James Rothman

Transcription

>> HELLO. WELCOME TO TODAY'S WEDNESDAY AFTERNOON LECTURE AT NATIONAL INSTITUTES OF HEALTH. THANK YOU FOR COMING. MY NAME IS JUSTIN TERASKA. I'M AN INVESTIGATOR IN THE LABORATORY OF MOLECULAR BIOPHYSICS IN THE NATIONAL HEART LUNG AND BLOOD INSTITUTE. IT'S MY PLEASURE TO INTRODUCE DR. JAMES ROTHMAN AS TODAY'S WALS SPEAKER. DR. ROTHMAN IS WALS PROFESSOR OF BIOMEDICAL SCIENCES AND CHAIR OF DEPARTMENT OF BIOMEDICAL AT YALE. HE'S ONE OF THE MOST INNOVATIVE AND INFLUENTIAL CELL BIOLOGISTS AND BUY CHEMISTS WORKING OFFER THE LAST FEW DECADES. DURING TENURES AT STANFORD, PRINCETON, INVENTORY DEBT R, COLUMBIA AN YALE, -- CLONE KETTERING AND YALE, THIS IS INCLUDED SEMINOLE DISCOVERIES RELATED TO HOW PROTEINS INSERT INTO MEMBRANES. HOW VESICALES TRAFFIC THROUGH THE CELL, AND HOW VESICALES FUSE WITH THE MEMBRANE, A PROCESS CALLED EXOCYTOSIS. ASIDE FROM THE BIOLOGICAL DISCOVERIES HIS LAB HAS INVENTED IMPORTANT METHODS INCLUDING IN VITRO RECONSTITUTION OF VESICAL TRAFFICKING PATHWAYS WHICH HAS REALLY ALLOWED THE COMPLEX STEPS OF VESICAL TRAFFICKING TO BE TEASED APART AT THEIR MOST FUNDAMENTAL LEVEL H. ADDITIONALLY DEVELOPED NOVEL PH FLUORESCENCE PROTEINS THAT ALLOW INDIVIDUAL SYNAPSES AND SINGLE VESICALES TO BE WATCHED IN LIVING CELLS IN REAL TIME. IN PARTICULAR, DR. ROTHMAN HAS HELPED TO ESTABLISH THE SNARE HYPOTHESIS OF MEMBRANE FUSION WHICH PROPOSES THAT THE CORRECT PAIRING OF ALPHA HELICAL PROTEINS ON TWO OPPOSED MEMBRANES DIRECTS AND CATALYZES THEIR FUSION. ALONG WITH THESE IMPORTANT DISCOVERIES DR. ROTHMAN MENTORED AND TRAINED MANY PROMINENT SUCCESSFUL BIOCHEMISTS AND CELL BIOLOGISTS. HE'S A MEMBER OF THE NAB NATIONAL ACADEMY, INSTITUTE OF MEDICINE AND RECIPIENT OF NUMEROUS AWARDS INCLUDING LASTER AWARD FOR BASIC SCIENCE AND CAVALI PRIZE FOR NEUROSCIENCE. HE WILL DISCUSS HIS RECENT WORK ON SNARES AND THE ACCESSORY PROTEINS THAT DIRECT THEIR FUSION. IN A TALK TITLED MOLECULAR MECHANISMS OF SYNCHRONOUS NEUROTRANSMITTER RELEASE. AFTER THE SEMINAR THERE WILL BE A RECEPTION IN THE LIBRARY SO PLEASE COME AND THEY'LL GIVE YOU AN OPPORTUNITY TO SPEAK MORE INFORMALLY WITH DR. ROTHMAN. JOIN ME GIVING A WARM WELCOME TO JIM ROTHMAN. [APPLAUSE] >> THANK YOU. THANKS FOR ARRANGING THE DAY, BEING A GREAT HOST AND ALSO SHARING WITH ME YOUR RECENT AND EXCITING WORK especially on novel methods used Ford measuring confirmational changes in proximity with fret. IT'SER EXCITING, I HOPE WE CAN COLLABORATE AS A RESULT OF THAT. PLEASURE TO BE HERE WITH YOU TODAY. I'M GOING TO AS THE TITLE SUGGESTS, TALK ABOUT THE PROCESS OF SYNCHRONOUS NEUROTRANSMITTER RELEASE. THERE'S BEEN A LOT OF PROGRESS OVER THE LAST FIVE YEARS I WOULD SAY ESPECIALLY IN BEGINNING TO UNDERSTAND THE MECHANISM BY WHICH THIS VERY IMPORTANT PHYSIOLOGICAL PROCESS OCCURS IN STRUCTURAL AND BIOCHEMICAL TERMS. SO WHAT I WOULD LIKE TO DO TODAY IS OFFER SOME HISTORICAL BACKGROUNDS TO THE THE PROBLEMS AND THEN AFTER THAT SHARE WITH YOU OUR CURRENT VIEW IN THE FORM OF A MODEL OF HOW A SYNCHRONOUS TRANSMISSION NEUROTRANSMITTER RELEASE MAY WORK. A STRUCTURAL BUOY CHEMICAL MODEL AND AFTER THAT SHOW YOU SOME OF THE EVIDENCE ACCUMULATED FOR THE MODEL OVER THE LAST TWO OR THREE YEARS ESPECIALLY. WHAT DO I MEAN BY SYNCHRONOUS NEUROTRANSMITTER RELEASE? IT'S ACTUALLY THE PHYSIOLOGISTS HAVE VARIOUS COMPLEX SCHEMES TO MEASURE IT AND WAYS OF DEFINING IT. TO ME IT'S VERY SIMPLE BUT FUNDAMENTAL CONCEPT IN NEUROSCIENCE WHICH IS WHEN THE ACTION POTENTIAL COMES DOWN THE END OF THE NERVE AND YOU RELEASE A NEUROTRANSMITTER ACROSS A SYNAPSE TO THE NEXT NERVE OR PERHAPS MUSCLE CELL, THE NEUROTRANSMITTER NEEDS TO BE RELEASED AT THE RIGHT TIME. WE DIDN'T WANT THE NEUROTRANSMITTER TO BE RELEASED ASYNCHRONOUSLY ON ITS OWN ACCORD BECAUSE IN THAT CASE IT'S A FALSE ALARM. YOU ALSO DON'T WANT THE NEUROTRANSMITTER TO GO NOT BEING RELEASED BECAUSE YOU MISSED AN IMPORTANT SIGNAL. IN FACT WHAT YOU WANT, YOU WANT THE NEUROTRANSMITTER TO BE RELEASED PRECISELY SYNCHRONOUSLY WITH THE ARRIVAL OF ACTION POTENTIAL AT SYNAPTIC TERMINAL. THE WAY THAT'S ACHIEVEED IS THROUGH MEMBRANE GATED CALCIUM CHANNELS, PROBABLY EVERYONE KNOWS THAT ARE LOCALIZED IN THE SYNAPTIC PRE-SYNAPTIC REGION THAT OPENED UP THE GATE FOR CALCIUM ENTRY AND IT ACTS AS A SECOND MESSENGER TO TRIGGER RELEASE OF NEUROTRANSMITTER STORED IN VESICALES. THE PROBLEM THAT WE HAVE TAKEN ON THAT I WOULD LIKE TO ADDRESS HERE IS NUMBER ONE HOW ARE THESE VESICLES RELEASED? HOW DO THEY FUSE WITH THE SURROUNDING MEMBRANE HAVING STORED THE NEUROTRANSMITTER WITHIN THEMSELVES. HOW DO THEY DO IT SO RAPIDLY? SO MUCH MORE RAPIDLY, ORDERS OF THE MAGNITUDE MORE RAPIDLY. THAN OTHER MEMBRANE FUSION PROCESSES THAT TAKE PLACE IN THE CELL. SO THOSE ARE THE ASPECTS BUILT INTO SIN CROW IN THISTY. IT'S OBVIOUSLY IMPORTANT AT A GROSS LEVEL. IN YOUR BRAIN IF YOUR NEUROTRANSMITTERS WERE RELEASED HEALTHER SETTLER, THERE WOULD BE NO POSSIBILITY OF C9 INFORMATION PROCESSING OR ANYTHING OF ANY REMOTE INTEREST. YOU WOULD HAVE A -- THINK ABOUT WHAT EXPERIENCE WOULD BE IF YOUR VESICALES ALL FUSED AS THEY SHOULD BECAUSE THE FUSION PROTEINS ARE CONSTITUTIVELY ACTIVE. THEY SHOULD FUSE AND RELEASE NEUROTRANSMITTER ALL AT ONCE SO THEN YOU HAVE EVERY NEUROTRANSMISSION TAKING PLACE AT ONCE WITHIN A SHORT PERIOD OF TIME, NO THOUGHTS WHATSOEVER OR PERHAPS EVERY THOUGHT YOU WOULD HAVE AND NOT BE ABLE TO COMMUNICATE TO EVERYONE ELSE, IT MIGHT BE AN INTENSELY TRANSFORMATIONAL EXPERIENCE BUT IT WOULD BE ONE THAT LASTS 10 MILLISECONDS. SO THAT CLEARLY DOESN'T HAPPEN. IN A MUCH MORE SUBTLE LEVEL THE SPEED OF SYNAPTIC TRANSMISSION IS VERY IMPORTANT FOR THE COMPLEX CIRCUITS THAT WE HAVE. IT TYPICALLY TAKES A FEW MILLISECONDS FOR A SIGNAL TO BE TRANSPORTED ACROSS A SYNAPSE OF WHICH THE RELEASE PROCESS, INITIATION OF IT TAKES TYPICALLY LESS THAN A MILLISECOND IN CENTRAL SYNAPSES. THAT'S ACTUALLY VERY IMPORTANT BECAUSE FROM THE TIME THAT A PRIMARY PIECE OF POTENTIAL COGNITIVE INFORMATION LIKE VISUAL FIELD AND AUDITORY PATTERN WHAT HAVE YOU, IS SENTENCED BY OUR INPUT OUTPUT DEVICES, IT HAS MAYBE 20 OR 30 MILLISECONDS FOR ALL THOSE PATTERNS COALESCE AT YOUR HIGHEST CENTERS HAVING BEEN TRANSMITTED THROUGH PERHAPS TEN OR 15 DIFFERENT SYNAPSES. I THINK WE'RE ALL FAMILIAR WITH SOME ANYWAY ARE OLD ENOUGH TO REMEMBER WHAT MOVIES USED TO BE LIKE WHERE THE FILM GOES BY AND YOU HAVE 35, THE MAGIC NUMBER FRAMES PER SECOND. WHY 35? BECAUSE IF IT'S FASTER THAN 35 IT LOOKS LIKE A CONTINUUM TO YOU. IF IT'S SLOWER THAN 35, YOU SEE SEPARATE PICTURES. HOLLYWOOD FOLKS WEREN'T SPEND THRIFTS THEY WERE LOOKING FOR PROFITS, SO THEY FIGURED OUT THAT THE GRANULARITY OF HUMAN EXPERIENCE IS ABOUT 25 OR 30 MILLISECONDS. SO YOU CAN GET BY WITH THE LEAST NUMBER OF PHOTOGRAPHIC FILM AT THAT SPEED. SO THAT 25, 30 MILISEDGES IS WHAT YOU HAVE THAT REPRESENTS SIN CROW IN THISTY AND THE JOB IS TO GET THROUGH 10, 15 SYNAPSES AND PLOW THROUGH THEM FROM HERE AND HERE UP TO HERE. FAST ENOUGH. HOW DOES THAT WORK IN MOLECULAR TERMS? THAT'S THE PROBLEM I WOULD LIKE TO THE ADDRESS TODAY. THE SOLUTION TO THIS PROBLEM HAS COME I WOULD SAY OVER SEVERAL DECADES, ACTUALLY MORE LIKE HALF A CENTURY, MANY WHICH CELL BIOLOGY AND NEUROSCIENCE OR NEUROPHYSIOLOGY AS IT WAS THEN CALLED DOVE TAILED TOGETHER, GONE APART COME BACK TOGETHER AND THERE'S MANY CRITICAL COMING TOGETHERS OF THESE TWO FIELDS. THE BEGINNING OF THIS FIELD IN FACT REFLECTED THAT. GOING BACK TO THE CLASSIC WORK OF FAT AND CAT WHO FOUND HOST SYNAPTIC POTENTIALS, THE