IT IS MY DISTINCT PLEASURE TO WELCOME DR. ERICA HOLZBAUR, SHE'S PROFESSOR OF BIOLOGY AT THE UNIVERSITY OF PENNSYLVANIA. SHE IS A CHEMISTRY AND HISTORY DOUBLE MAJOR FROM THE COLLEGE OF WILLIAM AND MARY AND HOLDS A Ph.D. IN BIOCHEMISTRY FROM PENN STATE AND WAS A POST DOCTORAL FELL FELLOW AT THE WORCHESTER INSTITUTE. SHE JOINED IN 1992 WHERE SHE'S NOW A FULL TENURED PROFESSOR. AS WELL AS STUDENT AND POST DOCTORAL FELLOW, SHE WAS A PIONEER AND IN CHEMISTRY AND REGULATION OF THE MOTOR DINING. AS A POST DOCTORAL FELLOW SHE MADE A SEMINAL DISCOVERY. SHE CLONED DINACTIN AND SHE'S DONE SINCE THEN BEAUTIFUL FUNCTIONAL WORK ON UNDERSTANDING HOW DINE ACTIN REGULATES THE CRUCIAL PROMOTER THAT LED HER TO FRUITFUL AND HIGH IMPACT COLLABORATIONS WITH NEUROLOGY NEUROLOGISTS THAT INCLUDING OUR OWN, WORKING ON MUTATIONS INVOLVED IN MOTOR NEURON DISEASE. I'M VERY EXCITED TO HOST THIS SERIES BECAUSE HER WORK SPANS FROM THE INTERNATIONAL CLASSIFICATIONRICATE MECHANISMS TO THE LEVEL AND THE MECHANISMS IN CELLS ALL THE WAY TO MOUSE MODELS, HER LABORATORY HAS BEEN AT THE FOREFRONT OF AUTOPHAGY, THE TOPIC SHE WILL DISCUSS TODAY. DR. HOLZBAUER HAS RECEIVED MANY AWARDS FOR HER WORK, AMONG THEM THE RECIPIENT OF THE PFIZER AWARD IN EXCELLENCE, KEITH PORTER FELLOWSHIP AWARD, PRESTIGIOUS JAVIS AWARD FROM NINDS. THANK YOU VERY MUCH FOR ACCEPTING OUR INVITATION. [ APPLAUSE ] >> THANK YOU SO MUCH. IT'S TERRIFIC TO BE HERE AND NERVE RACKING SINCE I HAVE NOT JUST ONE BUT TWO FORMER STUDENTS IN THE AUDIENCE AND QUITE A LOT OF COLLEAGUES, SO THANK YOU VERY MUCH FOR THE INVITATION. MY LAB HAS FOCUSED FOR QUITE A LONG TIME IN LOOKING AT THE ROLE OF MOTORS AND THE IRPT ACTION WITH CYTOSKELETON IN IT HOW THAT CONTRIBUTES TO CELLULAR FUNCTION AND THIS IS DRAWN BY JULIAN RIGHT THERE, AND SORT OF INCAPSULATES WHAT WE FOUND INTERESTED IN THE LAB, THE CHEMISTRY OF THE LAB, THE DINING, AND [INDISCERNIBLE] THAT'S WORKED ON AND [INDISCERE], AND THE ROLE IN MOVING ORGANELLES ALONG THIS SIDE THE CYTOSKELETON. SO TODAY I WILL TALK ALMOST EXCLUSIVELY ABOUT MY TRANSPORTS AND THE MOVEMENT OF THESE ORGANELLES ALONG THE TUBIALS. FOR THOSE THAT DON'T THINK ABOUT THIS MUCH, I WANT TO REMIND YOU OF THE SIGHT CYTOSK [INDISCERNIBLE] WITH THE PLUS END TO OUT. THESE ARE TRACKED MOTORS, SO THE MOTORS WILL TAKE THEM OUTWARD TO THE PERIPHERY OF THE CELL, AND [INDISCERNIBLE] WHICH WILL BE TALKED ABOUT THE MOST TODAY WILL MOVE CARGO FROM THE PERIPHERY OF THE CELL FOR A PROFESSOR. WE KNOW FROM DECADES OF WORK THAT THIS TYPE OF TRANSPORT IS ESSENTIAL FOR HOW EUKARYOTIC CELLS FUNCTION, BUT IT BECOMES INCREDIBLE LOAMACYY IMPORTANT IN THE CONTEXT OF THE NEURON. SO WE KNOW THAT NEURONS ARE CHARACTERIZED BY EXTENDED AXONS AND [INDISCERNIBLE] AS WELL AS OFTEN TIMES VERY DENSE DENDRITIC [INDISCERNIBLE]. THEY TRANSPORT ABSOLUTELY ALONG THESE [INDISCERNIBLE]. THE TO TELL YOU ABOUT THE SCALE FOR ANYONE THAT DOESN'T STUDY NEURONS IN HERE, THIS SCHEMATIC OF A NEURON SUGGESTS REASONABLABLENESS TO AXON BUT [INDISCERNIBLE] CAN HAVE QUITE REASONABLE AXONS. THIS IS DRAWN TO SCALE, HUMAN MOTOR NEURON WITH A CELL BODY HERE, EXTENDED AXON, FINALLY TO THE NEUROMUSCULAR FUNCTION DOWN HERE, SO THIS CORRESPONDS TO THE [INDISCERNIBLE] 20,000 TIMES LONGER THAN THE DIAMETER OF THE CELL. WHILE HE'S DOING THAT, LET ME REMIND YOU THAT WHAT'S INTERESTING ABOUT THIS PROBLEM IS THAT IN THE NEURONS MOST OF THEM GENERATE FROM NEW MATERIAL, AND LIPIDS IS OCCURRING IN THAT SOMA AND THAT HAS TO BE ACTIVELY DELIVERED TO THE TIP MUCH THE REASONABLE NEURON HERE, SO THESE PROTEINS, GO ON A JOURNEY THAT CAN TAKE FROM A FEW HOURS TO A FEW DAYS TO A FEW MONTHS IN SOME OF THE LONGEST NEURONS AND THE PROTEIN SYNTHESIS OF THE LOW TRANSPORT, SO WE ALSO HAVE THE COMPLIMENTARY PROBLEM IF YOU WORRY ABOUT CONTINUOUS IS PLIE, YOU HAVE TO WORRY ABOUT CLEARANCE SO YOU HAVE TO REMOVE AGGREGATED PROTEINS, DYSFUNCTIONAL ORGANELLES SUCH AS MITOCHONDRIA FROM THE DISTAL END OF THIS AXON FOR CLEARANCE BACK TO THE SELL BODY. YES? NO. MICROPHONE. ALL RIGHT CAN EVERYBODY HEAR? SHOULD I YELL LOUDER? SO WE HAVE IT CONTINUOUS HOUSEKEEPING OF SUPPLY AND CLEARANCE AND WE ALSO HAVE SPACIAL TEMPORAL SIGNALING SO, FACTORS SUCH AS NERVE GROWTH FACTOR HAD TO BE ACTIVELY TRANSPORTED FROM THE DISTAL END OF THE CELL BACK TO THE CELL STUDIES OF MULTIPLE ENDOCRINIA SO I WANT TO FOCUS ON TODAY LARGELY PUBLISHED WORK, I'M TRYING IT OUT ON YOU AND YOU CAN GIVE ME YOUR FEEDBACK WHERE WE'VE BECOME INTERESTED IN SPACIALLY SIGNIFICANT INFORMATION IN OF TRANSPORT IN THE NEURON. SO THERE'S OBVIOUSLY QUITE A LOT OF EXPERTISE HERE AT NIH IN TERMS OF SELECTIVE TRAFFICKING FROM--BETWEEN AXON AND DENDRITES AND A LOT OF THIS INVOLVES SPACIALLY SPECIFIC FILTERING AT THE AIS OR THE PREAIS SECTION OF THE NEURON. THE BUT THERE'S ALSO OTHER AREAS O THE NEURON WHERE YOU SEE SPACIALLY SPECIFIC REGULATION OF TRANSPORT, IN PARTICULAR, I WILL FOCUS TODAY ON THE SPECIFICITY OF THAT REGULATES TRANSPORT INITIALIATING IN THE DISTALAXON. SO THIS TRANSPORT INITIATING DISTALLY IN THE NEURON IS ALL DRIVEN BY CYTOPLAYS MIC DYNEIN AND IT'S THE MOTEAR WITH THESE BEAUTIFUL AAA DOMAINS THAT REGULATE THE ASSOCIATION OF THESE MICROTUBULE BINDINGS. SO WITH THE MIKE ON TUBULIN IS AN HDP