION OF THE PARATROPIC MEMBRANE COMPONENTS. SO WE BEGAN INVESTIGATING THIS BY DIFFERENTIALLY COMPARING THE PROTEINS THAT ARE NORMALLY OGLYCOSYLATEDDED IN THE WILD-TYPE CELLS THOSE THAT HAVE REDUCED OR LOST GLYCOSYLATION IN THE MUTANTS. AND SO WHAT WE FOUND IS 1 PROTEIN IN PARTICULAR THAT WAS VERY INTERESTING AND THIS IS TANGO 1. SO TANGO 1 IS NORMALLY OGLYCOSYLATEDDED AND THIS IS USING ANTES BODY SPECIFIC TO OGLYCOSYLATEDDED PROTEINS WHEN WE PULL DOWN TANGO 1 FROM WILD-TYPE PR CELLS AND THEN BLOT WITH THE NANO BODY THAT DETECTS AND WE WENT THROUGH THIS LITTLE SCHEME FOR A NUMBER OF DIFFERENT PROTEINS AND TESTING DIFFERENT PROTEINS BUT 1 OF OUR CRITERIA WAS IF THIS IS REALLY THE DIRECT TARGET OF PGANT 4 THAT'S MEDIATING THESE EFFECTS THEN WE SHOULD GET THE SAME PHENOTYPE IF WE KNOCK OUT THE PROTEIN SO WE DID THAT EXPERIMENT AND IN FACT,--OH, FIRST, OKAY, LET ME TELL YOU ABOUT TANGO FIRST. SO TANGO, TRANSPORT AND GOLGI ORGANIZATION, SO THIS IS A VERY INTERESTING PROTEIN. IT WAS ACTUALLY ORIGINALLY IDENTIFIED IN A DROSOPHILA CELL CULTURE SCREEN FOR GENES THAT WHEN MUTATED WOULD DISRUPT THE MUTATION OF A REPORTER CONSTRUCT. SO WHEN THIS GENE WAS MUTATED. THE REPORTER CONSTRUCT DIDN'T GET SECRETED PROPERLY AND IT WAS A SCREEN FOR THINGS THAT WOULD ALTER GOLGI ORGANIZATION SO THAT WAS FOUND IN DROSOPHILA TISSUE CULTURE CELLS, SUBSEQUENTLY THEY FOUND THAT THE MAMMALIAN LOG IS IN GENE, IT'S KNOCKED OUT IN MICE AND THEY DIE EMBRYONICALLY AND THEY HAVE DEFECTS, SO THE MODEL FOR HOW TANGO WORKS IS THAT--SO IT'S A TRANSMEMBRANE PROTEIN THAT'S PRESENT IN THE SECRET ORY APPARATUS AND THE LUMINAL PORTION OF BIND CARGO AND THE CYTOPLASMIC WILL BIND COMPONENTS SO THE MODEL IS IT WILL DIRECT CARGO INTO SECRET ORY GRANNUALS THAT ARE LARGE ENOUGH TO HOLD VERY LARGE CARGO SUCH AS COLLAGEN, OKAY? AND THAT'S THE MODEL FOR HOW THEY THINK TANGO IS WORKING. SO IF TANGO IN FACT A DIRECT TARGET OF PGANT 4 MEDIATING THE DEFECTS WE'RE SEEING IN OUR DROSOPHILA SYSTEM THEN IF WE KNOCK OUT TANGO WE SHOULD SEE THE SAME DEFECTS THAT WE SEE WITH PGANT 4. OKAY, AND THIS IS IN FACT THE CASE, SO AGAIN, WHEN NORMALLY YOU SEE THESE NICE GRANNUALS IN THE WILD-TYPE, YOU KNOCK OUT PGAUNT 4, THESE GET LARGE AND THE SECRETORY GRANNUALS DISAPPEAR, YOU KNOCK DOWN TANGO AND YOU GET THE SAME GRANULES. ADDITIONALLY GAB IN THE WILD-TYPE YOU HAVE A POLARIZED SECRETORY APPARATUS AND THIS IS A RAB 8 WHICH IS NORMALLY LOCALIZED WITHIN THESE PR CELLS THAT APICAL LOCALIZATION IS LOST WHEN YOU KNOCK DOWN PGAUNT 4 AND IT'S ALSO LOST WHEN YOU KNOCK DOWN TANGO 1. AND THE NEXT EXPERIMENT THAT WE DID WAS TO ADDRESS THE INTERACTION BETWEEN PGAUNT 4 AND TANGO. SO, IF TANGO AGAIN REALLY IS THE SUBSTRATE PROTEIN THAT IS MEDIATING THE PHENOTYPES WE SEE WHEN WE LOSE PGANT 4, 1 WOULD ARGUE WE MIGHT BE ABLE TO RESCUE THE PHENOTYPES BY OVEREXPRESSING TANGO. AND IN FACT WE CAN DO THAT. SO IF WE PUT TANGO