NEUROMUSCULAR JUNCTION IN CLASSICAL EXPERIMENTS FROM 1950s, IF YOU HAVE AN ELECTRICAL IMPULSE STIMULATING THE NERVE INNER INVESTIGATING THE MUSCLE, WHEN YOU GET SYNAPTIC TRANSMISSION THE MEMBRANE POTENTIAL OF THE MUSCLE CHANGES IN RESPONSE. IN YOU HAVE NO INPUT NO ACTION POTENTIAL, ON THE MUCH SMALLER SCALE THERE'S MINIATURE POTENTIALS OR MINIS THAT APPEAR AT FAIRLY LOW FREQUENCY BUT YOU CAN SEE THAT THEY'RE ABSOLUTELY MEASURABLE AND SEEM TO BE OF UNIFORM SIZE MEASURED AS A POST SYNAPTIC POTENTIAL. IN THAT CAPS THE IDEA TRANSFORMATIONAL IDEA THAT SYNAPTIC TRANSMISSION THE RELEASE OF NEUROTRANSMITTERS OKAY CANS IN A JUAN UNTIL FASHION WHICH THERE ARE INDIVIDUAL PACKETS OF NEUROTRANSMITTER SOMEHOW PREARRANGED STORED AT A NERVE ENDING SHOWN HERE. ABILITY THE SAME TIME IN 1950s MY PREDECESSOR AND FOUNDER OF CELL BIOLOGY AT YALE, THE FOUNDER TO LARGE DEGREE FIELD OF CELL BIOLOGY OBSERVED THESE MEMBRANE ENCLOSED VESICALES AT NERVE ENDINGS. HE CALLED THESE SYNAPTIC VESICALES. AND HE ALSO OBSERVED VESICALES OF MANY KINDS IN THE CELL AND CAME UP WITH THE IDEA THAT VESICALES ARE CAPABLE OF MEMBRANE FUSION. SUGGESTED THAT THESE SYNAPTIC VESICALES ARE IN FACT STORING THE NEUROTRANSMITTER, A JUAN UNTIL OF NEUROTRAN MITTER AND MADE THAT CONNECTION IN THE VESICAL HYPOTHESIS. THE IDEA IS THAT THESE VESICALES FUSE AFTER STIMULATION AND IT WAS CAPTURED DISCOVERED THAT THE ENTRY OF CALCIUM INTO THE -- THAT'S NEURONS SYNAPSES LIKELY PROVIDES THE IMMEDIATE TRIGGER. MORE MODERN WORK THROUGH THE 1970s, IN FACT CONDUCTED HERE AT NIH BY REECE AND HOUSER THIS VESICAL HYPOTHESIS RECEIVE STRONG SUPPORT WHERE THEY OBSERVE VESICALES LIKE THIS, IN FACT WERE SEEN FUSING WITH THE PRE-SYNAPTIC PLASMA MEMBRANE RELEASING THE NEUROTRANSMIT INTO THE SYNAPTIC CLEFT TO DIFFUSE ACROSS THE SYNAPSE. HERE THEN IS ONE OF THE EARLY COMING TOGETHERS OF CELL BIOLOGY AND NEUROPHYSIOLOGY IN THE VESICAL HYPOTHESIS. THIS VIEW THAT VESICALES CAN STORE COMPOUNDS FOR RELEASE FROM THE CELL, WHICH AS JUSTIN SAID GOES BY THE NAME OF EXOCYTOSIS BUT INDEPENDENTLY REACHED AND GENERALIZED BY PILATI AND COLLEAGUES. THERE ARE MUCH LARGER VESICLES THAT STORE INSULIN OR IN THIS CASE EXOCRINE SECRETIONS MANY THE PANCREAS THAT ARE READY TO BE RELEASED AND THEN ARE RELEASED. FOLLOWING A STIMULATION. WHAT THIS INTRODUCES IS A GENERAL CONCEPT WHICH IS VEST IT WILLS STORE PRODUCTS THAT NEED TO BE RELEASED RAPIDLY. MUCH MORE RAPIDLY THAN THEY CAN BE SYNTHESIZED OR LOCALIZEED TO THE SITE OF RELEASE. AND THAT THIS OCCURS PHYSIOLOGICALLY. IN NO PLACE IS THIS KINETIC DEMAND FASTER THAN IN THE BRAIN. FOR THE REASONS THAT I MENTIONED. PROBABLY SECOND FASTERS IS THE RELEASE OVINES -- FASTEST IS RELEASE OF INSULIN, SECONDS TO COUPLE OF MINUTES. MORE LEISURELY IS THE PROCESS OF MEMBRANE FUSION AS IT OCCURS WITHIN THE CYTOPLASM. AND INDEED WORK IN THE 1970s, 1980s AS WE BEGAN TO DISCOVER RANGE OF VESICALES THAT TRAVERSE THE CYTOPLASM CARRYING CARGO BY BUDDING AND FUSION FROM ONE COMPARTMENT TO ANOTHER WE NOW RECOGNIZE AT LEAST A DOZEN, SOME SPECIALIZED CELLS PROBABLY MORE TYPES OF VESICALES EACH SELECTS FOR A CARGO AND NEEDS TO DELIVER THAT BY MEMBRANE FUSION WE CAME TO REALIZE THIS PROCESS OF MEMBRANE FUSION IS REALLY GENERAL. NOT JUST WITHIN A SINGLE CELL CYTOPLASM THERE ARE PROBABLY TEN OR 20 IN COMPLEX EUKARYOTIC CELLS, TYPES OF MEMBRANE FUSION THAT OCCUR, BUT ALSO GENERAL BIOLOGY FROM PLANTS TO MICROBES EUKARYOTIC MICROBES AND TO HUMANS. SO WHAT AGAIN BY WAY OF BACKGROUND, THIS IS ALREADY BEEN INTRODUCED, WHAT I PERSONALLY FIND STILL QUITE ASTONISHING IS THAT THIS ENTIRE ARRAY OF PHYSIOLOGICALLY IMPORTANT MEMBRANE FUSION PROCESSES FROM THE SYNAPSE TO HORMONE RELEASE TO THE COMPARTMENTAL ORGANIZATION OF THE CYTOPLASM AND IT PROPAGATION IN CELL DIVISION, ARE ALL RELATE TO WORK THE HANDIWORK OF A SINGLE FAMILY OF PROTEINS. THIS FAMILY OF PROTEINS CALLED SNARE PROTEINS COMES IN A NUMBER OF VARIETIES BUT THEY HAVE IN COMMON PHICAL CHEMICAL MECHANISM FOR MEMBRANE FUSION. THERE ARE TWO TYPES OF SNARE PROTEINS PHYSICALLY COMPLIMENTARY TO EACH OTHER, WE CALL THEM V AND T SNARES. THEY EXIST IN -- THEY'RE LOCALIZED DIFFERENTLY WITHIN THE CELL. A V SNARE PARTNERS A T SNARE AND BRIDGES THE GAP BETWEEN TWO MEMBRANES, AS I'LL DESCRIBE IN STRUCTURAL TERMS IN A MOMENT NOT ONLY INITIATE IT IS PROCESS OF MEMBRANE FUSION, BILAYER FUSION BUT ACTUALLY FUSES THE BIOLAYERS. BUT THERE ARE MANY DIFFERENT TYPES OF V SNARES AND T SNARES IN A CELL. THEY'RE SHOWN HERE IN DIFFERENT COLORS. THE V SNARE ENCAPSULATED IN THE VESICLE THAT DEPARTS THE ENDOPALACE MIC RETICULUM CAN ONLY MATE WITH COGNATE T SNARE LOCATED AT THE ENTRY FACE OF THE GOLGI. THAT'S ONE OF THE SEVERAL MOLECULAR FEATURES, NOT THE ONLY FEATURE BUT A CRITICAL MOLECULAR FEATURE THAT DICTATES FUSION OF THIS VESICAL HERE, NOT THERE, NOT ANY PLACE ELSE AND ALLOWS SPECIFIC MEMBRANE TRAFFIC IN THE CELL. SIMILARLY THERE'S A DIFFERENT BUT STRUCTURALLY RELATED HOMOLOGOUS V SNARE PACKAGED TO VESICALES LEAVING THE EXIT OF THE GOLGI THAT FINDS ITS PARTNER IN THE PLASMA MEMBRANE THAT'S A DIFFERENT V AND T SNARE DEPENDING WHICH SURFACE OF THE CELL, APICAL OR BASAL LATERAL. I COULD GO ON AND ON BUT I WON'T. SO THE SPECIFIC PAIRING OF PROTEINS BETWEEN PARTNERED MEMBRANES INCLUDING THE RELEASE OF VESICALES AT THE CELL SURFACE AS A SPECIAL CASE BUT A VERY IMPORTANT SPECIAL CASE OCCURS. ALL THESE PROCESSES ACCEPT FUSION WITH THE PLASMA MEMBRANE ARE WHAT CELL BIOLOGISTS CALL CONSTITUTIVE PROCESS. THEY OCCUR AL THE TIME. THEY OCCUR THROUGHOUT THE CELL CYCLE. YES, THERE MAYBE REGULATION OF ONE SORT OR ANOTHER, NO DOUBT BUT THEY OCCUR ALL THE TIME. THIS PUTS THEM INTO A FUNDAMENTAL DISTINCTION WITH CERTAIN CLASSES OF FUSION WITH THE PLASMA MEMBRANE, EXOCYTOSIS, WHICH ONLY OCCUR SOME OF THE TIME WHEN SIGNAL IS PROVIDED AS IN THE RELEASE OF NEUROTRANSMITTER WHEN CALCIUM ENTERS SECONDARY TO THE ARRIVAL OF AN ACTION POTENTIAL. TIME COURSE HERE IS RELATIVELY LEISURELY FROM THE TIME A VESICAL DOCKS TO FUSION TYPICALLY TAKES TEN SECONDS TO A MINUTE DEPENDING ON THE SITUATION. THAT'S THE TIME FRAME. DIFFERENT THAN THE TIME FRAME IN SYNAPTIC TRANSMISSION. WHICH THE NEUROTRANSMITTER CAN BE RELEASED AS FAST AS 200 MICROSECONDS FROM THE TIMECAL YUM HAVES. SO THAT IS THE CONTRA DISTINCTION HERE. WHAT CONFOUNDS THIS FURTHER IS SNARE PROTEINS WHICH I'LL DESCRIBE IN INCREASINGLY GREATER DETAIL IN TERMS OF HOW THEY FUSE BILAYER ARE INTRINSICALLY POWERFUL FUSION PROTEINS THAT ARE ON RATHER THAN OFF. HOW CAN YOU HAVE A NEUROTRANSMITTER VESICAL SITTING RIGHT NEXT TO THE PRE-SYNAPTIC MEMBRANE WITH POTENT FUSION MACHINERY BY IN AND OF ITSELF IS ON NOT OFF AND SOMEHOW DOESN'T FUSE. BUT THEN BUT THEN AT THE RIGHT MOMENT IT FUSES RAPIDLY. SOME ANSWERS BEGIN TO COME FROM AN UNDERSTANDING OF THE UNDERLYING PRINCIPLE OF MEMBRANE FUSION. ENCAPSULATED BETWEEN THE TWO STRUCTURES. THE SNARE PROTEINS ARE ALPHA HELICAL BUNDLE PROTEIN, RELATIVELY SMALL, THEY HAVE TWO PARTS ALL A PART IN THE MEMBRANE, A TRANSMEMBRANE AND THEN THEY HAVE A HELIX FORMING SO CALLED SNARE MOTIF THAT IS IN THE CYTOPLASM. THE V?