FASHION HOWEVER WE KNOW FROM DECADES OF RESEARCH AGAIN THAT THE DYNEIN AND THERE'S INTERACTION BETWEEN THE INTERMEDIATE CHANGE HERE BYPASSING DIRECTLY TO THIS AS SHOWN BY CLAIR WATER MAN CHER IN MY LAB YEARS AGO AND THIS IS SHOWN MANY YEARS AGO AND I'LL TELL YOU A LOT ABOUT THIS DOMAIN, THE CAP SLIDE DOMAIN. TOGETHER THIS COMPLEX IS DRIVING THE RETROGRADE TRANCE PORT I'VE BEEN TALKING ABOUT IN THIS NEURONS. AND WE KNOW FROM MANY STUDIES IN MODEL ORGANISMS AS WELL AS HUMAN GENETICS JUST HOW IMPORTANT THESE MOTORS AND THIS MOTOR COMPLEX IS, SO IF WE LOOK IN THE SE FOR THE DYNEIN HEAVY CHAIN, WE CAN SEE THAT MUTATIONS IN THIS DOMAIN ARE RESPONSIBLE FOR AN ARRAY OF HUMAN DISEASES INCLUDING [INDISCERNIBLE], VARIOUS FORMS OF SPINAL MUSCULAR ATROPHY AND DEVELOPMENT AT DEFECTS ALSO COGNITIVE DYSFUNCTION HOWEVER, I WILL FOCUS TODAY ON MUTATIONS IN DINE ACTIN AND SPECIFICALLY IN THE AREA I A REA I WAS TELLING YOU ABOUT. --WE WERE ABLE TO SHE IN THIS MUTATION IS DEEP IN THE DOMAIN AND AS CAN YOU SEE WHEN I ROTATE THE STRUCTURE YOU CAN SEE IT'S EMBEDDED AT THE CORE DOMAIN OF THIS PROTEIN AND YOU WOULD EXPECT PERHAPS THIS SWITCH WOULD RESULT IN A DEFECT IN TRUCKURE AND PACKING AND THAT'S EXACTLY WHAT HAPPENS. IT CAUSES A DISRUPTION OF THE STRUCTURE THAT LEADS TO AGGREGATION IN THE NEURONS. HOWEVER WITH A FUNCTION WILL PERSPECTIVE WITH THE IDENTIFICATION OF A NOVEL SET OF MUTATIONS OF THE SAME DOMAIN, 11 AMINO ACIDS AWAY, IN 2009, WHERE HE FOUND MULTIPLE MUTATIONS IN THIS DOMAIN CAUSES VARIANT FORM OF NEURODEGENERATION CALLED PERRY SYNDROME. IT'S A VARYING FORM OF BARKIN SON'S DISEASE WITH A LATE ONSET AND RAPID PROGRESS, SO THE PATIENTS ONCE THEY SHOW SIGNS OF THE DISEASE ARE DEAD WITH TWO OR THREE YEARS. SO WHAT'S INTERESTING ABOUT THESE MUTATION SYSTEM THAT THEY'RE IN THE DOMAIN, BUT THEY'RE EXPOSED ON THE SURFACE, SUGGESTING THEY'RE GOING TO EFFECT BINDING INTERACF THE DOMAIN. SO THAT WAS REALLY INTERESTING AND MADE US REALIZE MORE THAN EVER THAT WE HAD TO INVESTIGATE THE ROLE OF THIS DOMAIN. SO WE STARTED OUT BY DOING A SIMPLE TRANSPORT ASSAY IN NEURONS, AND I'M SHOWING YOU HERE IS THE GANGLION NEURON IN CULTURE. THEY'VE BEEN TRANSFECTED WITH THE FLUORESCENT LAMP ONE CONSTRUCT SO CAN YOU SEE THE LYSOSOMES ALONG THE PROCESS, IF YOU LOOK IN A NORMAL AXON WHAT YOU'LL SEE WITH LAMP ONE, LABELED ORLG ANLES IS THIS TYPE OF BI-DIRECTIONAL MOTILITY ABOUT 60% RETROGRADE, 40% ANTERIOR GRADE AND ALSO STATIONERY ORGANELLES, WE CAN SHOW THAT THE P150 GLUTEIS THE WHOLE COMPLEX IS ESSENTIAL FOR TRANSPORT BECAUSE WHEN WE DEPLETE IT, WE SEE ABSOLUTELY NO MOTILITY. IMPORTANTLY WE CAN SHOW THIS IS NOT AN OFF TARGET EFFECT BUT FULLY EXPRESSING IT BUT BY EXPRESSIONING THE WILD-TYPE CONSTRUCT NOW WITH THIS AS AN ASSAY WE CAN DO THE EXPERIMENT WE WANT, AND THE CONSTRUCT THAT LACKS THE SAME CAP DOMAIN I'M TELLING YOU ABOUT. WHEN WE DO THAT, WE SEE NO MUSEUMTILITY DEFECTS SO DESPITE THE FACT THAT ALL THESE MUTATIONS HAVE BEEN IDENTEDDIFIED, THE MOTILITY IS NOT ALTERED WHEN THE DOMAIN IS MISSING. SO THIS I HAVE TO SAY, PHENOCOPIED EXACTLY WHAT THEY HAD SEEN IN S-TWO CELLS BUT WE WERE VERY SURPRISED THAT THIS DOMAIN DIDN'T SEEM TO BE REQUIRED FOR TRANSPORT ALONG THE Y OF THEAXON. SO LUCKILY MOUGHAMIAN WENT DEEPER AND TO LOOK AT THE P150 IN THE NEURON. SO HERE IS A NEURON EXPRESSING A SPACE FILLING MOLECULE WHERE YOU RATIO THOSE TWO AND YOU GET A CLEAR DISTAL LOCALIZATION WHEN THE CAP GLY DOMAIN IN THE EXPERIMENT. WHEN IT LACKS YOU CAN SEE IT GIVES THE EXACTLY THE SAME PATTERN AS GLYP AND JUST THE DISTALLIZATION SO FROM THIS EXPERIMENT WE HYPOTHESIZE THEN THAT THE DOMAIN IS REQUIRED FOR JUST A LOCALIZATION OF DYNACTIN. --SO TO TEST THAT ALL WE HAVE TO DO IS DISPLAY INTO THE NEURON, IN THESE NEURONS THAT ARE EXPRESSING THE LAMP ONE CONSTRUCT YOU CAN SEE IT'S RELATIVE LEE CHAOTIC SIGNAL. SO TO CLEAN UP THE ASSAY, WE FORM AN INITIAL PHOTOIN THIS REGION AND THEN IT'S FAIRLY EASY TO MEASURE PHLOX FROM THE END OF THE AXON INTO THIS PHOTOBLEACHED REGION. IF WE DO THAT AND THEN MAKE A CHIME O GRAPH INTO THOSE WE DON'T STUDY MOTILITY, EYE TIME O GRAPH IS WHEN YOU TAKE A FRAME FROM THE NEXT FRAME AND NEXT FRAME AND NEXT FRAME AND NEXT FRAME AND LINE THEM UP AND YOU HAVE THIS ORIENTATION, AND TIME ON THE Y-AXIS, WE CAN THEN LOOK AT THE INITIATION EVENTS WHERE OW CARGO IS INITIATING FROM THE DISTAL EPPED AND MOVING IT INTO THE AXON. WHEN YOU DO THAT, IN NEURONS THAT ARE KNOCKED DOWN FOR WILD-TYPE, AND RESCUED YOU GET YOUR BASELINE INITIATION RATE AND THEN YOU CAN PUT THAT IN NEURONS WITH THE DELTA CONSTRUCT. AND FROM THESE DATA YOU CAN SEE THERE'S A CLEAR DECREASE IN FLUX FROM THE TWISTAL END OF THE AXON IN NEURONS EXPRESSING THIS CONSTRUCT THAT WILL ASK THE CAP SLIDE DOMAIN. THE OH AND I JUST WANT TO EMPHASIZE THAT TOM LLOYD HAS VERY SIMILAR DATA IN VIVO SLIDE MODEL SO WE'RE CONNIDENT THAT THIS REQUIRED IN HAD NEURONS AND IN HAD VIVO IN THE FLY. I INTRODUCED THIS TOPIC BY SAYING THAT THEIR MUTATIONS IN THIS THE CAP SLIDE DOMAIN THAT CAUSE PERRY SYNDROME SO WE DO A SIMILAR EXPERIMENT RESCUED WITH THE CONSTRUCT OF THE MUTATION RATHER THAN A COMPLETE DEPLETION OF THE DOMAIN, WE SEE THE VERY SAME THING. SO THE PERRY SYNDROME IS ESSENTIALLY A