OVEREXPRESSION CONSTRUCTION IN A PGANT 4 RNAi BACKGROUND WE RESCUE THE FORMATION OF THE SECRETORY GRANNUALS AND WE--GRANULES AND WE RESCUE THE STRUCTURE OF THE APPARATUS AND THE SECRETION OF THE PARATROPIC MEMBRANE. OKAY. SO HOW IT PGANT 4 INFLUENCING TANGO? SO WE COMPARED TANGO IN GANT MUSEUMITANTS VERSES WILD-TYPE AND WE FOUND THAT THERE IS ACTUALLY 2 DIFFERENT FORMS OF TANGO, SO THERE IS--SHOWN HERE, AN INTACTED FORM OF TANGO AND THEN THERE IS THIS CLEAVED FORM AND SO IF YOU LOOK IN THE WILD-TYPE PRO VENT RICKULOUS, YOU SEE THE MAJORITY OF TANGO IS IN THE INTACT FORM AND JUST A BIT IN THE CLEAVED AND WE CAN EXPRESS IN AS A RATIO TO INTACT TO CLEAVED AND WHEN YOU KNOCK DOWN PGAUNT 4 EITHER BY RNAI IN THE PR CELLS OR BY CONVENTIONAL MUTATIONS, YOU DECREASE THE AMOUNT OF INTACT TANGO RELATIVE TO THE AMOUNT OF PORTION WE SEE HERE AND WE CAN RECAPITULATE THIS IN CELL CULTURE BY EXPRESSING TANGO IN DROSOPHILA CELL CULTURE. AGAIN YOU CAN GET THE INTACT VERSION OF TANGO AND THE CLEAVED VERSION AND WHEN YOU OVEREXPRESS PGANT 4, CAN YOU INCREASE THE GLYCOSYLATION OF THE INTACT VERSION AND INCREASE THE AMOUNT OF INTACT RELATIVE TO CLEAVED. AND THIS APPEARS TO BE SPECIFIC TO PGANT 4 AS EXPRESSION OF ANOTHER TRANSFERASE DOESN'T APPEAR TO CHANGE THE RATIO OF INTACT TO CLEAVED TANGO. OKAY, SO THIS SUGGESTING THAT THE ACTIVITY OF PGANT 4 AND IT IS CATALYTIC ACTIVITY BECAUSE WE DID CATALYTICALLY INACTIVE MUSEUM TACTS AND THEY DIDN'T RESCUE. THE CATALYTIC ACTIVITY OF PGANT 4 IS SOMEHOW PROTECTING IT FROM PROTEOLYSIS. SO WHEN WE LOOK AT THE SEQUENCE, IN FACT THERE ARE A NUMBER OF CLEAVAGE SITES SHOWN IN BLUE, VERY NEAR POTENTIAL SITES OF O-GLYCOSYLATION WHICH ARE SHOWN IN RED. SO WE NEXTED IS TANGO ACTUALLY SUBJECT TO THE MEDIATED PROTEOLYSIS AND WHEN WE INCUBATED CELLS EXPRESSING TANGO IN THE PRESENCE OR ABSENCE OF A FEWON INHIBITOR WE COULD CHANGE THE RATIOS OF INTACT CLEAVED TANGO SO WE GOT MORE--A HIGHER PERCENTAGE OF INTACT TO CLEAVED TANGO IN THE PRESENCE OF FEWON INHIBITORS INDICATING THAT A TINNING O IS SUBJECT TO PROTEOLYSIS. SO IF THIS IS HOW THINGS ARE WORKING, WE NEXT ASKED WHETHER OR NOT WE CAN RESCUE THE LOSS OF PGANT 4 BY KNOCKING DOWN FEWONS IN VIVO, OKAY? SO THERE ARE 2 MAJOR DROSOPHILAS THAT ARE EXPRESSED IN THE PRO VENT RICKULOUS SO WE PERFORM THOSE GENETIC EXPERIMENTS AND I'LL JUST KIND OF WALK YOU THROUGH THIS. OKAY. SO THE FIRST PANEL IS LOOKING AT AGAIN CLEAVED INTACT TANGO SO IF YOU LOOK AT THIS IS ESSENTIALLY CONTROL WILD-TYPE YOU CAN SEE YOUR INTACT TANGO AND VERY LITTLE CLEAVED IN THE PGANT 4 RNAi, VERY LITTLE INTACT TANGO AND A LOT MORE CLEAVED. IF YOU KNOCK DOWN D-FURON 1, IT LOOKS LIKE WILD-TYPE. THERE'S QUITE 8 BIT OF INTACT, VERY LITTLE CLEAVED. IF YOU KNOCK DOWN DFERON 2, LOOKS LIKE WILD-TYPE, IF YOU KNOCK DOWN PGAUNT 4, AND D-FEARON 1 TLOOKS LIKE THE PGAUNT 4 KNOCK OUT SO THIS--THE KNOCK DOWN OF FURON 1 DOES