úzBRE EMANATES PRIMARILY FROM THE VESICAL, THAT'S WHY IT'S CALLED THE V SNARE FOR VESICLE AND CONSISTS OF A SINGLE HELICAL PROTEIN CYTOPALACE MIC DOMAIN. THE T SNARE, STANDING FOR TARGET MEMBRANE VESICLE FUSE WITH THE PLASMA MEMBRANE, THE T SNARE CONTRIBUTES THREE HELICES, WHEN IT ASSEMBLES IT FORMS A FOUR HELIX BUNDLE THAT FOUR HELIX BUNDLE IS STABLE SO THAT IF YOU ISOLATE THIS PROTEIN, THIS COMPLEX FROM CELLS OR FROM BRAIN OR FORM IT ARTIFICIALLY WITH RECOMBINANT PROTEINS, YOU HAVE TO FIND AND OTHERS FOUND OF COURSE YOU HAVE TO HEAT THIS PROTEIN TO ALMOST 100-DEGREES, YOU HAVE TO BOIL THE WATER FOR IT TO DENATURE THAT'S HOW STABLE IT IS. THERE'S A VERY INTERESTING FEATURE. THESE PROTEINS WANT TO FOLD THE MAKE A FOUR HELIX BUNDLE BUT THEY CAN'T DO THAT WHEN BETWEEN TWO BILAYERS. IT'S FROM THIS THE PRINCIPLE OF FUSION FOLLOWS. THE V SNARE UNIQUELY ASSEMBLING FROM SYNAPTIC VESICAL. THE T SNARE IS ASSEMBLING FROM THE MAMA MEMBRANE. AS THESE TWO ZIPPER UP FROM MEMBRANE DISTAL END TERMINI TOWARDS THE MEMBRANE, THEY CAN ASSEMBLE BUT THEY CAN'T COMPLETELY ASSEMBLE. I HOPE EVERYBODY CAN SEE THAT. LEFT TO THEIR OWN DEVICES, REMOVED FROM MEMBRANE ENTIRELY, THEY DO FULL WILL ASSEMBLE. EVEN THE TRANS MEMBRANES FIND EACH OTHER. SO THEY WANT TO ZIPPER UP TO THIS TIGHT FOUR HELIX BOPPED L BUT THEY CAN'T. WHY CAN'T THEY? THEY TWO BILAYERS ARE SEPARATE. THE ONLY WAY THESE TWO BILAYERS BECOME ONE BILAYER CAN THE THEY ZIPPER. SO WHAT WE HAVE HERE IS THERMODYNAMIC LINKAGE DUE TOSER UK EXCLUSION OF TWO REACTIONS, THE FIRST REACTION IS A PROTEIN FOLDING REACTION. THE V SNARE ESPECIALLY IS A REASON DOCUMENT COIL AND THE T SNARE WITH THREE SUBUNITS IS LOOSELY AABLED BEFORE IT EPICOUNTERS THE V SNARE. IN THIS STATE THE SNARES ARE PARTIALLY OR COMPLETELY UNFOLDED. IN THIS STATE AFTER FUSION IN GOING FROM UNFOLDED TO COMPLETELY FOLDED THERE'S A LOT OF ENERGY POTENTIALLY RELEASED. IF IT'S MIXED IN A DETERGENT SOLUTION OR WATER, THEY WILL GIVE THAT ENERGY OFF AS HEAT. PLACED BETWEEN TWO BILAYERS IF -- IT'S -- THEY WILL PROVIDE ENOUGH ENERGY TO DO WORK ON THE BILAYER TO CAUSE THE BILAYERS TO FUSE. OBVIOUSLY WE DON'TNESS EVERY DETAIL ABOUT THAT TRANSMISSION AND THERE'S A LOT OF IMPORTANT WORK GOING ON, NOT THE LEAST FROM JOSH ZIMMERBURG AND OTHERS HERE. AND THERE ARE A NUMBER OF COMPETING MODELS BUT WHAT IS CLEAR IS THE ASSEMBLY OF SNARES CREATES AN INNER FORCE THAT PULL IT IS MEMBRANES TOGETHER AND THAT FORCE RESULTS IN OPENING OF A FUSION PORE TO RELEASE THE NEUROTRANSMITTER OR SOME EQUIVALENT CARGO. WE KNOW THIS OCCURS WITH ISOLATED SNARE PROTEINS. THE TIME COURSE IS MEASUREED IN A NUMBER OF LABORATORIES, IT'S LESS THAN 100 MILLISECONDS, IT'S TYPICALLY MORE THE AVERAGE MEASUREMENT IS PROBABLY 30 TO 50 MILLISECONDS. IF YOU HAVE ISOLATED PROTEINS WITH A V SNARE BILAYER THAT'S THE TIME COURSE THE SNARES WILL FUSE. THEY ARE EXTREMELY COMPETENT. RECENT STUDIES FROM (INDISCERNIBLE) LAB AND WE CONFIRMED THIS SHOW THAT A SINGLE SNARE COMPLEX THAT'S ASSEMBLING WE CALL IT A SNARE PIN, WILL BE SUFFICIENT AT LEAST AT A CERTAIN RATE ENERGETICALLY SUFFICIENT TO DRIVE THE FUSION OF A VESICLE WITH A BILAYER. THESE ARE ENERGETICALLY COMPETENT. SO WE HAVE MEASURED THIS INNER DIRECTED FORCE DIRECTLY USING THE SURFACE FORCE APPARATUS WHERE WE HAVE SNARES IN OPPOSITE MEMBRANES BRING TOGETHER WITH SUB-NANOMETER POSITION, PULL THEM APPEARED IN A DEFINED WAY AND MEASURE ADHESIVE FORCE. IT IS A FORCE AND ENERGY PERFECTLY CONSISTENT WITH THE IDEA THAT A SINGLE SNARE PIN IS ENERGETICALLY CAPABLE OF FUSING A LIPID BILAYER. FINALLY IN CASE SOMEONE ASKS AT THE END OF THIS FUSION PROCESS THERE'S AN ENZYME SYSTEM INVOLVING THE TRIPLE AATPASE CALLED NSF THAT UTILIZE A TP HYDROLYSIS TO SEPARATE SNARES UNFOLD AND ALLOW TO BE RECYCLED ENERGETICALLY TO THE HIGH ENERGY STATE OF UNFOLDED PROTEIN AND P TO INITIATE RECYCLING TO THE CORRECT DONOR COMPARTMENT. THIS IS OUR CURRENT UNDERSTANDING IN CARTOON LEVEL ANYWAY HOW MEMBRANE FUSION WORKS. TO MAKE IT MORE CONCRETE I'LL DRAW UPON A RECENT X-RAY CRYSTAL STRUCTURE OF RYAN HART AND COLLEAGUES, THAT SHOWS THE FOUR HELIC BUNDLE. THIS IS OF THE SYNAPTIC SNARE PROTEIN. BY WHICH OF INTRODUCTION THESE ARE THE PROTEINS THAT DO THE JOB TO RELEASE NEUROTRANSMITTER AT SYNAPSES. THE V SNARE CONSISTS OF A PROTEIN CALLED VAMP OR SYNAPTOBREVIN AND IT ORIGINATES IN THE SYNAPTIC VESICAL. THE T SNARE CONSISTS OF TWO PROTEIN, INTEGRAL MEMBRANE PROTEIN CALLED SYNTAXIN, AND SOLUBLE PROTEIN CALLED SNAP 25 WHICH CONTRIBUTES TWO OF THE HELICES. SO THE T SNARE HERE CONSISTS OF THREE HELICES, TWO CONTRIBUTED BY SNAP 25 IN GREEN, ONE CONTRIBUTED BY MEMBRANE PROTEIN SEN TAX AND PLASMA MEMBRANE INITIALLY AND THE OTHER THE VESICLE PROTEIN VAMP INITIALLY IN THE SYNAPTIC( VESICAL. I WANT TO DISTINGUISH THREE REGIONS, THERE IS A REGION CALLED THE BUNDLE REGION OR THE HELICAL BUNDLE REGION YOU CAN SEE IT'S CALLED THAT FOR OBVIOUS REASONS. SNAP 25 TERMINATES AT THAT POINT AND VAMP AND SYNTAX AND CONTINUE TO INTERACT IN A REGION CALLED THE LINKER REGION WHICH DOES NOT INCLUDE CONTRIBUTIONS FROM SNAP 25, THE LINKER BECAUSE IT CONNECTS THE FOUR HELIX BUNDLE TO THE MEMBRANE AND THEN YOU HAVE THE TRANSMEMBRANE DOMAIN OF V SNARE AND T SNARE. VAMP AND SYNTAXIN. THIS IS THE POST FUSION STATE THAT EXISTS AFTER THE FUSION. IF WE WANT TO UNDERSTAND THE MECHANISM OF FUSION MORE WE NEED TO UNDERSTAND FOR ABOUT STRUCTURE AS ASSEMBLING RATHER THAN AFTER ASYSTEMBLY BUT THIS PROVIDES OOH VERY IMPORTANT GUIDE. IMPORTANT LIT IT SHOWED SCRATCH AND SYNTAXIN CONTINUE THE INTERACT WITH A SERIES OF CONTACTS INTO THE BILAYER, EVEN AFTER FUSION. THIS ZIPPERING PROCESS EVIDENTLY PROCEEDS RIGHT THE WAY THROUGH THE FUSION PROCESS. NOW, IF I DIDN'T GET ANYTHING ACROSS HERE, I WOULD LIKE TO TAKE A MOMENT AND SUMMARIZE 25 YEARS OF MY LIFE IN THIS ONE SLIDE WHICH EXPLAINS HOW I THINK ABOUT THE PROBLEM. I HOPE NOBODY IS OFFENDED. THIS IS A READY'S HAIR PIN. ANY OF YOU WHO HAVE HAD THE EXPERIENCE OF TRYING TO SEPARATE THE TWO ENDS OF A LADY HAIR PIN WILL KNOW THAT IT TAKES WORK TO DO THAT. SO LET'S IMAGINE THIS IS A SNARE PIN ASSEMBLING BETWEEN TWO VESICALES EXPECT E START WHEN IT'S ASSEMBLED, NOW WE PULLED IT APART. HAVING PULLED IT APART, WE'RE GOING TO INSERT EACH END INTO A RUBBER BALL REPRESENTING TWO VESICALES OR IF YOU WILL, A SYNAPTIC VESICAL AND PLASMA MEMBRANE. NOW WHAT WE'RE GOING TO DO IS LET THEM GO. AS WE LET THIS GO WHAT HAPPENS? THE PIN BECAUSE IT WAS RESTORING FORCE INWARD DIRECTED FORCE THAT'S THE FORCE I'M REFERRING TO THAT OCCURS BETWEEN SNARE PINS. IT WILL -- WHAT WILL THIS PIN DO? IT WILL FORCE TOGETHER TWO RUBBER BALLS. THAT'S THE END OF IT IF IT'S RUBBER BALLS BUT IF THEY'RE NOT THEY HAVE A LIQUID LIKE CHARACTER WHICH IN PHICAL TERMS MEANS THEY HAVE SURFACE TENSION THAT CAN BE OVERCOME EXERTED BY THE PIN, THE SNARE PIN IN REALITY, THEN THE TWO BALLS WILL BE BLENDED INTO ONE WHICH ALLOWS THE PIN TO REACH ITS BROWN STATE AND P ITS MINIMUM ENERGY STATE. IN MY VIEW TRAINLY FUSION IS NOTHING MORE OR ANYTHING LESS THAN THIS. THERE ARE A LOT OF DEBATES ABOUT THE TRANSITION STATE WHEN THOSE TWO BALLS ARE ABOUT TO GO TOGETHER. AND I TEND TO LOOK AT THAT AS A STATISTICAL MECHANICAL DEBATE OF INTEREST. FROM A BIOLOGICAL POINT OF VIEW HOWEVER, OF GREATER INTEREST IS THE THERM MOW DYNAMICS THAT A PRE-CONDITION IS CREATED THAT MAKES THE FUSION INEVITABLE. THIS EXAMPLE IS NOT AN IDOL ONE, IN RECENT MONTHS MY COLLEAGUES GRABBED OUR HANDS ON INDIVIDUAL SNARED COMPLEXES AN LITERALLY PRIED THEM APART WITH OPTICAL TWEEZERS. SO HERE WHAT A COLLEAGUE IN CELL BIOLOGY IN HIS LAB AND WHAT WE HAVE DONE IS TO ATTACH A SMALL BEAD BY MOLECULAR MODIFICATION TO THE ASSEMBLED END OF A SNARE COMPLEX. WE ARTIFICIALLY CROSS LINK THE V AN T SNARE, THE SAME SNARES YOU SAW A MOMENT AGO, AND WE ARTIFICIALLY CONNECTED THEM AT THEIR MEMBRANE DISTAL END TERMINI. THE ATTACHMENTS WOULD BE HERE. THE ANCHORS