NULL, FUNCTIONAL NULL FOR THE CAP-GLY DOMAIN. SO THIS IS POSITIVE TO THE LAMP ONE MARKER AND THEY WANT TO GO TO THESE STUDIES AND ASK DID THIS DOMAIN FUNCTION AS A TRANSPORT OF OTHER CARGO? SO HE TESTED A DIVERSE ARRAY OF CARGOS, SHOWN HERE ARE THE TRANSPORT OF ORGANELLES LABELED FIVE OR TREK A OR TREK B. OR SIGNALING ALL REQUIRE THE CAP SLIDE DOMAIN SINCE WE SEE CLEAR DEFECTS OF FLUX WHEN WE RESCUE THE CONSTRUCT THAT LACKS THAT DOMAIN. CONNECTS WITH THE MITOCHONDRIA AND MITOCHONDRIA SHOWED A SIMILAR DEPENDENCE FOR THE CAPP GLY DOMAIN AND YOU LOOKED AT THE ATP POSITIVE AREAIERS AND THEY TOO DEPENDEDOT CAP SLIDE DOMAIN SO IT SEEMS ROBUST ACROSS CARGO, IF YOU'RE INITIAG TRANSPORT FOR THE DISTAL END OF THE NEURON, YOU REQUIRE THIS CAP-GLY DOMAIN, SO THAT SETS UP A VERY SIMPLE MODEL WHERE THIS CAPP-GLY DOMAIN WHERE THE MICROMOLAR AND THE NICE ROUGH ATOM TUBUAL WILL INITIATE AN INITIAL INTERACTION OF THE COMPLEX WITH THE MIKE O TUBIAL AND THAT THAT WOULD THEN ENHANCE THE EFFICIENCY OF INITIATION OF RETROGRADE PANZ PORT BUT WE WANTED TO GO DEEPER AND ASK HOW EXACTLY IS THIS WORKING AND HOW IT'S REGULATED AND THAT'S GOING TO BE WHAT I'M TALKING ABOUT FOR THE REST OF MY TALK. SO FIRST WE WANT TO USE SINGLE MOLECULE APPROACHES TO REALLY SHOW DEFINITIVELY THAT BINDING TO THE MICROTUBIAL BY THIS DOMAIN COULD ENHANCE TRANSPORT. SO WE TURN TO A SINGLE MOLECULE ASSAY, AND WE TURN TO A BEAUTIFUL REAGENT BY CHEN ZHANG, SHE TWEPPED A KNOCK-IN MOUSE THAT'S BEEN A FANTASTIC TIME STUDYING AND WE CAN ISOLATE THE NEURONS FROM THIS MOUSE AND WATCH THE LABELED GFP AND I WON'T BE SHOWING YOU THAT OR WE CAN PURIFY DINING FROM THIS MOUSE AND IT CO-PURIFIES WITH LABELED SUBUNITS AS SHOWN HERE, IT'S A CROSS GRADIENT WHERE WE FRACTIONATE SUPERINATENT FROM THE MOUSE, AND YOU CAN SEE WE CAN PURIFY THE DYNEIN ACTIN FROM THIS MICE THAT HAS TED GFP LABELED ASSOCIATED WITH IT. LET AND IN ORDER TO LOOK AT DYNACTIN, WE TURN TO RECOMBINANT CONSTRUCT, A TRUNCATED FORM THAT HAS THE CAP-GLY DOMAIN I'M TELLING YOU ABOUT CRITERIA THE THEN THE TRUNCATED TERMINAL END. AS EXPECTED, IT'S GOT A WEAK BINDING TO MICROTUBUALS AND DIFFUSES ALONG THE LATTICE AS SHOWN HERE. WELL, WE NICKED THE TWO, WE SEE THAT WE SEE RARE BUT INSTANCES OF COLOCALIZATION AND CO MOTILITY ALONG THE MICROTUBUAL IING THAT IN THESE DILUTED CONDITIONS WE ARE GETTING THE DYNACTIN COMPLEX. WE CAN THEN ASK HOW DOES THE BINDING OF THE DYNEIN TO THE MICROTUBULES DEPENDOT EDITION OF THIS RECOMBINANT P150 GLUTECONSTRUCT AS SHOWN HERE, WE LOOKING ASTERISKS MICROTUBULES AND SINGLE MOLECULES OF DYNEIN AND QUITE CLEAR FROM THESE INDIVIDUAL SNAPSHOTS IF WE MIX THIS WITH THE MOTIF, WE SEE MANY MORE BINDING OF THEM ALONG THE MICROTUBUAL AND WE DON'T SEE THAT IF WE MIX IN A CONSTRUCT THAT CAN BIND DYNEIN VIA THIS DIRECT BINDING INTERACTION BUT LACKS THE CAP-GLY DOMAIN, SO WE HAVE THE CAPACITY TO ENHANCE TRANSPORT ALONG THE MICROTUBULE AND IT'S SPECIFICALLY TO ENHANCE THE INITIAL ASSOCIATION OF THE MOTOR WITH THE MICROTUBULE. BUT AS MOST OF YOU KNOW IN THIS ROOM, BIOLOGY IS RARELY--ALWAYS COMPLICATED FACTOR. AND THE CASE OF DYNACTIN THE MAJOR COMPLICATED FACTOR IS THAT WE KNOW THAT THE SAME CAP-GLY DOMAIN CAN ALSO BIND EBs, EB-ONE AND THREE, AS WELL AS CLIP-170. THOSE WHO DON'T THINK OF MINDING PROTEINS WHAT THEY ARE, THESE ARE PROTEINS THAT CAN SPECIFICALLY TRACK THE GROWING MICROTUBUAL END, THE EB-1 AND THREE ARE THE CONONICLE TRACKING PROTEINS AND ALL YOU NEED IS EBs AND DYNAMIC MICROTUBULES TO FULLY RECAPITULATE THIS TRACKING. SO THIS IS WORK THAT WE PUBLISHED A NUMBER OF YEARS AGO AND A COLLEAGUE'S LAB HAD A BEAUTIFUL PAPER SHOWING THE SAME THING THAT EB-11 CAN BIND TOO AND TRACK WITH THE GROWING PLUS END OF THE MICROTUBUAL. SO IS THIS RELEVANT TO OUR MECHANISM OF PLUS-M INITIATION, WELL WE LOOKED AT THESE IN CULTURE AND IN NEURONS EXPRESSING GFP LABEL, THE EBTHREE AND YOU CAN SEE GROWING MICROUBULES ALL ALONG THE LENGTH OF THE AXON BUT THERE'S A PARTICULAR ENRICHMENT OF THESE DYNAMIC ENDS IN THE LAST 10-MICRONS. THE MOST DISTAL 10-MICRONS OF THE AXON. EVEN MORE TELLINGLY, IF WE DEPLETE EB-ONE AND THREE FROM THESE NEURONS, WE COMPLETELY LOSE THAT PLUS AND ENRICHMENT OF THE DYNACTIN THAT I WAS TELLING YOU ABOUT. SO INSTEAD OF HAVING THE STRONG RATIO OF IN THE CONTROLS, IF WE DEPLETE EB-1 AND THREE, WE LOSE THIS PLUS END SPECIFIC ITS. SO THAT SUGSUGGESTS THAT EBTHREE OR ONE MIGHT HAVE A ROLE IN GIVES US PLUS AND CONCENTRATION AND THAT'S PLUS ENHANCEMENT OR DISTAL ENHANCEMENT OF TRANSPORT INITIATION. SO WE WONDERED WHAT THE MECHANISM MIGHT BE. COULD IT BE THAT THE P150 TO THE DYNAMIC PLUS END? THE REASON WE THOUGHT ABOUT THIS MOST SIMPLE MODEL IS THAT STUDIES THAT WE HAD DONE AND AGAIN IN THEA LAB HAD ALSO DONE, CAN YOU TAKE A PROTEIN SUCH AS CLIP 170s WHICH HAS A WEAK AFFINITY AND THEY WILL INHERENT THIS SPECIFICITY, IF YOU MIX INTO THIS ASSAY, JUST BLACK EB-1 SO WE'RE MATT HOLT SEEING IT IN THESE MOVIES, ALL OF A SUDDEN YOU RECONSITUTE A VERY STRONG PLUS END TRACKER AND WE REASONED THAT P150, MIGHT HAVE A SIMILAR INTERACTION WITH EB-ONE THAT COULD LOCALIZE THIS MICROTUBUAL PLUS END BECAUSE LIKE P150, GLUTE, CLIP-170 AS NOT JUST ONE ABOUT TWO CAP SLIDES IN THE END TERMINAL SO BY ANALOGY