NOT APPEAR TO DO ANYTHING IN TERMS OF RESCUING THE CLEAVAGE OF TANGO, HOWEVER KNOCK DOWN OF DFEWON 2 RESTORES THE PERCENTAGES OF INTACT RELATIVE TO CLEAVE TANGO. SO IT LOOKS VERY MUCH LIKE WILD-TYPE AND WHEN WE LOOK MORPH LOGICALLY AT THESE GLANDS, AT THESE PR CELLS WHAT WE FIND IS THAT PGANT 4 RNAi IN THE PRESENCE OF KNOCK DOWN OF DFEWON 2 DOES IN FACT RESCUE MORPHOLOGY, AND THE APICAL LOCATION OF THE SECRETORY APPARATUS. SO OUR MODEL FOR WHAT'S HAPPENING HERE IS IN THESE CELLS THAT ARE SECRETING GOBS AND GOBS OF ALL KINDS OF PROTEINS THAT ARE COMPARE OPPOSITE BEHAVIORIAL PHENOTYPENTS OF THE PARATROPIC MEMBRANE. TAKE O WHICH IS PRESENT AGAIN IN THE SECRETORY APPARATUS IS OGLYCOSYLATEDDED BY PGANT 4 AND PROTECTED FROM FEWON MEDIATED CLEAVAGE BY DEFEWON 2. THIS ALLOWS PACKAGES OF THE CARGO INTO THE SECRETORY GRANULES AND THE FORMATION OF THE GRANNUALS AND THE SECRETION TO THE PARATROPIC MEMBRANE IN PGANT 4 MUTE ANTS, TANGO IS NOT GLYCOSYLATEDDED IT IS CLEAVED BY FEWON 2 AND SECRET ORY VESICLES NEVER FORM. OKAY? SO THIS IS THE FIGHTER IN VIVO DEMONSTRATION OF THIS INTERPLAY BETWEEN O-GLYCOSYLATION AND PROTEOLYSIS AND THE FIRST IN VIVO DEMONSTRATION THAT WE CAN POTENTIALLY RESCUE, DEFECTS AND GLYCOSYLATION BY MODULATING PROTEASE ACTIVITY AND THIS IS IMPORTANT BECAUSE NOW WE'RE GOING BACK TO FAMILIESIAL TUMOR KELSINOSEIS IN THE BEGINNING WHICH IS A DISEASE THAT'S CAUSED BY MUTATION IN 1 OF THESE ENZYMES, THE GALNT3 AND IN THESE PATIENTS THEY SUFFER FROM HYPER FOSTER NURSED FOCUSED ON TEEMIA, INCREASED BONE DENSITY, DEVELOP THESE SUBTERMAL CALCIFIED TUMORS THROUGHOUT THEIR BODY WHICH ARE INCREDIBLY PAIN EXCLUSIVELY THEY ALSO SHOW INCREASE SAID RATIOS OF CLEAVED FGF 23 WHICH IS A PHOSPHATE REGULATING HORMONE SO THE MODEL FOR WHAT'S HAPPENING IN THIS DISEASE IS THAT FGF23 IS NORMALLY OGLYCOSYLATEDDED BY T3 AND IT'S PROTECTED PRACTICES PROTEOLYSIS AND A LOT OF THIS HAS BEEN DEMONSTRATED INVITRO ASSAYS WITH PEPTIDES, BUT IT HASN'T SPECIFICALLY BEEN SHOWN IN VIVO YET, BUT IF WE'RE ABLE TO IDENTIFY THAT THIS IS ACTUALLY OCCURRING AND THE SPECIFIC PROTEASES INVOLVED OR PROTEASE INVOLVED, IT IMLIVES THAT WE MAY BE ABLE TO MODULATE SOME OF THESE BY MODULATING PROTEASE ACTIVITY AND THERE'S QUITE A BIT OF INTEREST IN FEWON INHIBITORS ANYWAY JUST BECAUSE OF VARIOUS TYPES OF BACTERIAL TOXICITY AND AND VIRAL INFECTIVITY THAT ARE ASSOCIATED WITH THEM SO THIS MAY HAVE POTENTIAL IMPLICATIONS FOR TREATMENTS EVENTUALLY. SO JUST IN THE LAST PART OF MY TALK, I WANT TO GO THROUGH AN IMAGING SYSTEM WE'VE SET UP TO LOOK AT SOME OF THESE SECRETION EVENTS IN REALTIME GIVEN THAT MOST OF THE EXPERIMENTS THAT WE'VE DONE HAVE POINTED US IN THE DIRECTION OF THESE--THIS MODIFICATION, IS REALLY AFFECTS SECRETION. WE WANTED TO HAVE A WAY TO IMAGE THIS IN REALTIME. SO, AS I'M SURE THIS GROUP KNOWS, THERE ARE MANY DIFFERENT STEPS INVOLVED IN SECRETION OF A PRODUCT