ARE REMOVED AND REPLACED BY A LINKER TO ONE BEAD AND LINKER TO THE OTHER ON VAMP AND SYNTAXIN. NOW WHAT WE CAN DO WHAT I ILLUSTRATED A MOMENT AGOND PULL ON THEM IN A DETERMINED WAY. WHAT WE OBSERVE IS AS WE DO THEY MELT LAYER BY LAYER. THE FIRST THING THAT MELTS IS THE LINKER LAYER. THE SECOND THING THAT MELTS IS HALF OF THE FOUR HELIX BOPPED L. AND NULL REVERSIBLY SO AS YOU PULL YOU GET WHAT A SINGLE CHANNEL FIZZ IDEAL GIST WOULD RECOGNIZE A SINGLE CHANNEL BEHAVIOR. BECAUSE THE LINKER HAS BEEN DESTABILIZED AND CERTAIN FORCE LEVELgÑ" FLUCTUATES BETWEEN OPEN CLOSED, OPEN CLOSED. AS IT OPENS THE DISTANCE BETWEEN THE TWO BEADS INCREASES AND THAT'S WHAT WE'RE MEASURING HERE. THIS WILL GO ON FOREVER UNLESS WE EXERT MORE FORCE AND WE GET TO THE NEXT FORCE LEVEL WE GO TO THE NEXT PORTION WHICH FLUCTUATES BECOME AND FORTH. ONE BEAUTY OF THIS EXPERIMENT BESIDES DEMONSTRATING THE BINARY LIKE SWITCH NATURE OF DOMAINS IS IT ALLOWS US TO MEASURE KINETICS WITH WHICH THIS FLIPPING BACK AND FORTH OCCURS. REMARKABLY ENOUGH FOR THE SYNAPTIC SNARE COMPLEX THE RATE OF REZIPPERRING HERE OCCURS WHAT WE ESTIMATE TO BE A DEIFYING CONTROLLED LIMIT. -- DIFFUSION CONTROLLED LIMIT. AS FAST AS THE V SNARE ZIPPERS IN AND P LAYS DOWN, BY DISPLACING WATER, THAT'S HOW FAST IT MOVES. THIS MACHINE IS NOT ONLY NEE JETTICLY SUFFICIENT FOR FUSION BUT -- ENERGETICALLY FOR FUSION BUT DESIGNED AS FAST AS PHYSICS ALLOW WHICH IS A WONDERFUL FACT WHEN WE CONSIDER HOW FAST SYNAPTIC VESICLE RELEASE HAS TO OCCUR, THE TWO HUNDRED MICROSECONDS. I WANTED YOU TO KNOW THAT THERE IS A PAUSE IN THE DISASSEMBLY. WE BUST THIS PART, BUST THAT PART AND IF WE PULL HARDER WHICH IS NOT SHOWN HERE WE PULL THE LAST PART CALLED THE END TERMINAL DOMAIN, LINKER DOMAIN, END TERMINAL DOMAIN IS LAST, THERE'S THE GUY THAT ASSEMBLES FIRST WHEN THE VESICAL STARTS DOCKING. THAT TURNS OUT TO BE VERY, VERY SLOW. IT'S VERY SLOW, MEASURED ACTUALLY RATE CONSTANT IS MEASURED ON THE ORDER OF AN HOUR. THE REASON IT'S SO SLOW IS BY BEING SO SLOW IT ALLOWS OTHER FACTORS TO KICK IN THAT CAN REGULATE THE DOCKING OF THE VESICAL. SO YOU HAVE FACTORS UPSTREAM AND YOU'LL SEE THAT REGULATE DOCKING OF THE VESICAL TO ALLOW IT TO ZIPPER THROUGH THE END TERMINAL DOMAIN. I'LL GIVE THE STORY AWAY NOW BECAUSE YOU'LL SEE THAT IN THE SPECIAL CASE OF THE SYNAPTIC VESICAL, THE VESICAL IS FROZEN AT THIS STAGE. HALFWAY ZIPPERED PUNT WAITED BY -- ONCE WAITED BY IONIC LAYER, THESE ARE HOPE TAB REPEATS, AND THOSE A CERTAIN IN EVERY SNARE COMPLEX IS REPLACED BY HYDROPHYLIC RESIDUE. THIS CAUSES AN INTENTIONAL PAUSE HALFWAY THROUGH THE ZIPPERING PROCESS. AND IN THE SYNAPSE THERE ARE OTHER PROTEINS AND I WILL DESCRIBE THEM TO YOU, THAT GRAB THE SNARE COMPLEX QUITE LITERALLY, AT THIS PAUSE POINT AND FREES IT UNTIL THE ACTION POTENTIAL ARRIVES, UNTIL CALCIUM ARRIVES. AND THAT IS REALLY NIFTY BECAUSE IT ALLOWS THE SNARE COMPLEX TO BE ACTIVATED FROM A VESICAL WHOSE FRAME IS FROZEN JUST BEFORE MEMBRANE FUSION CAN BE COMPLETED AT A DIFFUSION CONTROLLED LIMIT. THAT IS OUR PICTURE OF HOW THIS WORKS. TO SUMMARIZE THEN, THERE IS IN THE ASSEMBLED SNARE COMPLEX DISCREET DOMAINS, END TERMINAL, C TERMINAL DOMAIN, AND TRANSMEMBRANE DOMAIN. THEY HAVE DISTINCT FUNCTIONS. THE PURPOSE OF THE THE END TERMINAL DOMAIN WHICH ASSEMBLES FIRST IS TO DOCK THE VESICAL TIGHTLY TO THE PLASMA MEMBRANE. THE NEXT ASSEMBLY OF THE C TERMINAL DOMAIN IRREVOCABLY COMMITS THE VESICLE TO FUSE. THE ACTUAL FUSION OCCURS AS BEST WE CAN JUDGE SUMMARIZING WORK FROM MY LAB AND RYANHART YAN'S LAB HERE WHEN LINKER DOMAIN ASSEMBLABLES. THEN THE TRANSMEMBRANE DOMAIN IS BELIEVE TO HAVE ASSEMBLED BY THE X-RAY CRYSTALOGRAPHY. SINCE THE FUSION OF THE BILAYERS MEASURED BY ZIPPERING -- TRIGGERED BY LINKER DOMAIN ZIPPERING AND THE MIXING OF THE LIPID BILAYER AND THE INITIAL OPENING OF THE FUSION PORE, ALL THIS WOULD SEEM TO BE OVER SO IT WOULD SEEM TO BE NO ROLE NECESSARILY FOR THE ZIPPERING OF THE TRANSMEMBRANE DOMAIN BUT ACTUALLY NOTHING COULD BE FURTHER FROM THE TRUTH BECAUSE AS WE RECENTLY PUBLISHED, THE ZIPPERING OF THE TRANSMEMBRANE DOMAIN AFTER FUSION HAS OCCURRED, AFTER THE FUSION PORE JUST OPENS SEEMS CRITICAL BASED ON IN VITRO EMPERIMENTS FOR THE OPENING OF THE FUSION PORE, EXPANSION OF THE FUSION PORE. I WON'T REALLY HAVE TIME TO GO INTO THIS IN DETAIL BUT IF YOU'RE INTERESTED THIS WAS VERY REISN'TLY PUBLISHED IN SCIENCE, LAY SHI IS THE PERSON WITH THE MOST WORK ON THIS IN THE LAB. IT INVOLVES A NOVEL ASSAY IN WHICH WE MEASURE THE FUSION OF SNARE CONTAINING ARTIFICIAL VESICALES WITH V SNARE CON TAPING VESICALES WITH T SNARE CONTAINING NANODISC. IT OPENS A HOLE DURING THE FUSION PROCESS SO WE CAN MEASURE RELEASE OF CONTENT AS A PROXY FOR THE OPENING OF A FUSION PORE. WE ON SERVE IN NO INSTANCE IS THERE A BIG DIFFERENCE, YOU GET LIPID MIXING WITH OR WITHOUT THE VARIOUS MUTATIONS I DESCRIBE BUT GOING TO RYAN HART'S STRUCTURE WHEN WE MUTATE KNOWN CONTACTS IN HIS CRYSTAL STRUCTURE BETWEEN THE VAMP AND SYNTAXIN TRANSMEMBRANE ANCHORS WE GET SLOW OPENING OF THE FUSION PORE THOUGH FUSION OCCURRED WITH THE NORMAL SPEED. THE LINKER DOMAINS ARE INTACT, THE BILAYERS ARE FUSED. YOU CAN'T HAVE TOP LOGICAL FUSION WITHOUT SOME OPENING OF THE FUSION PORE SO YOU GET SOME RELEASE BUT THE RATE OF RELEASE IS DRASTICALLY INCREASED. IF YOU MUTATE THE NON-CONTACT RESIDUES WE'RE SHOWING ONLY A COUPLE HERE BUT WE HAVE DONE IT EXHAUSTIVELY, EVERY MOLECULAR CONTACT OBSERVED HERE IS IMPORTANT FOR OPENING OF THE FUSION PORE. SO WE'RE NOW TURNING TO PHYSIOLOGICAL SYSTEMS TO SEE IF THIS IS PHYSIOLOGICALLY RELEVANT BUT CERTAINLY SUGGESTS THAT EACH DOMAIN OF THE SNARE COMPLEXIN COLLUDING THE TRANSMEMBRANE HAS A CRITICAL ROLE. SO NOW I WOULD LIKE TO ENCAPSULATE THIS IN ONE MORE VIDEO. SO HERE WE HAVE NEUROTRANSMITTERS STORED IN A SYNAPTIC VESICAL. AND THE REASON I WOULD LIKE TO SHOW YOU THIS THOUGH IT'S PREPPY SHUTS, IT WILL OR YEN YOU TO WHAT'S NEXT WHICH IS A LITTLE MORE DIFFICULT. SO HERE WHAT WE HAVE IS THE T SNARE IN THE PLASMA MEMBRANE AND IN THE INITIAL STEP -- STATE WHERE THE VESICAL IS DOCKED THE END TERMINAL PORTION OF THE V SNARE VAMP IS HELIX. IT'S HELICAL STATE HALF ZIPPERED THROUGH THE HELICAL BUNDLE SO TO SPEAK HALFWAY FORMED. BUT THE REMAINDER OF THE V SNARE INCLUDING C TERMINAL HALF THAT WILL FORM THE BUNDLE AND LINKER PORTION HAS NOT YET ZIPPERED. WE BELIEVE FOR REASONS YOU'LL SEE THAT THAT IS THE POINT AT WHICH SYNAPTIC TRANSMISSION IS PAUSED TO ALLOW SIN CROW IN THISTY. SIN CROW IN THISTY RESULTS BECAUSE THE -- SIN CROW IN THISTY RESULTS BECAUSE IT IS ACCUMULATED AND THEREFORE BEEN RELEASED SYNCHRONOUSLY. IF THEY'RE IN THE ACCUMULATED AT A DEFINED STAGE THEY CANNOT BE RELEASED SYNCHRONOUSLY. IT'S FUNDAMENTAL TO THE PROCESS. NEXT THING THIS IS IN THE ABSENCE OF REGULATION, THE GENERAL FUSION PROCESS AS WE UNDERSTAND IT, IS THAT THE C TERMINAL PORTION OF THE VAMP ZIPPERS, THIS IS WHAT COMMIT TO FUSION. THEN THE LINKER ZIPPERS, AS THE ZIPPERS OPENS UP THE FUSION PORE INITIALLY BUT ONLY SO FAR. THEN FINALLY AS THE TRANSMEMBRANE ZIPPER, THIS DRIVES WE THINK IT PRODUCES A RADIAL FORCE SO WE DON'T REALLY KNOW THIS, IT'S SPECULATION, LITERALLY FORCE IT IS FUSION PORE OPEN. THAT'S OUR VIEW OF MEMBRANE FUSION FROM THE POINT OF VIEW OF THE PROTEINS. NOW I WILL LIKE TO COME BACK WITH BACKGROUND TO THE VERY SPECIFIC PROBLEM, HERE YOU HAVE A VESICLE, IT HAS IN VIVO A MUCH HIGHER CONCENTRATION OF V SNARES THAN ANY IN VITRO SYSTEMS. WE MIGHT TYPICALLY WORK WITH FIVE, TEN VAMPS IN AN