WE THOUGHT THE GGLUED WITH COULD BYPASSEDDED. HOWEVER TO OUR DISAPPOINTMENT AND MANY TRIES WE FOBBED WE COULDN'T RECONSITUTE THAT INTERACTION, EVEN THOUGH OUR CONTROLLING EXPERIMENTS WORK, WE ALWAYS GOT THE PLUS END SPECIFIC LABELING OF HIKE ROUGH ATOM TUBUALS WITH CLIP ONE SECTIONAL ANALYSIS THAT WE CHANNELED BEFORE WE DID NOT SEE THAT WITH THE P150 GLUED. THERE WAS A PAPER THAT WAS DEPENDENT ON RECRUITMENT OF A P151 STRUCTURALLY TO MT PLUS ENDS AND INTERACTION WAS WEAK AND EASILY COMPETED OFF. INSTEAD WE HAVE TO INVOKE AN INDIRECT ASSOCIATION. SO I TOLD THAT YOU CLIP 170 HAD CAP GLY DOMAINS THAT'S SUFFICIENT TO CAUSE DIRECT TRACKING OF THE PLUS END. WHAT I DIDN'T TELL YOU IS THE C-TERMINAL PART OF CLIP-170 CAN DIRECTLY BYPASSED TO THE CAP GLY TO THE GLUED. SO INSTEAD OF THE MOST EASY EXPLANATION WHICH IS THAT IT'S BINDS DIRECTLY TO THE MICROTUBULE, THAT THE P-50 WOULD BIND EB-1 AND IT'S THE MICROTUBUAL SLIDES IN, WE NEED TO INVOKE, THIS HERE THAT THIS THERE'S AN INDIRECT MECHANISM THAT EB-ONE THAT IN TURN COULD RECUTE P150 GLUED. NOW THE QUESTION IS: IS THERE EVIDENCE FOR THIS INDIRECT MECHANISM, NOT JUST IPT GREATER VITRO BUT IN NEURONS? SO WE CAN DO THE SAME TYPE OF EXPERIMENT THAT I TOLD YOU ABOUT, BEFORE, WHERE WE CAN DEPLETE CLIP-170 TO NEURONS AND CARRY THAT TO CONTROL NEURONS AND ASK THERE IS A PROBLEM WITH TRANSPORT INITIATION? AND AS CAN YOU SEE FROM THIS QUANTITATION HERE, THERE'S NO CHANGE IN FLEX ALONG THE MIDAXON IN THIS EXPERIMENT, SO CLIP-170 IS NOT REQUIRED FOR TRANSPORT OF ORGANELLES ALONG THE SHAFT OF THE AXON, BUT IT IS REQUIRED TO GET THE MOST EFFECTIVE INITIATION OF TRANSPORT FROM THE DISTAL END. SO THAT BRINGS US UP TO WHAT'S PUBLISHED. LET WE ENDED UP WITH A MODEL PUBLISHED IN 2013 WHERE WE HYPOTHESIZED THAT EB-ONE AND THREE ARE DISTALLY ENRICHED IN MICROTUBE Y'ALLS IN THE DISTAL AXON AND I SHOWED THAT DATA AND THEN CLIP 170 CAN RECRUIT THE DINING ACTIN AND POTENTIA WILY CARGO BOUND, AND THAT GIVES US THIS ENHANCED INITIATION FROM THE DISTAL EDGE. SO THIS IS I NICE MODEL BUT OBVIOUSLY RAISES MANY QUESTIONS SUCH AS HOW IS THIS SPECIFICALLY REGULATED, HOW IS IT TUNED TO THE FUNCTION OF THE NEURON AND THAT'S WHAT I'M GOING TO TELL YOU ABOUT FOR THE REST OF THE TIME. IT'S NO--SURPRISE TO THE ROLL OF MACAQUE LAB THAT THE ANSWER INVOLVES MICROTUBUAL MODIFICATION AND IT WOULD BE NO SURPRISE TO KNOW THAT IT INVOLVES THE SPECIFIC REGULATION OF CLIP 170. SO I'M SHOWING HERE CLIP-ONE SEIVET AS AN ELONGATED MOLECULE WITH THE TANDEM DOMAIN HERE AND I DIDN'T MENTION BUT IT HAS A LONG FOIL SECTION WHICH FORMS THE DIMER IN VIVO AND INVITRO. WHAT HER LAB HAS SHOWN IS THAT THIS MOLECULE IS AUTOINHIBITTED THAT THE HEAD CAN FEEDBACK INDIRECTLY AND INTERACT WITH THE TAIL AND MORE IMPORTANTLY, FOR WHAT I'M TELLING YOU ABOUT THAT, HEAD TO TAIL INTERACTION IS TIGHTLY REGULATED BY PHOSPHORYLATION. SO IF YOU HAD THE NONPHOSPHORYLATED FORM PARTICULARLY HERE, TO FORM CLIP A IN OUR MUTANT CONSTRUCT, YOU GET THE EXTENDED FORM, THE ACTIVE FORM AND IF THEY'RE MUTATED TO ACIDIC AMINO ACID, YOU GET THE CLOSED FORM AND HERE'S SOME IMAGES WITH A CLOSE AND OPEN FORM THAT YOU PREVIOUSLY SHOW. SO HOW DOES THIS EFFECT THE LOCALIZATION OR ACTIVITY IN THE CELL, YOU CAN FIRST OF ALL LOOK AT COMMON TELES THIS IS JUST IN WILD-TYPE FORMS YOU CAN SEE THE COMET TAILS AT THE END OF THE MICROTUBUALS AND THE CLOSED FORM MUCH SHORTER TAIL, AGAIN REPEATING PREVIOUS WORK. WHY IT'S INTERESTING TO US BECAUSE WHEN WE LOOK AT WHERE THE GLUTEIS, ALONG THESE COMET TAILS WE SEE PRELIMINARY EVIDENCE OF TARGETING SYSTEM O LING EVEN IN CELLS SPECIFIC AT THESE PLUS ENDS THAT ARE POSITIVE FOR CLIP 170, SO WE CAN SEE THESE APPARENTLY STRUCTURES AT THE END OF THESE LIP 170-A-MICROTUBUAL M. AL TERNATIVELY IF WE EXPRESS A FORM OF CLIP 170, OF P-150, GLUE WITH THE TEARING MUTATION I TOLD YOU ABOUT, THE LOSS OF FUNCTION MUTATION, WE SEE NOW RECRUITMENT OF THE P-150 TO THE PLUS END. SO USING FORM TO GO DEEPER AND TO LOOK AT THESE STRUCTURES IN MORE DETAIL, THESE ARE IN CELLS EXPRESSING THE GLUED, WE CAN START TO SEE THE PARTICULAR STRUCTURES ALONG THESE ENDS THAT ARE NOT SEEN FROM THE CELLS EXPRESSING THE PERIMUTE ANT AND OF ANY WHEN WE LOOK AT RELATIVE DIAMETER OF THESE STRUCTURES, THEY HAVE WHAT WE WOULD EXPECT FOR TRANSPORT VESICLES AND AN AVERAGE DIAMETER OF ABOUT A HUNDRED NANOMETER. SO, NOW WE HAVE A PROPOSED MECHANISM AND ONE WAY TO SHOW THAT IS TO RECONSITUTE INVITRO FROM COMPONENT PART SO WHAT WE WANTED TO DO WAS SEE IF WE COULD RECONSITUTE AN INITIATION MECHANISM INVITRO IN A TURF MICROSCOPE SO HERE WE'RE ADDING THE VESICLES AND I'LL TELL YOU ABOUT THEM IN A MINUTE. EB-1 AND CLIP 170 AND ASKING HOW WE RECONSITUTE THIS SPECIFICITY THAT QUEY SEE AT THE TIP OF THE NEURON. --THAT WE SEE AT THE TIP OF THE NEURONS EMPLOYED SO FIRST NEURON 18 VESICLES WE COULD DO THIS WITH MOUSE, OR IN THIS CASE A MOUSE THAT WE MADE A FEW YEARS PRIOR, WE CAN MAKE MOUSE MODINATE IT AND SEPARATE IT ON THE STEP GRADIENT TO GET A STRAIKS THAT'S HIGHLY ENRICHED IN LATE ENDOSOMES AND LYSOSOMES. OF NOTE THEY HAVE A HUNDRED NANOMETERS IN OUR STORAGE UNITS EMPLOYED WHAT WE LOOK AT PURIFY FOR THESE VESICLES FROM BRAINS WE SEE THEY'RE POSITIVE FOR THE MOTORS, THE