PROPERTILY SO YOU HAVE TO SYNTHESIZE THE PRODUCT. IT HAS TO BE APPROPRIATELY MODIFIED, IT HAS TO MOVE THROUGH THE SECRETORY APPARATUS, HAS TO BE PROPERTILY PACKAGED, THE VESICLES HAVE TO MOVE TO THE RIGHT PART OF THE CELL, THEY HAVE TO FUSE WITH THE MEMBRANE AND RELEASE CONTENTS IN AN APPROPRIATE FASHION, IT'S DIFFERENT STEPS, COMPLEX PROCESS SO AGAIN WE TURNED BACK TO DROSOPHILA AND WE'RE USING THE DROSOPHILA SALIVARY GLANDS TO SET UP THE SYSTEM TO IMAGE REALTIME SECRETION EVENTS IN A LIVING ORGAN SO THIS IS WORK BY AGAIN DUY TRAN IN THE LAB. MAJOR SECRETORY CELL IN THE FLY, PUMPING OUT HUGE AMOUNTS OF PROTEINS SO IT'S COMPOSED OF SECRETORY CELLS AND DUCTAL CELLS AND THESE AT LATE DURING THIS TIME THERE'S A SMALL PULSE OF THE MOLTING HORMONE THAT INDUCES EXPRESSION OF MANY JEEPS AND ALL OF THE JEEPS THAT ARE GOING TO BE PACKAGE EXPDZ EVENTUALLY SECRETED AND THEN LATER ON, THERE'S A LARGER PULSE OF THE DYSONE THAT TRIGGERS EVERYTHING AND PACK CALVERTED AND SO DUY HAS DONE IS ISOLATE GLANDS PRIOR TO THIS SECOND ECDYSONE PULSE AND HE TAKES THESE OUTS AND CULTURES THEM EXVIVO AND THEN EXVIVO ADDS THE DYSEWN AND THEN IMAGES THE SECRETION EVENTS THAT ARE OCCURRING IN REALTIME. OKAY, AND HE'S TAKEN ADVANTAGE AGAIN OF THE MANY TOOLS ARE THE DO SOVILLA COMMUNITY HAS DEVELOPED OVER THE YEARS AND 1 OF THESE TOOLS IS A GFP LABELED SECRETORY PROTEINS DEVELOPED BY ANDRE'S LAB SO THIS IS 1 OF THE MAJOR GLUE PROTEINS THAT'S PACKAGED AND THEN SECRETE WIDE THE PULSE, SO THIS IS GFP LABELS AND WHAT IT ALLOWSITOUS DO IS TO FOLLOW--ALLOWS US TO DO FOLLOW THE FORMATION OF THE GRANNUALS AND THEN FOLLOW THEM AS THEY FUSE AND THEN DUMP THEIR CONTENTS INTO THE LUMEN OF THAT GLAND. THERE ARE ALSO FLIES THAT EXPRESS LABELED ACTINS SO THAT ALLOWS US TO FOLLOW ACTIN DYNAMICS SO ACTIN IS NORMALLY ALONG THE APICAL MEMBRANE OF THE GLAND AND WE CAN WATCH AS THE GRANULES FUSE AND ARE COATED AND COLLAPSE AND RELEASE CONTENTS THERE IS A FLY THAT EXPRESSES LABELED MICE IN AND THAT ALLOWS US TO WATCH AND STUDY MICE AND DYNAMICS AS SECRETION IS OCCURRING. AND THEN WHAT WE CAN ALSO DO IS ONCE THE GLANDS ARE HARVESTED AND WE CAN PUT A DYE IN THE LUMEN AND THIS ALLOWS US TO VISUALIZE IS THE EXACT MOMENT WHEN THIS FUSION PORT OPENS AS THE GRANNUAL FUSES WITH THE MEMBRANE AND THAT'S ACTUALLY QUITE IMPORTANT. OKAY. AND SO THIS IS JUST A STILL IMAGE TO SHOW YOU THE SECRETORY GRANGRANNUAL IN THE CELLS SO THIS IS A SALIVARY GLAND CELL RIGHT HERE AND SO ALL OF THESE GREEN CIRCLES ARE THE SECRETORY VESICLES THAT ARE FILL WIDE THIS GFP LABELED GLUE THAT I TOLD YOU ABOUT. AND SO, WHAT WE CAN DO IS WE CAN IMAGE SECRETION ASK REALTIME USING THIS. SO THIS IS--JUST TO ORIENT YOU, THIS IS THE DISTAL TIP OF A SALIVARY GLAND, OKAY? THIS IS A CELL THIS, IS A CELL THIS, IS A CELL THIS, IS THE NUCLEUS. THIS IS THE LUMEN WHICH HAS NOTHING IN IT RETIRED NOW AND ALL OF THESE DOTS THAT YOU SEE IN