ARTIFICIAL VESICAL OF THIS SIZE IN VITRO. BUT THE V SNARE HERE WAS PRESENT AT 70 COPIES PER VESICAL. THE T SNARE SYNTAXIN IS PRESENT AT PERHAPS ONE COPY FOR EVERY THOUSAND PHOSPHOLIPID MOLECULES IN THE ARTIFICIAL BILAYERS WE USE. HERE IT'S PRESENT IN CLUSTERS THAT ARE ALMOST PURE WITH RESPECT TO SYNTAXICSN ACCORDING TO THE LATEST WORK. REMARKABLY ENOUGH THESE VESICALES STAY WHERE THEY ARE, THEY DON'T FUSE, EVEN THOUGH IF YOU TAKE SAME PROTEINS OUT OF THE SYNAPSE, YOU PUT THEM INTO BILAYERS AT LOWER CONCENTRATIONS, WITHIN 50, 100 MILISECONDS THEY HAVE FUSED. I HAPPEN TO BE A BELIEVER IN PHYSICS. I HAPPEN TO BELIEVE THAT PHYSICS DOESN'T DISAPPEAR WHEN YOU BUT MOLECULES TO A CELL. THE BIOPHYSICS REPRESENTS THE GROUND TROOP OF WHAT A PROTEIN CAN DO. THAT GROUND TROOP DOESN'T CHANGE WHEN PROTEINS ARE PLACED IN THE CELL. I HAVE TO CONCLUDE FROM THIS THAT THERE'S A CLAMP THAT LOCKS THE EXOCYTOSIS PROCESS AND BLOCKS ABOUT 50 -- BLOCKS IT PROBABLY ABOUT A MILLISECOND OR LESS BEFORE THE RELEASE PROCESS. WHAT IS THAT CLAMP? OVER A TEN YEAR PERIOD WE TRIED TO FIND OUT WHAT THAT CLAMP WAS. IN ABOUT 2005 WE DISCOVERED WHAT IT WAS. WAS A PROTEIN DISCOVERED BY TOM PSEUDOOFT CALLED COMPLEXIN, AS THE NAME SUGGESTS IT'S A COMPLEX PROTEIN. SOMETIMES NAMES NEVER FALL EWE EVEN IF TERRIBLE NAMES. IT WAS COMPLEXIN BECAUSE IT FORMED A COMPLEX WITH A SNARE MENTION AND NOBODY ELSE KNEW WHAT IT IS AT THE TIME IT WAS NEEDED FOR SYNAPTIC NEUROTRANSMITTER RELEASE, WE WON'T HAVE TIME TO GO INTO THIS, IT HAS POSITIVE ROLES AN NEGATIVE ROLES, IT IS BOTH AN ACTIVATOR AND INHIBITOR. BUT MOST IMPORTANTLY I WOULD LIKE TO CONCENTRATE ON ITS ROLE AS CLAMP OR INHIBITOR. WHICH WE FOUND WHEN WE ADDEDDED COMPLEXIN AS A CANDIDATE CLAMP TO FUSION SYSTEMS THAT CONTAIN DEFINED VAMP SYNTAXIN SNAP 25, THAT OTHERWISE FUSED. IF YOU ADD COMPLEXIN AT HIGH ENOUGH CONCENTRATION THEY DIDN'T FUSE. WITH THAT THAT MEAN? NOT NECESSARILY ANYTHING BUT IF WE ADD CALCIUM SENSOR BACK AND ONLY THEN IF WE ADD CALCIUM COULD WE RESTORE FUSION IN THIS ARTIFICIAL SYSTEM. THAT SUGGESTED THATN%" PLEXIN COULD BE THE CLAMP. TOM PSEUDOOFF AND OTHERS SINCE HAVE MUCH MOVE PHYSIOLOGICAL EMPERIMENTS THAT SHOW THIS BEYOND A DOUBT AND I'LL SHOW YOU A COUPLE SUCH EXPERIMENTS TODAY. TOM AND JOSEPH SOLVED THE X-RAY LISTAL STRUCTURE OF COMPLEXIN IN THE EARLY 2000s, THE HELICAL PROTEIN THEY ARE PROBABLY THE ONE THING YOU NEED TO KNOW ABOUT IN THIS ENTIRE FIELD. IN VESICAL TRAFFICKING IT'S MOSTLY ABOUT HELICES INCLUDING TETHER PROTEINS. COMPLEXIN BINDS TO THE OUTSIDE OF THIS HELIC BUN L AND THE MEMBRANE PROXIMAL HALF. IMPORTANTLY THIS IS THE STRUCTURE OF COMPLEXIN WHEN IT IS BOUND TO THE FULLY ASSEMBLED COMPLEX WHICH ONLY OCCURS AFTER FUSION IT DOES NOT NECESSARILY INFORM US THEREFORE ABOUT THE ROLE THAT COMPLEXIN PLAYS DURING THE FUSION PROCESS. WHERE IT ACTS AS A CLAMP. WE ADDED COMPLEXIN TO THE SURFACE FORCE APPARATUS EXPERIMENT, A PIVOTAL EXPERIMENT HERE, FRED PALSA AND I AND HIS LABORATORY IN PARIS, (INDISCERNIBLE) WE FOUND COMPLEXIN CREATE AS NEW STATE WHICH THE SNARE COMPLEXES FROZEN IN THE HALF ZIPPERED STATE, 50% AS BEST WE CAN. SO WE KNEW IT DID SOMETHING IMPORTANT AND IT SOMEHOW COULD BE THE CLAMP. SO THE NEXT QUESTION WAS HOW DOES COMPLEXIN CLAMP? AS I WARNED YOU, I'M GOING TO ILLUSTRATE THIS BY A MODEL AND THEN SHOW YOU THE X-RAY CRYSTAL WORK IN THE CONTROLS, THAT STAND BEHIND IT. OUR PRESENT UNDERSTANDING ARE REACHED BY KAREN RYANSH AN X-RAY CRYSTALOGRAPHER DEPARTMENT OF YALE IN MY LABORATORY SHOWN HERE. THIS IS THE HALF ASSEMBLED SNARE COMPLEX WHERE THE INTERVENTION BY COMPLEXIN WE BELIEVE BEGINS. THE COMPLEXIN HELIX ACTUAL HI HAS TWO PARTS. THE CENTRAL HELIX AND THE ACCESSORY HELIC. THE CENTRAL HELIX IS ENDOWED WITH THE ABILITY TO BIND VAMP AND SYNTAXIN AND BINDS TO THE ASSEMBLED SNARE PIN. IN FACT, IT BINDS TO PORTIONS THAT ARE PRESENT BUT ONLY PRESENT WHEN THE SNARE COMPLEX IS HALF ZIPPERED. SO BEFORE THIS STATE, THERE'S NO BINDING BY COMPLEXIN. WHEN VN AS WE CALL IT IS HALF ZIPPERED TO THE IONIC LAYER THAT I MENTIONED, THEN THE BINDING SITE IS CREATED FOR THE CENTRAL HELIX. WHEN THAT HAPPENS, THE COMPLEXIN BINDS. IT BINDS IN SUCH A WAY TO LEAVE THE ACCESSORY HELIX POINTING OUT. THE CENTRAL HELIC BINDING TO THE SNARE COMPLEX IS VERY IMPORTANT. BECAUSE IT MEANS THAT YOU WILL ALWAYS HAVE A COMPLEXIN THERE. BY THE WAY, REMEMBER I MENTIONED THAT THIS INITIAL BINDING OF VN TO THE T SNARE IS VERY, VERY SLOW. ONE OF THE FACTORS THAT DRASTICALLY ACCELERATES IT IS THE ACCESSORY HELIC. SO THIS DESIGN ENSURES THAT THE ACCESSORY HELIC THE COMPLEXIN THE LOADED AT THE TIME THE VESICAL FIRMLY DOCKS. SO YOU ARE POSITIONING YOUR CLAMP IN ORDER TO MAKE SURE PRE-POSITIONING TO MAKE SURE IT'S THERE AT THE RIGHT TIME. THE ACCESSORY HELIX IN FACT WILL BE DOING THE CLAMPING. IF OUTTAKE IT OFF, SOME EXAMPLES, YOU DON'T GET CLAMPING. IF YOU MUTATE YOU DON'T GET CLAMPING. YOU NEED ACCESSORY HELIX FOR CLAMPING. YOU ONLY NEED THE CENTRAL HELIX FOR THE ACTIVATION OF NEUROTRANSMITTER RELEASE. THE NEXT THING THAT HAPPENS IS THAT THE ACCESSORY HELIX DOES ITS JOB BY REACHING ACROSS AND GRABBING A SECOND SNARE PIN. SO IT CLAMPS IN TRANS. IN THIS WAY ONE SNARE PIN THAT'S ASSEMBLED CLAMPS ANOTHER, MUTUALLY INHIBITING EACH OTHER. LET'S LOOK AT THIS FROM THE TOP VIEW IN WHICH WE NOW LOOK AT THE PROTEINS LYING LIKE A SANDWICH BETWEEN THE PLASMA MEMBRANE BELOW AND SYNAPTIC VESICLE ABOVE. THE FIRST THING THAT HAPPENS IS COMPLEXIN BIND BY CENTRAL HELIX. LEAVING ACCESSORY HELICES FREE TO BEHIND BETWEEN COMPLEXES. THE SEQUENCE OF THE ACCESSORY HELIX IS HOMOLOGOUS TO THE SEQUENCE OF THE V SNARE IN THE SAME REGION. WHICH IS VERY ELEGANT. 'S AN HONNARY V SNARE. IT GOES RIGHT IN HERE AND BINDS THE T SNARE A IF IT WERE A V SNARE BUT P IT'S NOT. BY BINDING THERE ITW V SNARE FROM FURTHER ASSEMBLING. THAT UNFOLDED PORTION OF THE V SNARE CANNOT CONVERT TO A COIL BECAUSE TO DO SO IT NEEDS TO DISPLACE THE COMPLEXICSN. SO THERE'S A TUG OF CAR RESULTING IN CLAMPING SO IF THE ACCESSORY COMPLEXIN HELIX IS REMOVED AS ILLUSTRATED HERE THE BLOCK ZIPPERING IS REMOVED AND FUSION WILL PROCEED. BUT THAT DOESN'T OCCUR BECAUSE THE ACCESSORY HELIX IS CLAMPING, THIS CLAMPING OCCURRING BETWEEN TWO SNARE PINS. AS YOU CAN IMAGINE THIS ACCESSORY HELIX, THERE'S NOTHING TO PREVENT IT FROM FORMING, FROM RECRUITING ANOTHER SNARE PIN. AND ANOTHER ONE TO GIVE RISE TO WHETHER WE CALL A ZIG ZAG ARRAY OF -- WHICH IS A HIGHLY COOPERATIVE STRUCTURE AND THAT GIVES RISE WE BELIEVE TO SIN CHRONICITY. ACCORDING FADING A LARGE NUMBER OF SNARES IN ONE STRUCTURE THAT IS ALL OR NONE T HAS THE ALL OR NONE QUALITY OF COOPERATIVE STRUCTURE, IT ALL WILL COME APART AT ONCE, THERE'S A CLEAR PREDICTION FROM THE STRUCTURE AND THIS WILL LEAD TO THE SYNCHRONOUS RELEASE OF A VESICLE. SO WE THINK THAT ULTIMATELY IS WHAT SIN CHRONICITY COMES FROM. THIS IS HOW WE REGARD CLASPING TO OCCUR. HOW DOES ACTIVATION éñ OCCUR? NOW WHAT WE HAVE GOTTEN TO IS A CLAMPED VESICAL THAT NEEDS TO BE RELEASED WHEN CALCIUM ENTERS. CALCIUM WHEN CALCIUM ENTERS THE SENSOR FOR CALCIUM IS THE PROTEIN SYNAPTIC TAGUMEN DISCOVER AS A VESICAL COMPONENT BY TOM SUDHOFF. IN YEARS OF PAINSTAKING WORK MUCH CONTROVERSIAL BUT NOW WIDE UNDERSTOOD TO BE CORRECT TOM WENT TO SHOW IT IS INTACT THE CALCIUM SENSOR FOR SYNCHRONOUS NEUROTRANSMITTER RELEASE. AND PROBABLY THE MOST PERSUASIVE EXPERIMENT IS A STRUCTURE ACTIVITY EMPERIMENT WHERE HE MUTATED THE CALCIUM BINDING SITES IN SYNAPTOTAGMIN TO RATCHET CALCIUM BINDING