MOTORS, PLUS CONSISTENT WITH A ENDOSUCH AS LAMP ONE AND OTHER MARKERS SUCH AS RAB FIVE. IMPORTANTLY THEY HAVE LOW LEVELS OF CLIP-170 SO WE'RE ABLE TO VARY THE TYPE OF CLIP-170 WE USE IN THE ASSAY. SO THE ASSAY THEN IS TO MIX THESE PURIFIED GFP LABELED VESICLES. UNLABELED PURIFY IN RECOMBINANT IN EB-ONE AND CLIP 170. SO WE'VE BEEN EXPERIMENTING WITH DIFFERENT WAYS TO MAKE CLIP-170 BUT EXPERIMENTS I WILL TELL YOU ABOUT ARE ALL USING CLIP 170 THAT'S EXPRESSING [INDISCERNIBLE]. ONCE WE USE THESE CELLS THESE WILL GIVE US A BRIGHT SPAIBL LABEL AND THEN WE CAN USELESS USE THAT TAG TO QUANTITATIVELY ADD THE IDENTICAL AMOUNT OF THE PROTEIN EITHER THE WILD-TYPE OR THE DIFFERENT MUTE ANTS TO OUR SARKS ASSAYS. SO WE CAN THEN NICK THESE UP INTO A CHAMBER AND LOOK AT THE BINDING EVENTS OF THESE VESSELS TO THE MICROTUBULES TO THE CORPS SLIP TO THE BIOTIN LINKAGE. IN IF ORDINANCE NUMBERER TO UNAMBIGUOUSLY ANALYZE THESE DATA IN AN UNBIASED WAY, WE USE THE ANALYZERS UTRACK APPROACH WHERE WE'RE TAKING MULTIPLE AND MATCHING THEM, SEGMENTING THEM INTO THE MICROTUBUAL OR BACKGROUND AND THAT WAY WE CAN MAP THE PARTICLES AND CALCULATE PLANNING RATES AS WELL, AND VELOCITIES NOT JUST FOR A FEW PARTICLES FOR THOUSANDS OF PARTICLES. SO WHAT DO WE SEE WHEN WE DO THIS ASSAY. SO HERE ARE TIMER GRAPH SHOWING DIFFERENT EVENTS WHEN WE HAVE OUR VESICLES MIXED WITH BLACK EB ONE AND NONTRANSVECTED CELL LIAISON SADE SO OUR BACKGROUND CONDITION. CAN YOU SEE A NUMBER OF BINDING EVENTS, SHORT AND LONG, MOTILITY BUT GIVING--YOU DIRECTIONAL MOTILITY AS WELL AS DIFFUSIVE MOTEL IL. LET IF WE PUT CLIP 170 IN, WE CAN PREPARE EITHER THE MIMIC FOR THE EXTENDED STAY, THE ACTIVATED STAY OR A MIMIC FOR THE CLOSE STATE. WHAT YOU CAN SEE IS THAT THERE'S NO MAJOR DIFFERENCE IN THIS THE RATIO OF SHORT TO LONG EVENTINGS. THERE'S NO SWITCH IN TERMS OF FROM DIFFUSE, MORE, LESS DEFUSIVE OR LESS,S OR MORE PERSPECTIVE, THE MAJOR CHANGE IS THAT WE SIMPLY SEE MORE EVENTS. SO THAT'S QUANTITATED DOWN HERE, WE SEE MORE THAN A TWO FOLD INCREASE IN THE LANDING RATES, WHEN CLIP 170 IS IN ACTIVE FORM AS COMPARED TO AN ABSENCE OF EXOGENOUSLY ADD CLIP-170 OR WHEN WE ADD THE CLOSED CONFIRMATION. STRIKINGLY HOWEVER, WE SEE ABSOLUTELY NO CHANGE IN THE DWELL TIME ALONG THE MICROTUBULES, OUR EXPOTENTIAL CLIPS ARE ESSENTIALLY THE SAME INDICATING WE'RE EFFECTING LANDING RATES BUT NOT WELL TIME IN THESE ASSAYS. SO THAT'S INVITRO, CAN WE SEE WHAT WE HAVE THE NEURON. AND I TOLD YOU ABOUT THIS BEFORE WHERE WE'RE LOOKING FOR VESICLES INTO THE AXON SHARE. WE'VE DONE THIS UNDER A RANGE OF CONDITIONS WHERE YOU USE THE MEMBERS OF THE COMMITTEE CONDITION, DEPLETION OF ENDOGENOUS CLIP 170, RESCUE WITH WILD-TYPE CLIP 170 OR RESCUE WITH EITHER THE NONPHOSPHORYLATABLE FORM OR FOSTER NURSED NO MAGNETIC. THE QUANTITATION TELLS YOU WHAT YOUR EYES ARE SEEING IF YOU LOOK AT THESE CHIME O GRAPHS, DEPLETION OF CLIP BON 70 AS I SHOWED YOU BEFORE GIVES A SUBSTANTIAL DECREASE IN THE END OF THE AXON RESCUE WITH WILD-TYPE CLIP 170 RESTORES THE EFFECT. RESCUE WITH THE NONPHOSPHORYLATABLE, NONAUTOINHIBITTED FORM RESCUES THE DEFECT AND EFFICIENCY BUT THE AUTOINHIBITTED PHOSPHO MAGNETIC FORM CAN'T RESCUE. SO JUST AS WE EXPECTED BOTH INVITRO AND IN CELLS, THE CLIP 170 IS ABLE TO ENHANCE AND RECRUITMENT OF THE DYNACTIN AND THE COMPLEX TO THE MICROTUBULE END AND IT DOES SO IN A WAY THAT'S RIGGULATED DIRECTLY FOR PHOSPHORYLATION, SO OUR MOTTO FOR THIS PART OF THE TALK IS CLIP 170 AS WE EXPECT IS ABLE TO ENHANCE THE REG--THE RECRUITMENT OF DININE AND DYNACTIN DOWN TO CARGO AND THIS REQUIRES THE ACTIVE FORM OF CLIP 170 AND IS INHIBITED BY THE CLIP 170 IS AUTOINHIBITTED. SO THAT MAY PARTIALLY EXPLAIN THE TRANSPORT INITIATION EFFICIENCY THAT WE'RE SEEING IN CELLS, BUT WE WONDERED IF THERE WAS ANOTHER COMPONENT TO THE SPACIAL SPECIFICITY OF INITIATION. HERE WE TURN TO THE FIELD THAT'S FINALLY COMING INTO IT'S OWN, THE RADIO UNDERSTANDING OF HOW MICROTUBULE MODIFICATIO ARE EFFECTING TRANSPORT WITHIN THE CELL. OUR ATTENTION TURNED TO THE POSSIBLE TIARAS ROSETTAINATION OR DETIARAS ROSETTAINATION STATE OF MICROTUBULES IN PARTICULAR BECAUSE OF LITERATURE GOING BACK FOR A NUMBER OF YEARS SAYING THAT AS YOU MOVE ALONG THE AXON TOWARDS THE GROWTH CONE, YOU'RE GETTING TO AN AREA THAT'S HIGHLY EHED IN TIARAS ROSETTAINATE, TIARAS ROSETTAINATED AS OPPOSE TO C-TIARAS ROSETTAINATED MICROTUBUALS. IN TERMS OF CAP SLIDE PROTEINS WE KNOW THIS REALLY MATTERS BECAUSE THIS TIARAS ROSETTA NATION RESDUE FITS RIGHT INTO THE BINDING SITE FOR TUBE LYNN FOR THESE CAP-GLY PROTEINS. SO EVERYTHING ABOUT THESE PREVIOUS STRUCTURAL STUDIES PREDICTED TO US THAT THE TIARAS ROSETTA NATION, DEROSETTA NATION STATE OF TUBE LYNN WOULD HAVE A TO TENTIAL EFFECT ON TRANSPORT INITIATION. SO JUST AS I SAID, IT'S BEEN KNOWN FOR A WHILE THAT THE ENDS OF MICROTUBUALS ARE ENRICHED IN THE TUBE LYNN BUT WE WANTED TO TAKE ADVANTAGE OF THE ENHANCED RESOLUTION OFFERED BY SUPER RESOLUTION HERE, HERE IT'S MICROSCOPY IF YOU LOOK AT THE MICROTUBULES IN THE AXON, AND YOU LOOK THE DETIARAS ROSETTAINATION IN THE TUBE LYNN, AND YOU CAN SEE THIS CLEAR ENRICHMENT RELATIVE TO THE BETA TUBE LYNN WHERE AS IF YOU LOOK INAXON SHIFT OR CLOSER TO THE CELL, YOU SEE THE OPPOSITE