THE CYTOPLASM FOR THOSE LITTLE GROUND SECRETORY GRANULES CONTAINING THE GLUE SO YOU WILL SEE THESE WHEN I PLAY THE MOVIE THAT THESE WILL MOVE TO THE SURFACE, FUSE WITH IT AND DUMP THE CONTENT IN THE LUMEN AND THIS WILL EXPAND WITH THE GREEN GLUE. AND SO WE CAN IMAGE ALONG WITH THAT, WE CAN IMAGE THE DYNAMICS SO NOW THE GREEN IS LABELED ACTIN AND AGAIN THIS IS THE APICAL SURFACE, THIS IS THE SALIVARY GLAND CELL AND THE ACTIN IS LABELED IN GREEN. SO WHAT YOU SEE HERE IS THE APICAL SURFACE NORMAL HAS ACTIN, THE SECRETORY VESSELS BEFORE THEY FUSE DO NOT. SO YOU WILL SEE THE VESICLING COME DOWN HERE, FUSE WITH THE MEMBRANE, COCOATED WITH ACTIN AND COLLAPSE AND RELEASE CONTENTS. SO WE CAN WATCH ACTIN DYNAMICS IN REALTIME AS THESE VESICLES COME IN AND FUSE WITH THE PLASMA MEMBRANE WITH THE APICAL MEMBRANE. OKAY AND WE CAN IMAGE ACTUALLY INDIVIDUAL VESICLE FUSION EVENTS SO NOW THIS IS AGAIN THE GREEN IS ACTIN SO ACTIN ALONG THE APICAL SURFACE. IN THIS CASE, THE RED IS THE LUMEN THAT'S BEEN FILLED WITH THE LABELED DEXTRAN AND WHAT YOU WILL SEE IS YOU'LL SEE A VESICLE COME IN HERE AND FUSE, BE FILLED WITH THE RED DIE BECAUSE THE FUSION PORES OPEN AND THEN IMMEDIATELY BE COATED WITH ACTIN AND COLLAPSE AND RESOLVE. DID YOU SEE THAT? HERE'S ANOTHER EXAMPLE, SO THIS IS JUST AGAIN LUMEN FILL WIDE DYE. THIS IS RED IS NOW THE LABELED ACTIN AND AGAIN YOU WILL SEE A GRANULE COME IN HERE, FUSE, FUSION PORE OPENS, DILUTION HAPPENS, COATED WITH ACTIN. OKAY AND SO WHAT THIS ALLOWS US TO DO IS TO PUT THIS IN THE SEQUENCE. SO WE CAN TELL FROM THESE MOVIES THAT IN FACT, THE FUSION PORE FORMS FIRST BECAUSE WE SEE THE DYE FROM THE LUMEN LEAKING INTO THESE SECRETORY GRANNUALS BEFORE WE SO THE COAT FORMING SO FUSION FORMS FIRST AND THEN ACTIN COATS THE VESICLE AND MEDIATES ITS COLLAPSE. WE CAN ALSO PUT ACTIN AND MICEIN IN A SEQUENCE, 2 BY IMAGING IN THE SAME SLIDE SO AGAIN YOU'LL SEE A SECRETORY GRANNUAL COME IN HERE AND GET COAT WIDE ACTIN AND THEN RIGHT AFTERWARDS GET COATED WITH MICEIN. --MYOSIN. AND THEN AGAIN WE CAN PUT THIS IN A SEQUENCE, SO ACTIN RECRUITMENT PROCEEDS MYOSIN RECRUITMENT AND THIS IS THE SAME AS ROBERTO WYGANT HAS SHOWN AND DURING SECRETION. SO THEN ULTIMATELY WHAT WE WANT TO BE ABLE TO DO IS TO ASK QUESTIONS ABOUT THE TRANSFERASES AND SOME OTHER PROTEIN WEES KNOW NOW ARE TARGETS AND WE'RE IN THE PROCESS OF DOING THAT, BUT JUST TO KIND OF GIVE YOU A FLAVOR FOR IT, THIS A KNOCK DOWN OF 1 OF OUR TRANSFERASES SO BEFORE I PLAY THIS MOVIE, CAN YOU SEE THERE ARE SOME OBVIOUS DIFFERENCES FROM THE WILD-TYPE SITUATION, RIGHT? SO, THE FIRST THING IS, THESE GRANNUALS LOOK A LITTLE IRREGULAR, RIGHT? SO FORMALLY IN WILD-TYPE SITUATION, THE GRANNUALS ARE FAIRLY ROUND AND EACH IN SIZE, HERE WE HAVE GRANNUALS THAT ARE VERY KIND OF ABNORMAL IN MORPHOLOGY AND SHAPE AND SIZE. ADDITIONALLY, WE HAVE GRANNUALS THAT ARE RED AND THIS PROBABLY RUNS A KISS AND RUN