UP OR DOWN IN TERMS OF BINDING CONSTANT, PUT IT INTO A MOUSE IN PLACE OF NORMAL SYNAPTOTAGMIN GENE AND RATCHET UPPER DOWN SENSITIVITY AT SYNAPSES ACCORDINGLY. SO THAT DOESN'T TELL US HOW CALCIUM FUSION OCCURS BUT IT TELLS US IT DOES OCCUR BY MEANS OF THIS MOLECULE. SO HOW DOES SYNAPTIC -- SYNAPTOTAGMIN SENSE CALCIUM? I'M MOVING FROM WHAT I REGARD AS FACT TO SPECULATION. ONE THING THAT IS A FACT, THOUGH IS SYNAPTOTAGMIN IS A TIGHT BINDER OF MEMBRANES, IT HAS AN ALOETHATIC LOOP THAT INSERTS TO THE BILAYER AND INSERTS WHEN CALCIUM IS BOUND AT THE BINDING SITES. THE CALCIUM BINDING SITE CONSISTS OF A COMPOUND ASPAR TICK ACID RESIDUES. THERE ARE APARTIC ACID RESIDUES THAT BIND CALCIUM IONS AN COORDINATE IT WITH PIP-2 THAT IS ON THE MEMBRANE. SO THE ACTUAL CALCIUM BINDING SITE IS A SANDWICH OF CALCIUM BETWEEN THE TWO SITES. NOW, THE COST OF DOING BUSINESS FOR THATCAL YUM BINDING SITE IS THIS ALOEPHATIC LOOP WHICH MUST BE INSERTED BECAUSE IT'S THE ONLY WAY THE CALCIUM BINDING SITE CAN BE SATISFIED. SO SYNAPTOTAGMIN INSERTS. MUTATIONS IN SYNAPTOTAGMIN THAT ABROGATE THE INSERTION OR LIKEWISE DON'T ACTIVATE SYNAPTIC TRANSMISSION. ONE OR ANOTHER WAY EVERY MODEL HAS TO ACCOMMODATE THIS AS AN IMPORTANT ELEMENT OF SYNAPTIC TRANSMISSION. THE OTHER THING SYNAPTOT ACTIONGMIN DOES IN LARGE STUDIES IS PERTURB MEMBRANES IN ONE OR ANOTHER WAY. THE INITIAL EVIDENCE FROM HARVEY MCMAHAN, TAGMIM IS ADDED IN EXCESS TO LIPSOMES IT WILL TUBE LATE THEM IN THE PRESENCE OF CALCIUM. THAT REQUIRES INSERTION OF THIS REGION. PROBABLY LESS THAN MEGA DOSES IT DOESN'T TUBE LATE BUT PHYSIOLOGICALLY PERTURBS IT AND EVERY LAB WHO STUDIES THIS HAS FOUND SYNAPTOTAGMIM ACCELERATES THE RATE OF LIPSOME FUSION WHEN YOU HAVE SNARES DRIVING IT IN A CALCIUM DEPENDENT WAY. IT DOESN'T FUSE BY ITSELF BUT WILL ACCELERATE PROBABLY A FACTOR OF 10 TO 100. BUT VERY SUBSTANTIALLY THE RATE OF LIPID FUSION, LIPID MEMBRANE FUSION. WE DON'T UNDERSTAND EXACTLY HOW THAT WORKS, PROBABLY IT'S BEEN CHANGING MEMBRANE INTENTION ACCORDING TO BRUNGER AND OTHERS BUT IT'S AN IMPORTANT FACT. SO IT'S A CALCIUM SENSOR AND AXEL RANT. HOW DOES SYNAPTOTAGMIN FIT TYPE THIS STORY? THIS IS THE CUTTING EDGE OF THE FIELD. BUT WE KIND OF KNOW HOW SYNAPTOTAGMIN BINDS TO THE SNARE COMPLEX. WE KNOW THIS LARGELY FROM THE WORK OF ALEX BRUNGER, I'LL SHOW YOU MORE DETAILS. WHEN IT BINDS IT BINDS ONE PER SNARE PIN AND WE IMAGINE IT BINDS CALCIUM FROM THIS ARRAY BOUND SYNAPTOTAGMIN. WHEN IT BINDS CALCIUM IT INSERTS TO THE BILAYER. WE IMAGINE FURTHER THIS IS OUR HYPOTHESIS, WHEN I'LL SHOW YOU SIDE VIEW WHICH IS MORE EXPLANATORY. SO NOW YOU SEE THE SYNAPTOTAGMIN BINDING. WHEN CALCIUM ENTERS AND BINDS TO SYNAPTOTAGMIN WILL CAUSE REARRANGEN'T ONE WAYER ANOTHER WHICH WE BELIEVE EXERTS MECHANICAL FORCE ON THIS ARRAY AND REMOVES A SNARE PIN THERE BY UNCLAMPING. THAT'S OUR MODEL, THE AMOUNT OF EVIDENCE IS RELATIVELY MINIMAL. AND BUT IT IS IN FACT A SIMPLE POSSIBLY. NOW, WHERE DOES THIS COME FROM? I'LL TRUE TO BUZZ THROUGH THIS MUCH MORE QUICKLY SEEING THE HOUR. WHAT KAREN AND I AND COLLEAGUE ESPECIALLY DANIELLE CUMEL WHO DID THE WORK HERE AND BRILLIANT GUY IS TO SOLVE THE CRYSTAL STRUCTURE OFR>Ñ4 SNARED COMPLEX. FOR ALL REASONS I SHOWED YOU WHEN VAMP IS HALF ZIPPERED THAT'S WHEN WE THINK ALL THE ACTION IS OCCURRING FOR CLAMPING. I EXPLAINED WHY THAT IS. PROBLEM YOU HAVE IS YOU CAN'T GET A CRYSTAL STRUCTURE OF A DISORGANIZED STATE LIKE A FUSION INTERMIT OR HALF ASSEMBLED SNARE COMPLEX. THE NEXT BEST THING YOU CAN DO WHICH IS WHAT WE DID IS PRODUCE A STABLE HALF ZIPPERED SNARE COMPLEX BY USING JUST THE END TERMINAL PORTION OF VAMP AN LEAVING THE REST OUT. SO THAT PRODUCES A STRUCTURAL KNEW METIC OF HALF ZIPPERED SNARE COMPLEX. WE WERE ABLE TO CRYSTALLIZE THAT IN COMPLEX WITH COMPLEXIN, NOT SYNAPTOTAGMIN. NO ONE GOT ONE TO MY KNOWLEDGE. WE SOLVED THAT STRUCTURE. AND WE GOT SOMETHING THAT WAS MARKEDLY DIFFERENT FROM WHAT SUDHOFF AN RIDDO FOUND. THEY FOUND THAT COMPLEXIN LIES AT ALPHA HELIX IN THE GROOVE BETWEEN SYNTAXIN AND VAMP CONTACTING BOTH OF THEM, THIS IS THE SEN TRILLION HELIX PORTION, ACCESSORY HELIX HAD NO CONTACTS WITH THE SNARE COMPLEX BUT HELIX RUNS PARALLEL TO THE SNARE COMPLEX. THAT'S NOT AT ALL WHAT WE FOUND. WE FOUND THE OPEN CONFIRMATION. IN CONTRAST TO THE OTHER CONFIRMATION POST FUSION REPRESENTING THE FULLY ZIPPERED SNARE COMPLEX WE CALL CLOSED. OPEN GOES OFF AS 45-DEGREES. THE COMPLEXIN HELIX IS ISOMORE FIXED. 'S THE SAME HERE AND HERE EXCEPT FOR THE WAY IT LIES ON THE SNARE COMPLEX, IT'S BASICALLY THE SAME. BUT NOW GOES OFF AT 45-DEGREES. THAT POSED A PROBLEM FOR US BECAUSE I WAS TELLING YOU THE -- ALL THE GENETICS SAID THE ACCESSORY HELIC DOES CLAMPING. BUT WHAT'S CLAMPED IS THE ASSEMBLING OF MEMBRANE PROXIMAL WOULD BE HERE, PORTION OF THE SNARE COMPLEX ASSEMBLED LAST, HOW CAN THIS HELP -- HOW CAN THIS INTERFERE WITH ZIPPERING OF THIS WHEN THEY'RE ACTUALLY DIVERGING AWAY FROM EACH OTHER. THE ANSWER COMES FROM THE CRYSTAL PACKING. WHEN WE LOOK AT LAYER OF CRYSTAL WE SEE A SNARE PIN, THE MEMBRANE ANCHORS WOULD BE AT THIS END, THE SYNAPTIC VESICAL WOULD BE ABOVE AS IN THE CARTOON, THE PLASMA MEMBRANE IN THE PLAIN BELOW, HERE IS THE HALF ZIPPERED VAMP. NOW WHAT YOU SEE THE COMPLEXIN CENTRAL HELIX LAUNCHING AN ACCESSORY HELIX, BUT THAT ACCESSORY HELIX IN THE OPEN CONFIRMATION ACTUALLY BINDS IN THE SAME GROOVE THIS V SNARE WOULD IF IT CONTINUES TO ZIPPER. EXACTLY AS I CARTOONED IT. THIS IS THE BASIS FOR THE ZIG ZAG ARRAY AND IDEA OF INTERMOLECULAR CLAMPING AS THE PRINCIPLE FOR CLAMPING OF SYNAPTIC TRANSMISSION TO ALLOW SIN CHRONICITY. ANY CRYSTALOGRAPHER TELLS YOU THIS IS GOOD BUT HOW DO YOU KNOW THIS ISN'T A CRYSTALZATION OAR FACT? I HAD NEVER HEARD OF A COUNTRYALZATION ARTIFACT. -- CRYSTALZATION ARTIFACT. I THOUGHT AS SOON AS YOU GET A CRYSTAL STRUCTURE EVERYBODY CONGRATULATES YOU. PUBLISHES YOUR PAPER. NO, THEY SEND YOU BACK HOME THAT'S WHERE WE BIOCHEMISTS START COMING IN TO THE PICTURE BECAUSE HOW DO WE KNOW THIS DIDN'T OCCUR AS A RESULT OF THE CRYSTALLIZATION? WE KNOW. HOW? BECAUSE WE CAN ISOLATE COMPLEXES IN SOLUTION AND WE OBSERVE TO BEGIN WITH THIS OPEN CONFIRMATION EXISTS. THIS WAS WORK BY CRYSTAL KUMAR AND DANIEL RADOFF IN MY LAB. NO DETAILS GIVEN THE TIME. WE USE FRET ENTER INTERMOLECULAR FRET. WE FORM COMPLEXES OF COMPLEXIN AND THE SNARE COMPLEX. AND PUT A THREAT ACCEPTTOR ON SNAP 25 ON THE BASE OF THE COMPLEX AND TWO POSITIONS O MORE. IN THE COMPLEXIN ACCESSORY HELIX, WE PLACE THE COGNATE FLUORESCENT PROBE. THIS ALLOWS US TO READ THE DISTANCE THROUGH FRET, THE SIGNAL WILL GO UP IN THE CLOSE CONFIRMATION ON OPEN CONFIRMATION. AS YOU CAN SEE WE OBSERVE VERY STRONG FRET SIGNAL WHEN WE HAVE THE FULL LENGTH VAMP HERE BUT WHEN WE HAVE THE FIRST 60 RESIDUES OF VAMP THE HALF ZIPPERED STATE NOW WE GET OPEN. I HOPE THAT'S HOW DO WE KNOW THESE CONFIRMATIONS ARE WHEY THEY SAY? WE CAN TAKE THE FRET NUMBERS AN CRUNCH THE NUMBERS. AND ACTUALLY WE PREDICT WITHIN 10% SYSTEMATIC THE CRYSTAL COORDINATES OF THESE -- THIS POSITION, THIS POSITION, THIS POSITION AND THAT POSITION RELATIVE TO THAT POSITION IN THE TWO CRYSTALS TWO TYPES OF CRYSTALS OPEN AN CLOSE IN THREE FORMS. SO WE'RE VERY CONFIDENT THAT THIS IS THE NATURAL STATE AND SOLUTION, THEREFORE THE CRYSTALZATION DID NOT CAUSE THE OPEN STATE, THE OPEN STATE HAD TO BE ACCOMMODATE MISDEMEANOR THE CRYSTAL. THE OTHER POTENTIAL CONCERN IS HOW DO WE KNOW THIS TRANSINTERACTION BETWEEN THE ACCESSORY HELIX AND T SNARE AT V SNARE BINDING SITE OCCURS INDEPENDENT OF CRYSTAL? WE KNOW THAT BECAUSE WE CAN MEASURE IN SOLUTION NOW THAT WE KNOW TO LOOK FOR IT. AGAIN, THIS IS PUBLISHED IN 2011, IN NATURE STRUCTURAL MOLECULAR BIOLOGY, I NEED TO UPDATE THE SLIDE. IF YOU MIX COMPLEXIN AND P THE SNARE COMPLEX WITH A HALF ZIPPER ED SNARE EXACTLY WHAT WENT INTO THE CRYSTAL BUT WITH ITS COMPLEXIN ACCESSORY HELIX SITE PRE-BOUND AN PRE-BLOCKED BY CENTRAL HELIX, I MEANT CENTRAL HELIX BINDING SITE PRE-BLOCKED SO ONLY THE HYPOTHETICAL TRANSSITE INVOLVING THE TERMINAL T SNARE IS AVAILABLE. WE GET STOIC METRIC BINDING. 