PICTURE WHERE YOU'RE SEEING A DE-ENRICHMENT FOR DISCIPLINARY ROSETTAINATED OR ENRICHMENT FOR DETIARAS ROSETTAINATED ALPHA TUBE LYNN. MORE IMPORTANTLY WE CAN TAKE A MEASURE FROM THE AXON TIP IN WHICH WE SEE THE TIARAS ROSETTAINATED TOMB LYNN IN OUR NEURONS IN CULTURE AND YOU CAN SEE THIS EXACT SAME TOMAIN TEND TO 20 MICRONS FROM THE TIP WHERE WE'RE MEASURING OUR TRANSPORT INITIATION. SO COULD WE ALSO RECONSITUTE THIS THE SPECIFICITY INVITRO. SO AGAIN WE STARTED GFP LABELED VESICLES, ISOLATED FOR MOUSE FRAME AND IN THIS CASE WE MIXED THISEM WITH EITHER KIROSETTAINATED OR DETIARAS ROSETTAINATED TUBE LYNN. AND WE GOT THIS FROM OUR COLLABORATOR, CARSON YANKEE'S LAB WHO'S ABLE TO ISOLATE SELECTIVELY, DISCIPLINARY RESINATED OR TIARAS ROSETTAINATED INVITRO TO GIVE US TWO DIFFERENT POPULATIONS AND YOU CAN LABELLIZE THESE DIFFERENTIALLY WITH TWO DIFFERENT COLOR LABELS. WE CAN THEN PUT THIS INVITRO, IN OUR ASSAY AND SEE IF THE VESICLE SHOW A CLEAR PREFERENCE FOR BINDING FROM ONE TO THE OTHER. SO THIS IN THIS CHAMBER WE HAVE THE TIARAS ROSETTAINATED MICROTUBULES AND THIS KIND OF VIOLET COVER, THE MICROTUBUALS IN THE RED COLOR AND THE VESICLES IN GREEN AND WHAT CAN YOU SEE, AS THIS MOVIE PLAYS IS A VERY CLEAR SPECIFICITY OF THE VESICLES FOR THE TIARAS ROSETTAINATED MICROTUBULES AND NOT FOR THE DETOY PROSINATED MICROTUBULES. JUST TO MAKE SURE IT'S NOT AN ARTIFACT, WE SWITCH THE COLORS, BUT THE TUBULES ARE LABEL INDEED A DIFFERENT COLOR AND AGAIN YOU CAN SEE THE CLEAR SPECIFICITY OF THE INTERACTION FOR THE MICROTUBULES AND THE LACK OF SPECIFICITY FOR THE DETIARAS ROSETTAINATED TUBULES IN THE CHAMBER. SO AGAIN TURNING TO THE ANALYSIS, AND IF YOU COMPARE QUALITATIVELY WHAT YOU'RE SEEING, AGAIN, IT'S NOT A CHANGE IN THE NUMBER OF SHORT VERSES LONG EFFECTS WHEN YOU COMPARE MOVEMENT ALONG DETIARAS ROSETTAINATED VERSES TIARAS ROSETTAINATED TUBE LYNN BUT A CHANGE IN THE FREQUENCY OF EVENTS. SO, A VERY ROBUST CHANGE IN THE LANDING RATE AS SHOWN HERE WITHOUT ANY CHANGE IN THE DWELL TIME AS SHOWN HERE. AGAIN WE'RE ABLE TO FIT THESE TWO VERY SIMILAR EXPOTENTIAL SO TS GIVES A MODEL WHERE WHERE IT EFFECTS THE BINDING OF THE LABELED VESICLES WITH OUR DYNEIN DYNACTIN MOTOR AND I TOLD YOU TWO STORIES, OF THE TWO WE SAW THE SPECIFICITY FAR THE TIARAS ROSETTAINATEDDED TUBE LYNN SO YOU MIGHT WONDER HOW THESE FIT TOGETHER OR AT LEAST WE DID. SO WE THEN ASKED CLIP 170 IS KNOWN TO HAVE A PREFERENCE FOR TIARAS ROSETTAINATED MICROTUBUALS, IS THAT ENOUGH TO OVERRIGHT THIS INHIBITION OR NOT? SO HERE WE'RE MIXING THE ACTIVATED ELONGATED FORM OF CLIP 170 WITH A NONPHOSPHORYLATED AMINO ACID AND IN THIS FRAME OR CHAMBER WHICH I'M SHOWING YOU THE FOSTER NURSED FOCUSED ON GENETIC WHICH WE PREDICT IN THE CLOSE STATE, WHAT YOU CAN SEE IS VERY ROUGH ATOM DUST MOVEMENT AS WE WOULD EXPECT OF THE-ELONGATED CLIP 170 ALONG THE TIARAS ROSETTAINATED MICROTUBULES BUT WHEN WE LOOK AT THE FORM THAT WAS PREDICTED TO BE AUTOINHIBITTED WE SEE EVEN THOUGH IT HAS A STRONG AFFINITY FOR TIARAS ROSETTAINATED MICROTUBULES, WHEN IT'S AUTOINHIBITTED WE SEE VERY LITTLE BINDING EVENTS AND THAT'S SHOWN--FOR THIS TIKULES THIS IS OVERRIDDEN BY THE FOSTER NURSED FOCUSED ON METIC STATE, THE CLOSED DATA, THE CLIP 170 SO THAT GIVES US THEN TWO INTERACTING MODELS AND THE AXON, ONE A SPACIAL EFFECT OF THE LOCALIZED MODIFICATION STAGE, THE CYTOSKEL TIN WITH THE TIARAS ROSETTA NAGS THAT ENHANCES BINDING THEM DISTALLY AND OVER LAYING THAT IS SOMETHING THAT MAY GIVE TEMPORALLY REGULATABLE EFFECT ON TRANSPORT, THE OPEN OR CLOSED STATE OF CLIP 170 SO YOU'RE GOING TO OVERRIGHT THE AFFINITY FOR HIGHER OSCILLATION AND HAVE A LOWER AFFINITY OF INTERACTION AND IF IT'S EXTENDED YOU'LL GET THE HIGH AFFINITY WITH THE END OF THE MICROTUBUAL. ANOTHER THING TO SHOW THE MECHANISM IS TO RECONSITUTE IT INVITRO. ANOTHER WAY THE MODELERS KEEP TELLING US IS TO MODEL WITH A MINIMUM MODEL BASED ON EXPERIMENTALLY DERIVED VALUES. AND IF WE KNEW ABOUT THIS TRANSPORT INITIATION TO DEVELOP A NODDLE OF IT. SO WE JUST PERFORMED A SIMPLE MONTY CARLOW STIMULATION OF THE VESICLE DIFFUSION AND CAPTURE OF THE NEURONS AND KEY CALCULATE IN THE GROWTH HOME AND DIFFUSING IN THIS THE TWO DIMENSIONS UNTIL THEY ENCOUNTER A MICROTUBE ALTHOUGH. WE USE THE MASK OF THE NEURONS WE EXPERIMENTALLY DERIVED AND THE MICROTUBULE AND WE MODELED ACTIVE CLIP-170 AS EXTENDED 135-NANOMETER MOLECULES IS ENSEL WE KNEW THE DIMENSIONS FROM THE INVITRO WORK AND WE MODELED INTO OUR AND THE TIARAS ROSETTAINATION AND THE DETIARAS ROSETTAINATION AS WE OBSERVED EXPERIMENTALLY. ALL OF WHICH ARE MEASURED EXPERIMENTALLY AND WE USE CAME FROM STUDIES OF THE PREVIOUS GRAD STUDENT IN THE LAB, BUT YOU'LL SEE THE MODEL IS RELATIVELY STABLE OVER A RANGE CONSITANCE IT'S HIGHLY EXPERIMENTALLY DRY AND THE DENSITY OF VESICLES ARE BASED ON EXPERIMENTAL OBSERVATIONS OF LAMP ONE VESICLES IN THE DISTALAXON. SO THAT'S AUTOINHIBITTED OR THE ACTIVATED THAT AND THE EXTENDED CLIP A IS A HIGHER FLUX, WHAT'S PARTICULARLY INTERESTING TO US IS THAT THE FLUX THAT'S SPIT OUT BY OUR MODEL IS NOT DISSIMILAR FROM THE WHAT WE VERY MUCH IN THE RIGHT BALL PARK FOR PREDICTING WITH OUR MODEL, THE FLUX WE SEE IN THE NEURONS IN VITRO. HERE'S AN EXAMPLE OF OUR SIMULATION WHERE WE'RE