EVENT WHERE THE GRANULE FUSED AND GOT THE DYE FROM THE LUMEN AND THEN WASN'T ABLE TO COLLAPSE AND DISSOLVE AND DETACHED AND MOVED BACK INTO THE CYTOPLASM AGAIN. OKAY, AND SO, AGAIN THIS IS THE GREEN IS THE SGS 3, GFP CARGO AND THE RED IS THE LUMEN, THE DIE THAT'S IN THE LUMEN SO WE'LL JUST WATCH WHAT HAPPENS HERE. AND SO WHAT YOU SEE IN THIS MUTANT IS YOU SEE A LOT OF TOPS AND FLASHES INDICATING THAT GRANNUALS ARE FUSING WITH THE MEMBRANE AND IN MANY CASES THEY STAY RED AND DON'T PROPERLY RESOLVE AND YOU'LL ALSO NOTICE THIS LUMEN NEVER REALLY EXPANDS, NEVER REALLY BECOMES GREEN SO THERE ARE OBVIOUS OBLIGATIONS ARATIONS IN SECRETION WHEN WE KNOCK OUT THIS PARTICULAR TRANSFERASE, BOTH IN--WE BELIEVE THE FORMATION OF THE VESICLES TO BEGIN WITH, BECAUSE THEY'RE ABERRANTLY FORMED AND ALSO IN THEIR EVENTUAL COLLAPSE AND SECRETION. OKAY, SO FROM THIS SYSTEM, WE'VE GOT AN IMAGING SYSTEM WHERE WE CAN ACTUALLY IMAGE VESICLE FORMATION SO AS THEY FORM WHICH WE'VE DONE WHICH IS REALLY KIND OF INTERESTING TO WATCH ALSO, VESICLE MOVEMENT, THE FUSION PORE FORMATION, ACTIN AND OTHER FACTOR RECRUITMENT, MYOSIN RECRUITMENT AND COLLAPSE, AND BECAUSE WE'RE WORKING IN DROSOPHILA SOV ILLEGALS AWE CAN RAPIDLY ANALYZE THE GENES WE'RE INTERESTED IN AND NOT ONLY CAN WE DO THIS IN FLIES THAT ARE EXPRESSING DOUBLE STRANDED RNA IN VIVO BUT DUY SET UP A SYSTEM WHERE WE PERFORM EXVIVO RNAi GLANDS IN CULTURE. SO WHAT HAVE WE LEARNED SO FAR, WE'VE BEEN ANAL TO PUT THINGS IN KIND EVER A SEQUEBS SO WE KNOW THE FUSION PORE FORMS FIRST, THE ACTIN COAT IS RECRUITED, THE MYOSIN COAT AND THEN VESICLE COLLAPSE, AND WE ALSO KNOW I DIDN'T HAVE TIME TO SHOW YOU TODAY, WE KNOW THAT RACKETIN IS ESSENTIAL FOR THIS PROCESS, THAT MYOSIN IS ESSENTIAL AND THAT A NUMBER OF THE PGANT, ARE INVOLVED IN BOTH VESICLE FORMATION AND EVENTUAL SECRETION. OKAY. SO JUST TO SUMMARIZE WHAT I'VE GONE THROUGH TODAY. I'VE SHOWN YOU THAT THIS CONSERVED PROTEIN MODIFICATION IS IN FACT ESSENTIAL FOR DEVELOPMENT IN THE FLY. I'VE GIVEN EXAMPLES WHERE IT'S INVOLVED IN INFLUENCING THE COMPOSITION OF THE EXTRA CELLULAR MATRIX BY MODULATING THE SECRETION OF SPECIFIC PROTEINS SO IN THE DROSOPHILA WING THAT WOULD HAVE RESULTING EFFECTS ON CELL ADHESION DURING DEVELOPMENT AND IN MAMMALS, AFFECTING CELL PROLIFERATION DURING SUBMANDIBULAR GRAND DEVELOPMENT AND WE'RE OF COURSE IN THE PROCESS OF LOOKING AT THE ROLE OF THIS MODIFICATION IN MANY OTHER DEVELOPMENTAL PROCESSES. AND THEN ALSO, WE HAVE SHOWN THAT THE GLYCOSYLATION INFLUENCES SECRETION BY SPECIFICALLY MODIFYING ESSENTIAL COMPONENT OF THE SECRET ORY APPARATUS AND MEDIATED CLEAVAGE AND PROVIDING THE FIRST IN VIVO DEMONSTRATION FOR THIS I DIDN'T THINK-YANG BETWEEN O-GLYCOSYLATION AND PROTEOLYTIC CLEAVAGE. AND THEN WE'VE DEVELOPED A SYSTEM FOR IMAGING THE SECRETION EVENTS IN REALTIME IN A LIVING SECRETING ORGAN. SO NONE OF THIS WOULD HAVE BEEN POSSIBLE