15 MICROMOLAR BINDING CONSTANT, NKT BINDING ENERGY, A NUMBER WORTH REMEMBERING. THAT IS CLOSELY LINKED TO THE ACTIVATION FOR MEMBRANE FUSION. NOW VERY IMPORTANTLY, WE MADE MUTATIONS -- I CAN SEW -- WE MADE MUTATIONS IN ACCESSORY HELIX INCREASE OR DECREASE THE BINDING ENERGY HERE. YOU CAN SEE HERE POINT MUTATION IN THE ACCESSORY HELIX RIGHT HERE THAT ELIMINATES BEHINDING >> SO WHAT DO WE PREDICT? NORMAL GENE THIS GENE TESTED PHYSIOLOGICALLY SHOULD CLAMP BETTER THAN NORMAL AND SHOULD REDUCE SPONTANEOUS FUSION THIS IS WHAT WE SEE IN A CONTROL ANIMAL, WHAT THEY SEE REALLY, IT OCCURS T A COUPLE PER SECOND MORE OR LESS AS DESCRIBED. IF YOU KNOCK OUT COMPLEXIN THE RATE IS UP TO PREVENT SPONTANEOUS RELEASE. A CLAMP P IF YOU REMOVE IT IF YOU REMOVE THE CLAP THAT PREVENTS THE SPONTANEOUS RELEASE, WHAT HAPPENS TO SPONTANEOUS RELEASE? GOES UP. THAT'S EXACTLY WHAT HAPPENS HERE. COMPLEX,N IS THE CLAMP. IF WE MUTATE RESIDUES THAT FACE THE INSIDE CONTACTING REGIONS FOR THE T SNARE, WHEN WE DO THAT, THEN OR IF WE DISRUPT THE HELIX NOW ACTUALLY -- WE PUT IN THE HUMAN COMPLEXIN WE ACTUALLY HAVE NO AFFECT. EVEN THOUGH IT'S THERE, IT'S THERE AT THE SYNAPSE, IT DOESN'T CLAMP. IT CLAMPS NOT QUITE AS WELL. WE'RE TRYING TO UNDERSTAND THIS AS DROSOPHILA GENE IF WE PUT IN ACCESSORY HELIX MUTANT THAT HAS A FIVE TIMES HIGHER BEHINDING ENERGY WE GET FIVE TIMES LOWER SPONTANEOUS RELEASE. SO TO SUMMARIZE THEN, THIS INTERACTION IN THE CRYSTAL STRUCTURE THAT WE BELIEVE IS THE STRUCTURAL BASIS OF CLAMPING, THAT INTERACTION MUST BE CONTROLLING SPONTANEOUS RELEASE BECAUSE AS WE RATCHET UP AND DOWN THAT INTERACTION BY THE SAME LOGIC AS TOM SUDOFF RATCHETING UP AND DOWN CALCIUM BINDING THAT RATCHETS UP AND DOWN SYNAPTIC NEUROTRANSMITTER RELEASE, THAT'S WHY IT'S THE CALCIUM SENSOR. THIS MUST BE THE CLAMPING INTERACTION IF WE RATCHET UP AND DOWN CLAMPING GETS RATCHETED UP AND DOWN IN PHYSIOLOGICAL SYSTEM. SO THERE'S NO DOUBT THIS TRANSINTERACTION THAT GIVES RISE TO THIS ZIG ZAG ARRAY FROM THE COUNTRYAL STRUCTURE IS THE STRUCTURAL BASIS OF CLAMPING WHICH MUST THEN OCCUR IN THE HALF ZIPPERED STATE. SO THIS IS A VERY IMPORTANT EXPERIMENT I THINK FOR THE ENTIRE CASE AND FOR THE ENTIRE MODEL. DO WE KNOW THAT THE ZIG ZAG ARRAY EXISTS IN THE WAY IT DOES IN THE CRYSTAL COURSE? WE DOPE KNOW. WE'RE INVESTIGATING. IT MAYBE MORE TRUNCATED. MAYBE INVOLVES TWO SNARE COMPLEXES. I DON'T KNOW AT THIS POINT. BUT THAT INVOLVES CLAMPING INVOLVES TRANSINTERACTION SEEMS CLEAR. HIGH CONCENTRATION HERE AS THEY ARE IN THE CRYSTAL. IN FACT, WHEN WE CALCULATE ESTIMATE WHAT IT'S LIKE IN THE SPACE HERE IT'S ACTUALLY LOWER THAN THE CRYSTAL THAN IN THE SPACE. WHERE YOU HAVE TWO CLOSELY OPPOSED MEMBRANES SO IT'S LIKELY THAT WE HAVEN'T PROVEN THIS ARRAY IS SOME SIGNIFICANT EXTENT PROBABLY 10, 15 COPIES OF THE SNARE PIN WE'RE GUESSING. A VERY IMPORTANT POINT A VERY IMPORTANT POINT IS THAT THIS IS THE PRE-FUSION STATE IN THE HALF ZIPPERED STATE, THIS IS WHAT EVERYBODY IMAGINES THE FUSION PORE MUST LOOK LIKE, IT HAS TO BE CIRCULAR, IF IT HAS MULTIPLE SNARES THEY HAVE TO BE ARRANGED WHETHER BY INTENT OR NECESSITY. SO -- AND THE OPEN STATE HERE, OF THE COMPLEXIN OCCURS MUST GO THROUGH A TRANSITION THEN BETWEEN OPEN AND CLOSE, THERE MUST BE A TRANSITION TO START AGAIN BETWEEN OPEN AND CLOSED STATE OF COMPLEXIN BECAUSE THIS IS WHAT WE GET WHEN IT'S IN THE CLAMP STATE AND THIS IS WHAT WE SEE OR WHAT IS SEEN IN THE POST FUSION STATE. SOMEWHERE IN THERE THERE IS A SWITCH. NEW YORK CITY I THINK YOU CAN SEE IN ORDER FOR THIS VAMP TO CONTINUE TO ZIPPER THAT ACCESSORY HELIX HAS TO GET MOVED OUT WAY. THAT'S OBVIOUS. BUT WHEN THAT OCCURS, THERE IS NOTHING THEN WHEN THAT HAPPENS THERE'S NOTHING TO PREINVENTORY THIS ZIPPERING. IF YOU WERE TO TAKE ALL THESE OPEN STATES AN CONVERT TO CLOSED, I THINK YOU CAN SEE THAT THE ZIG ZAG ARRAY CAN NO LONGER FORM. THE ZIG ZAG ARRAY THE ONLY FORM IF THE OPEN CONFIRMATION AND THE CLOSED THE ACCESSORY HELIX IS SATISFIED WITH ITS OWN SNARE COMPLEX AND NO LONGER CAN INTERACT WITH ANOTHER ONE. SO THIS OPEN TO CLOSE SWITCH IS CLEARLY THEN WHAT WILL BE DRIVING THE ACTIVATION PROCESS. I WON'T HAVE TIME TO GO INTO THIS, IT'S IN OUR PAPERS IF YOU'RE INTERESTED BUT IT TURNS OUT FOLLOWING RESIDUE 60 IN VAMP, THERE'S RESIDUE 1, THERE'S 60, BETWEEN 60 AND 67 THERE ARE TWO VERY SPECIAL TURNS OF THE HELIX. THAT CONTAIN THREE ASPAR TICK ACID RESIDUES. THOSE ACTUALLY FORM AN INTERACTION WITH THE ACCESSORY HELIX HERE. THAT INTERACTION CANNOT OCCUR WHEN THOSE TWO TURNS ARE NOT YET FOLDED. WHEN THOSE TURNS ARE FOLDED, THEY THEN CREATE THE BINDING SITE THAT IS DIFFERENT BETWEEN THIS STRUCTURE AND THIS STRUCTURE AND THEY PULL THE COMPLEXIN ACCESSORY HELIX DOWN. WHAT HAPPENS IN FACT WE THINK IS THAT WHEN ANY ONE OF THESE ACCESSORY HELICES FLUCTUATES OUT LONG ENOUGH, THIS VAMP SAY THIS ONE HERE ZIPPERS, WHEN IT ZIPPERS TO HELICES THAT CREATES A BINDING SITE, WITH COMPLEXIN, COMPLEXIN IS SWITCHED FROM OPEN TO CLOSED WHEN IT DOES THAT IT RETRACTS FROM THIS GUY. WHEN THIS RETRACTS THEN THAT VAMP GETS ZIPPER AND PULLS DOWN THIS GUY. LIKE A HOUSE OF CARDS IT GOES DOWN. WE HAVE TESTED THAT MUTATING THE APARTIC ACID RESIDUE AND WHEN WE DO THAT YOU GET IN VAMP AND YOU GET PERFECTLY GOOD FUSION IN AND OF ITSELF BUT YOU LOSE AT LEAST IN OUR IN VITRO SYSTEM THE ABILITY TO ACTIVATE FROM CALCIUM. WE'RE TESTING THAT IN A PHYSIOLOGICAL SYSTEM IN NEUROMUSCULAR JUNCTION THAT'S OUR CURRENT VIEW. SO I JUST WANT TO LEAVE YOU THEN WITH OUR HYPOTHESIS OF HOW ACTIVATION MAY WORK WHICH IS LARGELY BASED ON COMBINING WHAT I HAVE SHOWN FROM OUR LAB AND VERY ELEGANT WORK THAT AXEL BRUNNER AN COLLEAGUE AT STANFORD USING FRET EXTENSIVELY TO MEASURE POSITIONING OF SYNAPTOTAGMIN ON THE SNARE COMPLEX. IT'S MISSING A CRYSTAL STRUCTURE BOUND TO A SNARE COMPLEX IN ANY STATE OF ASSEMBLY. EVEN HIGH RESOLUTION PICTURE. IF ANYBODY HAS AMBITION IN THIS FEEL THAT'S HELPFUL TO HAVE. BUT BASED ON HIS MOLECULAR DYNAMIC MODEL AND BIOCHEMICAL DATA CONSISTENT WITH THIS, IT APPEARS THAT SYNAPTOTAGMIN BINDS TO THE SNAP 25 SIDE OF THE ASSEMBLING SNARE PIN. IT BINDS ONE PER SNARE PIN. STOICHIOMETRY, ONE ACTIVATOR PER SNARE PIN. IT BINDS TO THE SNAP 25 FACE. THERE ARE FOUR HELICES IN THE BUNDLE, TWO ON ONE SIDE, TWO ON THE OTHER. TWO ON ONE SIDE ARE THE TWO FROM SNAP 25. THEY SEEM TO BIND THIS SYNAPTOTAGMIN. THE OTHER TWO IS WHERE THE PLANT SIDE WHERE THE CLAMPING OCCURS, THAT IS WHERE VAMP AND SYNTAXIN ARE WHERE THE ACCESSORY HELIX. THE CENTRAL HELIX TO ALL THE ACTION THAT I HAVE JUST BEEN SHOWING YOU IS OCCURRING ON THE OPPOSITE SIDE OF THE SNARE BUNDLE WHERE WHERE SYNAPTOTAGMIN BINDS. WHEN IT BINDS THIS WAY CONVENIENTLY THIS IS SHOWN FACING THE VESICAL MEN BRAIN IT FACES THE MEMBRANE AND WE DONE KNOW IF ANY (INDISCERNIBLE) SO ALL THAT NEEDS TO OCCUR WHEN CALCIUM ENTERS IS FOR THIS SYNAPTOTAGMIN TO BE ORIENTED BY THE MEMBRANE. I HAVE SHOWN YOU ANYTHING FRAMEWORK THAT GOT REMOVEDDED