SHOWING THAT THE MAJOR CLIP IS ESPECIALLY TO AND MICROTUBUAL YOU CAN SEE THAT THE MICROTUBULE DOES NOT FILL THAT AND THIS IS THE OVER THE NEXT HOUR, CLIP A IS EXTENDING THESE AND THAT HAS A MAJOR EFFECT IN THE DECREASE OF THE SEARCH TIME FOR THE VESICLES TO ENCOUNTER THE MICROTUBULES. SO THAT'S SHOWN HERE IF WE JUST MEASURE THE MEDIAN TO THE MICROTUBUAL CAPTURE BETWEEN THE MODEL WHERE WE RUN AN AUTOINHIBITTED FORM OF CLIP 170 VERSES AN ACTIVATED CLIP 170. WE SEE A VERY SUBSTATIAL DECREASE IN THE TIME TO CAPTURE. AS I MENTIONED, WE STARTS WIDE AN EXPERIMENTALLY DERIVED DIFFUSION CO EFFICIENT BUT IF WE LOOK AT THE BEHAVIOR OF THE MODEL OVER EIGHT RANGE OF DIFFUSION, CO EFFICIENTS WE FIND THAT THE--RESULT IS ROBUST ACROSS ALL THOSE CO CERTAINLY--CERTAINLY FICIENTS. AND I THINK WHAT MAY BE THE MOST TELLING IS IF WE LOOK THE SCATTER PLATOF ALL OF OUR RESULTS FROM ALL OF OUR SIMULATION, WHAT YOU SEE UP HERE IN RED IS VESICLES THAT NEVER REACH THE MICROTUBULE OVER THIS TIME COURSE AND THE TIME COURSE OF OUR STIMULATION SPACE IS 20 MINUTES SOPHISTICATEDY A STRIKING VESICLE COULD NEVER REACH A MICROTUBULE UNDER THESE CONDITIONS UNLESS WE PUT IN AN EXTENDED CLIP 170 STRUCTURE. SO THIS IS TELLING US THAT BASED ON MODELING WE HAVE A GOOD READOT REGULATION OF DISTAL AXON AND DEPEND END BUT IT INVOLVES EB ONE OR THREE, CLIP 170 AND ALSO THIS GRADIENT TIARAS ROSETTAINATED FOR DETIARAS ROSETTAINATED TUBE LYNN ALONG THE AXON. FOR THOSE OF YOU THE TRUE NEUROSCIENCES IN THE AUDIENCE, YOU WONDER WHAT EFFECT ALL THIS HAS ON THE REALITY OF THE MUTATIONS WE PUT ON THE DISEASE, I WANT TO REMIND YOU, WHICH TYPE OF NEURONS ARE REALLY EFFECTIVE IN THIS DISEASE, THERE ARE THESE REALLY INTERESTING MIDBRAIN PROJECTION NEURONS WOULD BE VERY DIVERSE AXONAL ARBORS SO MANY DIFFERENT ENDS AND FURTHER THE DISEASE DOES NOT INITIATE TO THE 50S WHICH IS LOOKING LIKE A YOUNG AGE FOR ME NOW, BUT COULD BE CONSIDERED A LATE ONSET DISEASE. SO WHAT WE'RE THINKING IS THAT OVERTIME WITH AGING THE MIKE LO TUBULE DENSITY MAY BE DECREASING IN THESE AXONAL PROJECTIONS BUT THAT'S BEEN POPULA IN THIS THE LITERATURE AND ALTERNATIVELY WE MAY BE GETTING A CHANGE IN THE EFFECTIVENESS OF THE ACTIVATION OF CLIP 170 AND THESE FACTORS MAY BE ENOUGH TO DEPRESS THE INITIATION OF A TRANSPORT SPECIFICALLY FROM THE DISTAL END AND MOVE TOWARDS THE CELL STOMA, THE IMPORTANT THING IS THAT THIS IS AN EXPERIMENTALLY MANIPULATABLE AND HOPEFULLY MANIPULATABLE PHENOMENA, AND IF IT COMES DOWN TO THE STATE OF ACTIVATION OF CLIP 170, HOW EFFECTIVE YOUR TRANSPORT IS, SOMETHING THAT COULD BE MANIPULATED AND SMALL MOLECULES CAN EFFECT THE ACTIVATION STATE OF CLIP 170 SO POTENTIALLY BY DRILLING DEEP INTO THE MECHANISM, WE HAVE A PATHWAY TO POTENTIAL TREATMENT FOR THOSE EFFECTED BY THESE MUTATIONS. NOW GRAPTED THERE ARE VERY FEW PEOPLE IN THE WORLD EFFECTED BY PERRY SYNDROME BUT IT'S POSSIBLE THAT IMPROVING THE EFFICIENCY OF TRANSPORT FROM THE AXON, WILL HAVE MORE BROAD BASED APPLICATION TO PERHAPS OTHER FORMS OF THESE IN PARKINSON'S DISEASE, THAT'S HIGHLY SPECULATIVE BUT SOMETHING WE'RE INTERESTED IN THIS WORKING ON IN THE FUTURE. SO, I HAVE TO GIVE CREDIT WHERE IT'S DUE. I DON'T DO ANYTHING EXCEPT CHEER PEOPLE ON, ANYMORE. THIS WORK WAS ALL INITIATED IN MY LAB BY ARMEN WHO'S NOW A STUDY OUT, AND SWATHI IS NOW WORKING ON A PROJECT OF OPTOGENETICS AND JEFF DID THE ABSOLUTE OVERWHELMING MAJORITY OF WHAT I TOLD YOU ABOUT, IT'S A FANTASTIC MDPh.D. STUDENT IN MY LAB, OTHER PEOPLE I WANT TO POINT OUT ESPECIALTILY TO CLAIR AND MARCO, WE'VE BEEN TOGETHER SINCE THE BEGINNING WE'RE ESSENTIALLY MASHED AND I WANT TO THANK NIH FOR MY FUNDING, THEY'VE BEEN VERY GENEROUS AND WE'RE VERY APPRECIATIVE AND HOPE TO KEEP UP THE WORK WITH THEIR SUPPORT. SO THANK YOU ALL FOR YOUR ATTENTION. [ APPLAUSE ] >> LOOKS LIKE CLAIR HAS A QUESTION. >> SO WHAT WE DID AND I DIDN'T--YES. >> [INDISCERNIBLE] >> SO WE MIMICKED THE MICROTUBULE STRUCTURE WITH DYNAMIC INSTABILITY TO QUEER REALLY QUERY THE SPACE AND ENHANCE THE CAPTURE, IT MIGHT ENHANCE THE CAPTURE BUT NOT ALL OF THE CAPTURE BUT THAT'S A GREAT IDEA. >> [INDISCERNIBLE]. >> OH THAT'S A COOL IDEA. THAT WOULD BE GREAT. NOW WHAT I WANT TO DO IS TRY TO USE DRUGS AND SEE IF WE CAN TUNE IT AND THEN THE NEXT PHASE WILL BE REALLY COOL, SORRY TO GET DEEP INTO THE WEEDS BUT HOW ACTIN MAY ACT, SO WE CAN ACTIVATE AND LOOK AT ACTIN AND HOW THIS ACTIN SO STAY TUNED. ANOTHER QUESTION. >> [INDISCERNIBLE]. >> THERE'S ONE BACK HERE. >> [INDISCERNIBLE]. O SO THAT'S ABOUT WHAT CLAIR WAS JUST ASKING, I DON'T THINK WE WENT OUR OUR GMPP RESULTS BUT IT WOULD BE INTERESTING--I DON'T THINK SO BUT WE NEED TO TRY IT. THAT'S SOMETHING WE DEFINITELY NEED TO TRY. >> [INDISCERNIBLE]. >> THAT WAS A FANTASTIC QUESTION, SO TO ANSWER YOUR FIRST ONE WE DIDN'T SEE FRAIRCHG ACCUMULATION BUILD UP VESICLES WHEN WE DEPLETED CLIP 170 OR ED-THREE, WE DID NOT SEE ACCUMULATION AT THE DISTAL END. THIS IS VERY MUCH AN EFFICIENCY EFFECT NOT A REQUIRED EFFECT. YOU STILL GET INITIATION EVEN WITHOUT THIS MECHANISM. HOWEVER IN DROSOPHILA, TOM LLOYDS LAB HAS SHOWN THAT THEY DO SEE OVER LONGER TIME PERIODS, THEY DO SEE DISTAL ACCUMULATION. SO I FORGOT-THE SECOND PART WAS OH DENDRITIC CELL RIDES WHICH