AT ALL WITHOUT MY ABSOLUTELY FANTASTIC GROUP OF PEOPLE IN MY LAB. SO I ABSOLUTELY HAVE TO ACKNOWLEDGE EVERYTHING THEY'VE DONE. DUY TRAN AS I MENTIONED SPEAR HEADED THE WHOLE RNAi SCREEN AND HAS DEVELOPED THE IMAGING SYSTEM. REBECCA MANDT IS A POST DOC IN THE LAB WHO HAS BEEN WORKING WITH DUY, AND ISOLATING VESICLES TO DETERMINE THEIR COMPONENTS. SUENA JI IS WORKING ON ANOTHER DROSOPHILA DEVELOPMENT TRANSFERASE. LIPING ZHANG, IS WORKING ON THE PRESS WORK RIGHT NOW AND E TIAN IS A LEADER IN MAMMALIAN SECTION OF THE GROUP IN WHAT WE FIGURED OUT IN THE FLY INTO THE MAMMALIAN SYSTEM AND THEN OF COURSE I WANT TO RECOGNIZED AND THANK MY MANY COLLABORATORS, MATT HOFFMAN WHO IS THE SALIVARY GLAND IN NIDCR AND ROBERTO WHO IS THE SECRETION EXPERT AND THE NIDCR COMMUNITY IN GENERAL. EVERYBODY'S VERY INTERACT AND I HAVE GREAT TO WORK WITH. LANCE WELLS AND JAE-MIN LIM, AT A HELPED WITH THE PGANT 4 PROJECTS AND THEN I REALLY HAVE TO THANK THE NIH DROSOPHILA COMMUNITY AND THE GLYCOBIOLOGY COMMUNITY. ABSOLUTELY TREMENDOUS GROUPS OF PEOPLE, SO HELPFUL, ALWAYS ANSWERING ALL KINDS OF QUESTIONS, QUESTIONS THAT ARE IBT--INTEGRATE TELEGENT AND NOT SO INTELLIGENT. ALWAYS PROVIDING SO MANY REAGENTS AND EXTREMELY COLLABORATIVE AND COLLEGIAL AND IT'S BEEN MY PLEASURE TO WORK HERE AND DO SCIENCE HERE. SO WITH THAT I'LL ANY QUESTIONS. [ APPLAUSE ] IF YOU HAVE QUESTIONS PLEASE GO TO THE MICROPHOBE BECAUSE WE ARE BROADCASTING TO MANY DIFFERENT POINTS. >> SO KELLIE THE TANGO WORK IS REALLY, REALLY BEAUTIFUL AND I'M WOBDERRING SINCE TYPE 4 COLLAGEN IS 1 OF THE LARGE OF THE POLYMERS THAT HAVE TO BE SECRETE INDEED THE TACT FORM, IF YOU THINK THAT THIS IS GOING TO BE A GENERAL PHENOMENA FOR SUCH LARGE POLYMERIC COLLAGEN MOLECULES AND SO, WILL THEY HAVE THEIR OWN SIGNATURE TRANSFERASE. >> YEAH, I HOPE SOMENT AND I THINK THAT--IT'S BEEN PUSHED A LOT THAT TANGO IS RESPONSIBLE FOR COLLAGEN SECRETION AND I ACTUALLY THINK IT'S RESPONSIBLE FOR MORE THAN THAT BASED ON OUR WORK AND THEY MAY VERY WELL BE OTHER PROTEINS LIKE TANGO THAT ARE BINDING TO SPECIFIC TARGETING SYSTEM O AND MEDIATING THEIR PARTICULAR TYPE OF ENGULFMENT IN THESE VESICLES THAT NEED TO GO TO A CERTAIN AREA OF THE CELL. SO YEAH, I THINK THIS IS GOING TO BE SOMETHING THAT'S GOING TO TURN UP IN MANY DIFFERENT SYSTEMS. AND I DO THINK THERE ARE OTHER--WE HAVE EVIDENCE THAT THERE ARE OTHER TRANSFERASES THAT ARE MODULATING TANGO, 2. >> KELLIE IT WAS A BEAUTIFUL TALK AND YOU OBVIOUSLY DEVELOPING A WHOLE TOOL BOX OF TOOLS TO BE ABLE TO ANSWER SPECIFIC QUESTIONS. HAVE YOU STARTED TO ADDRESS WHETHER YOU CAN--WHETHER THE TRANSFERASES ARE INTERCHANGEABLE? I MEAN IN YOUR TANGO SYSTEM, YOU PUT IN A PROTEASE OR COULD YOU PUT IN ANOTHER TRANSFERASE TO TRY TO RESCUE THAT. >> SO WE'VE DONE A COUPLING OF THOSE EXPERIMENTS AND A COUPLE DIFFERENT SYSTEMS WHERE YOU TRIED TO RESCUE THINGS. WE HAVEN'T GONE GONE THROUGH THE ENTIRE FAMILY AND PUT EVERYTHING BACK IN EACH 1 OF OUR MUTANTS THAT WE HAD, BUT THE FEW WE TRIED WE HAVE NOT BEEN ABLE TO RESCUE, INDICATE SOMETHING SPECIFICITY, WHETHER OR NOT THERE'S A NUMBER OF THE FAMILY WE PUT BACK IN, WE DON'T KNOW THE ANSWER TO THAT YET BUT IT DOES SPEAK TO SPECIFIC ETICS AND BIOCHEMICALLY, WE KNOW EACH 1 OF THESE ENZYMES AS A SLIGHTLY DIFFERENT PREFERENCE FOR WHERE IT ADDING G ELNECK SO MR. IS PLAUSIBILITY THERE TOO. >> SO WITH THE QUESTION OF EXOCYTOSIS TO BE AFFECTED SIMILARLY? >> CAN YOU REPEAT THAT? >> YOU SEEM TO GET BOTH THE CONSITTATIVE THERE AND THE REGULATOR EXOCYTOSIS TO BE AFFECTED VERY SIMILARLY IS THAT HAPPENING IN THE PACKAGING STAGE OR THE ACTUAL EXOSIGNIFY TO THETIC STAGE? I DON'T KNOW AND THE TRUTH IS FROM THE INGI SHALL TANGO STUDIES DEMONSTRATED THEY FOUND TANGO BECAUSE IT AFFECTED CONSITTATIVE SECRETION, LIKE SECRETION OF A REPORTER AND AND THEN ALSO DISRUPTED GOLGI APPARATUS BUT THEN THEY LOOK AT IT IN THE MAMMALIAN SYSTEM AND ALL KINDS OF PACKAGING, I THINK TANGO IS DOING MULTIPLE THINGS AND I THINK IN DIFFERENT SITUATIONS, DEVELOPMENTALLY, I THINK IT'S GOING TO BE RESPONSIBLE FOR DIFFERENT THINGS SO THE OTHER THING ABOUT TANGO IS IT IS EXPRESSED UBIQUITOUSLY SO IT'S EXPRESSED IN ALL CELLS BUT NOT ALL CELLS MAKE SECRETORY CELLS, THEY'RE NOT ALL DOING THIS TYPE OF POLARIZED SECRETION, SO WE THINK THAT THE ACTIVITY IS MODULATE INDEED CELLS POSSIBLY BY THE ADDITION OF THESE GALNACs TO STABILIZE IT MORE OR DO SOMETHING RELATIVE TO WHAT IT NEEDS TO DO IN A PARTICULAR SECRETORY CELLS. >> SO YOU MAY HAVE MENTIONED THIS AND I MAY HAVE MISS TODAY BUT YOU SHOWED THAT INTERFERON 2 IS INVOLVED IN THE ABSENCE OF GLUE MARIOUSS ON KOIGZ IF YOU KNOCK OUT INTERFERON 2, DO YOU SEE THE DEFECT IN THE GLYCOSYLATION OR IS THE PROTEOLYSIS ESSENTIAL FOR THE DEFECT AND SECRETION? >> SO WE DID THAT CONTROL. SO IF WE KNOCK OUT CADHERON 2, WE KNOCK IT OUT IN THE PR CELLS. >> NO FUNCTIONAL CHANGE? >> NO FUNCTIONAL CHANGE. NO FUNCTIONAL CHANGE. >> UNDERTHE IMPRESSION THAT TANGO WAS A COX 2 BINDING PROTEIN AND RESTRICTED MAINLY TO THE ER, HAVE YOU THIS SPACIAL ISSUE WITH THE GLYCOSYLATEDDED TRANSFERASE, GOLGI HERE, HOW DO YOU EBVISION TANGO GETTING TO THE GOG SCHERECYCLING OR? --GOLGI AND RECYCLING OR? >> IN MAMMALS IT LOOKS LIKE E. R. EXIT SIGHTS BUT IN OURS WE SEE QUITE A BIT OF OVERLAP WITH GOLGI SO I DON'T THINK IT'S QUITE RESTRICTED SPACIALLY AS PEOPLE THINK IT IS. I DON'T THINK IT'S JUST E. R., E. R. EXIT SITES, I THINKOOSE MORE--THERE'S SOME OF IT IN THE GOLGI, SOME OF IT IN THE E. R. AND IT'S CLEARLY O-GLYCOSYLATEDDED SO HERE WE HAVE IN ADDITION TO THE I. P. EXPERIMENTS WE HAVE MASS EXPERIMENTS THAT ARE MAP THANKSGIVING SO IT IS CLEARLY IN CONTACT WITH THE ENZYMES SO IT'S CLEARLY IN THE GOLGI. AND WE ALWAYS SEE THESE ENZYMES IN THE GOLGI IN OUR HANDS, WE DON'T SEE THEM IN THE E. R. >> THANK YOU. >> [ APPLAUSE ]