IS--DEBT RIGHTS SO THAT'S REALLY COOL. WE JUST STARTED LOOKING AT DENDRITES FOR TRAFFICKING BUT I'LL BE HAPPY TO TALK TO YOU ABOUT IT. IT'S A SEPARATE STORY AND WE VICTORY LOOKEDDA THIS THE IN PARTICULAR. THE THIRD QUESTION WAS ALONG THE SHAFT WHAT'S HAPPENING? WELL, I THINK VERY DIFFERENT THINGS ARE GOING ALONG ON THE SHAFT BECAUSE THE MICROTUBULES ARE VERY DENSE THERE SO IF YOU DO OFF A MICROTUBULE, HAVE YOU A HIGHER PROBABILITY OF REBINDING. EVENT GIVEN THAT THERE ARE MECHANISMS THAT HELP YOU REBYPASSEDMENT SO I DIDN'T SHOW YOU THE DATA FOR LIST ONE WHICH IS A FACTOR THAT HELPS DININE MAINTAIN A TYPE FINDING STATE IN THE MICROTUBULE. EVEN IN THE PRESENCE OF NUCLEOW TIDE IS FOUND. SO IF WE KNOCK DOWN CLIP ONE, WE SEE EFFECTIVE DISTAL INITIATION, SO IT'S VERY DIFFERENT THAN THE SPECIFIC MECHANISMS THAT I'M TALKING ABOUT. SO OUR HIGHWAY POT SIS IS IN THE CASE OF GLY-ONE IS A RESET BUTTON THAT IF YOU FALL OFF, IT WILL BIND AT SOME FREQUENCEET AND ALLOWS TO REBIND AT A HIGH LOCAL RECONSTITUTION AND THAT'S OUR WORKING MODEL AND WE HAVE EVIDENCE THAT WE WANTED, ACTIVITY DISRUPTING DISEASE AND THEN YOU SEE MORE PAUSING CONSISTENT WITH THAT, SO, IT COULD BE A GOOD THING MOST OF THE TIME BUT IF YOU ARE GETTING TOO MUCH, YOU WILL STOP TWO OFTEN. >> [INDISCERNIBLE]. >> IT COULD BE WE HAVEN'T SEEN EVIDENCE OF THAT IN OUR WORK. I FORGOT THE SLIDE WHERE WE QUERIED DIFFERENT COMPONENTS HERE AND I DON'T HAVE AT A GOOD ANSWER IT TO YOUR QUESTION. I CAN'T THINK OF A GOOD EXAMPLE FROM OUR OWN WORK THAT REALLY SHOWS THAT. WHAT WE CAN DO IS REPLICATE THE VESICLE SPECIFIC EFFECT WITH PURIFIED P150, AND THAT'S A SLIDE I'M MISSING BUT NOT KINNIESIAN OR DYNEIN, THAT'S SAYING IT'S ALMOST THE CAP-GLY FURTHER AND IF WE BLOCK THE DOMAIN, WE CAN BLOCK THE VESICLE WITH THE MICROTUBULE SO FOR THESE, IT'S DEPENDENTOT CAP SLIDE DOMAIN, THE OTHER CARGO WE SHOULD LOOK AT BUT ALL THE CARGO WE SAW WAS ALSO DEPENDENT ON THIS. >> [INDISCERNIBLE]. >> I THINK THAT ACTIN IS ABSOLUTELY INVOLVED HERE. I WANT TO GET TO MODEL NEXT IS THROUGH A ACTIN NET IN HERE AND LOOK AT MICROTUBULES GOING IN THERE AND LOOKING AT THAT SO DO I THINK IT'S INVOLVED THERE BUT YOU HAVE TO GET EFFECTIVE MOVEMENT FROM THE ACTIVE TO THE MICROTUBULE HERE SO MAYBE THAT'S WHY YOU NEED IT AS A SPECIALIZED SYSTEM TO HELP YOU COMPETE WITH THAT. YEAH, YOU AS YOU KNOW WE HAVE DONE A LOT OF INVITRO RECONSTITUTION CROSS FILAMENTS AND ALL TO LOOK AT RELATIVE EFFECTS OF MOTORS SO IN THOSE CASES, DYNEIN COMPETES WELL IF YOU HAVE A HIGHER CONCENTRATION ON THE CARGO. >> I HAVE A COUPLE OF QUESTIONS [INDISCERNIBLE] >> YEAH. >> [INDISCERNIBLE] >> YEAH, YEAH, SO WE DIDN'T SEE ANYTHING LIKE GRANULAR LIKE THAT, SO IT PRETTY MUCH LOOKED VERY CLEARLY ENRICHED AND THEN WHEN YOU GO BACK, NOTHING REALLY LEFT OUT BUT I WILL GO BACK AND LOOK AT IMAGES AND DOUBLE CHECK THAT TO ANSWER THAT QUESTION. >> [INDISCERNIBLE]. . >> RIGHT, RIGHT. NO, IT'S A [INDISCERNIBLE] PEP SID ACE, THAT'S A GOOD QUESTION, I'LL HAVE TO LOOK AT CONTROLS WE DID FOR THAT BUT I DO KNOW THAT WE DID NOT SEE ANY EFFECT ON A RANDOM OTHER BINDING PROTEIN SUCH AS KINIESIN AND DYNEIN IS ALSO, WE'RE NOT DESTROYING IT BUT WHETHER WE'RE ALTERING IT, I WILL LOOK BACK AT THE DATA BEFORE AND BLANKETLY SAY THAT WE KNOW THAT. --BEFORE WE BLANKETLY SAY THAT WE KNOW THAT. >> [INDISCERNIBLE] >> I'D SAY VERY LITTLE AND SOMETHING WE NEED TO DO IS ALL THIS IS GOING UPSTREAM AND SAY WHAT'S RIG REGULATING THAT IS I'M FORGETTING THE AUTHORS AND ALL THE CELLS SO LAST YEAR OR THIS YEAR SHOWING THAT THEY THOUGHT THAT MOST OF THE CLIP 170 WAS OFF AND ASSOCIATE WIDE ACTIN MOST OF THE TIME AND WE WOULD PROBABLY AGREE SO THAT SUGGESTED A TEMPORALLY SPECIFIC ACTIVATION OF CLIP 170, SO IT COULD BE JUST WAY MORE INTERESTING THAN THIS, THAT YOU GET UPTAKE OF A TROPEIC FACTOR AND A RECYCLING ENDOSOME AND THAT ACTIVATES TRANSPORT BACK TO THE CELL BODY WHICH WOULD BE A FANTASTICALLY INTERESTING MECHANISM, HOWEVER, THERE ARE USELESS IS A CONTINUOUS BASELINE AND EVERYTHING I'M SHOWING YOU IS ON, NOT EXOGENOUS GROWTH FACTORS OR SOMETHING COMING IN BUT THE ENDOGENOUS CARGO SHOW THE SAME THING SO THERE HAS TO BE SOME SORT OF ENDOGENOUS ACTIVATION MECHANISM THAT HAS ENOUGH INITIATION SO THAT YOU'RE NOT BUILDING THINGS UP WITH THE TYPE WHICH WOULD BE BAD FOR THE NEURONS SO THERE'S BOTH--THERE'S A BASELINE LEVEL OF ACTIVATION AND THEN THIS ACTIVATES BY UPSTREAM DINE ACES, MULTIPLE ONES HAVE BEEN SHOWN TO BE ABLE TO ACTIVATE CLIP 170 BUT WHICH ONES ARE RELEVANT, I THINK NOBODY REALLY KNOWS OR WE DON'T KNOW YET. SO THAT'S THE NEXT QUESTION. SCERNIBLE] >> PKA, GENERIC, SO I THINK TELL BE MORE INTERESTING THAN THAT BUT WE HAVE TO LOOK AT THAT. >> [INDISCERNIBLE] >> YEAH, THAT'S A GREAT QUESTION, BECAUSE IF IT'S A NEURODEVELOPMENT ALT DISEASE, THEN'RE YOU WOULD SAY OKAY, YEAH, BUT IT'S NOT MOST PATIENTS DISCIPLINARY FROM DEPRESSION AND METABOLIC CHANGES SO I WOULD SAY WE DON'T KNOW ENOUGH ABOUT IT BUT IT'S AN INTERESTING QUESTION TO GET AT. NOTHING WE'VE DONE IS SHOWN IT'S CARGO SPECIFIC ISSUES BUT IF WE CAN DRILL N THIS CLIP 170 ACTIVATION AND FIGURE OUT WHAT THE KINASE IS MADE, WE WILL FIND A SPECIFIC PATH WAY. >> IF THERE ARE NO MORE QUESTIONS, THANK YOU [ APPLAUSE ]