FROM. THE SIMPLEST MODEL CARRIES THIS OUT WE FORMED WITH TWO NANODISABLES WHICH ZIPPER TO A LARGE DEGREE BUT CAN'T FUSE BECAUSE OF CONSTRAINTS. WE PUT SYNAPTOTAGMIN ON IT AND WE HAVE DONE STOP FLOW MEASURING ON A SCALE OF MILLISECONDS IN FACT THE INSERTION OF THIS LOOP OF SYNAPTOTAGMIN TRIGGERED BY CALCIUM AND ASKED WHETHER IT REMAINS BOUND TO THE SNARE COMPLEX AS IT INSERTS. BECAUSE IF SYNAPTOTAGMIN IS GOING TO ACTIVATE BY PHYSICALLY DOING WORK ON THE SNARE COMPLEX REMOVING IT FROM THE PLAIN, IT'S PLAINER ARRAY STRUCTURE CLEARLY NEEDS TO HANG ON DURING THIS INSERTION PROCESS AND THAT IS IN FACT THE CASE. HERE WE LOOK AT FLUORESCENCE ORING INSESSION OF ALOETHATIC LOOP BETWEEN A AND V DOMAIN AND SNAP 25 WHICH BECOMES ABSOLUTELY INVARIANT. BY THE WAY ALSO FOR THOSE WHO MAYBE AFICIONADOS THE A AND B DOMAIN DON'T MOVE WITH RESPECT TO EACH OTHER DURING THIS POWER STRUGGLE SO THERE IS A POWER STROKE WHICH SYNAPTOTAGMIN INSERTS TO THE BILAYER SHOWN GENETICALLY TO BE NEEDEDTOR PASSAGE OF CALCIUM SIGNAL TO THE SNARE. IT'S PHYSICALLY BOUND TO THE SNARE AND BASED ON THIS IN VITRO WORK MAINLY THE ONLY TENABLE MODEL BECAUSE THE SYNAPTOCONFIRMATION A AND B DON'T CHANGE THEIR DISTANCE, A MECHANICAL FORCE IS EXERTED AND WE CAN'T PROVE TODAY THAT THAT MECHANICAL FORCE IS TRANSDUCED INTO REMOVING THE ACCESSORY HELIX FROM THE T SNARE BEHINDING SITE THAT CAUSES THE INHIBITION BUT OBVIOUSLY CAN SEE THAT THAT'S A VERY TENABLE HYPOTHESIS THAT'S THE ONE WE'RE FOCUSED ON. IT'S GENERAL TECHILY AND KINETICALLY. THIS IS MY LAST SLIDE. THE ACTIVATE ENERGY TO REMOVE A SNARE PIN CAN REQUIRE BREAKING TWO INTERACTIONS. IT'S ENTERACTING WITH ONE SNARE PIN AND IN DISARRAY WITH ANOTHER. THIS INTERACTION CAN BE BROKEN BY SO FOR THIS INTERACTION THAT WILL BREAK TO THIS ONE OR 10 KT WILL BREAK THIS ONE. IF YOU PULL OUT IF YOU HAVE A FLUCTUATION IF YOU HAVE 10-KT ONLY YOU GET FLUCTUATIONS HERE OPT ORDER OF ABOUT TWO MICROSECONDS ACCORDING TO SINGLE MOLECULE TYPES OF CALCULATIONS. DOWN HERE ACCORDING TO SOMEBODY OR OTHER RELATIONSHIP WHICH MY PHYSICIST COLLEAGUE IS TELLING ME ABOUT AND WHICH I MIGHT ONCE HAVE UNDERSTOOD. SORRY TO BE BLAH SAY ABOUT THAT BUT THE PHYSICISTS AGREE THE FLUCTUATION OCCURS IN 2 TO 20 MICROSECONDS, NOT LONG ENOUGH TO BE PERSISTENT. SO THAT ALLOWS DETERMINED ACT INVESTIGATION BY REMOVING TWO OF THESE POINTS WHEN YOU REMOVE TWO, WHEN YOU LOOK YOU'LL SEE THE WHOLE THING FALLS APART LIKE DOMINOES BUT IF YOU REMOVE IT TAKES 20 KT TO CAUSE ACTIVATION. THE SIMPLEST HYPOTHESIS IS THAT THIS+ THE ACTUAL ENERGY RELEASED WHEN IT BINDS CALLS YUM PLUS PIP AT RELEVANT CONCENTRATION 45 KT. IS IT FULLY AVAILABLE TO TO BE CHANNELED? NO, DID NOT KNOW THAT, WITH WE DONE KNOW THAT BUT AT LEAST IT'S ATENABLE HYPOTHESIS. AND KINETICALLY TENABLE AS WELL. SORRY FOR THE BREATHLESS FINISH. I WOULD LIKE THE JUST THANK THE COLLEAGUES WHO ARE MOST CRITICALLY IMPORTANT FOR THIS WORK, THE EXPERT CRYSTALOGRAPHY DANIELLE KUMEL AND CAPE RYAN ISH PROFESSOR IN YALE CELL BIOLOGY DEPARTMENT, THE ELECTROPHYSIOLOGY THAT WAS SO IMPORTANT ESTABLISHING THE PHYSIOLOGICAL RELEVANCE AT MIT. AND WORK IN THE FIRST INSTANCE COMPLEXIN CLAMPS IN A DIFFERENT STATE THAT GIVES US THE CLUE HOW TO DESIGN THE MOLECULE FOR THE CRYSTAL STRUCTURE FOR THE PHYSICIST,'S IMPORTANT IN ALL COLLABORATIONS. MORE RECENTLY THE OBSTACLE WORK WITH ANOTHER PROFESSOR AT YALE AND FOLKS FROM MY LAB I MENTIONED I BELIEVE AS I HAVE GONE THROUGH THE WORK IN THEIR PARTICULAR CONTRIBUTION. SO THANK YOU ALL VERY MUCH. [APPLAUSE] >> WE'LL TAKE QUESTIONS AFTER THE CALL CLEARED OUT PEOPLE WHO NEED TO GO RIGHT AWAY. >> I ONLY HAPPY TO DO IT HERE OR GO TO THE RECEPTION. MAYBE HARDER TO DO THAT. >> QUESTIONS. >> THANK YOU. VERY, VERY EXCITING AND ALSO IT HAPPENS IN THE IMMUNOLOGICAL EXCEPT FOR SLOWER PACE. MY QUESTION AND MAYBE SUGGESTION IS YOU MENTIONED THE END TERMINUS IS ACTUALLY STARTING THE WHOLE GAME FOR STRUCTURE OF AND SIGNAL TO THE END TERMINAL. IS THERE ANY INFORMATION KNOWN ABOUT THE END TERMINAL COMPOSITION FOR GROUP THAT IT'S -- >> WHAT'S THE QUESTION THEN? >> THE COMPOSITION OF THE END TERMINAL AND THE C TERMINAL, CHANGES AND THE CHARGE -- >> WE SHOULD DISCUSS PRIVATELY BUT COMPOSITION OF THE AMINO ACIDS ISN'T LIKELY TO CHANGE, IF THAT'S WHAT YOU MEAN. IS THERE ANOTHER QUESTION? >> OKAY. >> YES. >> IT SEEMS LIKE ONE OF THE CRITICAL THINGS IN THE MEASUREMENT OF THE SYNCHRONOUS PHENOMENA AND TIME DEPENDENT ARE THE TOOLS THAT YOU USE AND X-RAY COUNTRYALOGRAPHY ARE A SNAP SHOT THAT HAPPENS OVER A PERIOD OF TIME. SOME SECONDS OR NANOSECONDS. IF YOU HAD A WISH LIST OR -- OF A NEW TOOL THAT YOU COULD USE OR MAYBE IN EXISTENCE, WHAT DOES THAT IT WILL LOOK LIKE? TWEEZERS IS THERE ANYTHING KNOWN)/P ABOUT THE LIPID ENVIRONMENT -- AND HOW THAT MIGHT ASSIST. THISES WHO FUSION PROCESS? >> GOOD QUESTION. ASIDE FROM PIP IS A BEING ASIDE. THAT IS ABSOLUTELY REQUIRED PHYSIOLOGICALLY, REQUIRED IN VITRO SYSTEMS FOR THE MAXIMUM EFFECT, IT IS VERY,VERY IMPORTANT. OTHER THAN THAT WE'RE UNFORTUNATELY LEFT WITH THE IMAGINATION THE SNARES ARE LARGELY UNCARING ABOUT THE SPEED OF LIPIDS, IT WILL GO UP OR DOWN BUT NOT DETERMINING. THERE'S SOMETHING IMPORTANT IN THE CLUSTERING THE T SNARES SEEM TO BE IN MANY DIFFERENT CELLS IN SO CALLED LIPID RAS OR DOMAINS THAT ARE CHOLESTEROL DEPENDENT, YOU DON'T KNOW ENOUGH ABOUT THEM. SO I DON'T WANT TO SAY -- I ACTUALLY TO THINK THEY'RE QUITE IMPORTANT, MAYBE THE (INAUDIBLE) ITSELF IS A LIPID RAS. THERE'S SUGGESTIONS ALONG THOSE LINES SO THERE IS A VITAL IMPORTANCE BUT THE -- NEITHER IN VITRO SYSTEMS NOR THE CRUDE TOOLS AVAILABLE, I DON'T MEAN TO SAY GENETIC IS CRUDE BUT RELATIVE TO THE TYPE OF PHYSIOLOGY THAT KNOCKING SOMETHING OUT, IT'S ALL RELATIVELY PROVEN SO WE DONE KNOW. I WILL LIKE TO KNOW. >> DO YOU HAVE ANY INSIGHT TO WHAT HAPPENS IN ASYNCHRONOUS RELEASE WHICH MAYBE IMPORTANT FOR INSULIN SECRETION IN >> DOES NOT REQUIRE SYNAPTOTAGMIN. I DON'T HAVE PERSONAL KNOWLEDGE, MY IMPRESSION FROM THE LITERATURE IS THAT SYNAPTOTAGMIN IS NOT THE CALCIUM SENSOR, MY IMPRESSION IS THERE'S SOME SENTIMENT THERE'S A RELATED PROTEIN CALLED DOCK 2 THAT MAYBE THE CALCIUM SENSOR BUT MY OTHER IMPRESSION IS CONTROVERSIAL THERE. IS A CALCIUM SENSOR. THAT'S CLEAR. >> WHAT IS COMPLEXIN DOING IN THAT SITUATION? >> I DON'T KNOW. I WOULD PREDICT PROBABLY NOTHING. I WOULD GUESS IT'S NOT INVOLVED. IN FACT, YOU CAN GET CALCIUM DEPENDENT -- THE NICEST EXPERIMENT HERE, THERE'S A LOT OF NICE EXPERIMENTS BUT TO MY MINE THE NICEST IS IN VITRO EXPERIMENT WITH PURE PROTEINS BY THOMAS SULNER LAST YEAR BECAUSE IT -- EWE ABSOLUTE CONTROL OF EVERYTHING. WHAT HE SHOWS IS IF YOU LEAVE COMPLEXIN OUT BUT YOU HAVE STILL HAVE PIP 2 SYNAPTOTAGMIM AND SNARES YOU GET VESICAL DOCKING AND RELEASE THAT'S CALCIUM DEPENDENT. BUT THE AMOUNT OF FUSION IN THE ABSENCE OF CALCIUM GOES UP VERY DRAMATICALLY, THE AMOUNT OF VESICAL FUSION THAT YOU GET WHEN YOU ADD CALCIUM IS -- BRINGS YOU UP TO 100% BUT LESS -- FAR TO GO BECAUSE YOU FUSED 20%. SO THERE'S LESS RELEASE IF YOU WANT TO THINK ABOUT IT THAT WAY. INSTEAD OF IT GETTING DONE LESS THAN SECOND IT DRIBBLES OUT 10, 20 SECONDS, NO FUSION IN THE ABSENCE OF CALCIUM, TOTAL HI CLAMPED. 100% RELEASE AND ALL WITHIN THE FIRST SECOND. ASYNCHRONOUS GOES TO SYNCHRONOUS. ASYNCHRONOUS DEPENNING HOW IT IS, YOU CAN GET THAT WITH SYNAPTOTAGMICS N ALONE. IT'S LIKELY AN IMPEDIMENT TO MEMBRANE FUSION BY SITTING BETWEEN THE TWO MEMBRANES. SO IT SLOWS THE TERMINALS. LET'S ADJOURN TO THE LIBRARY AND THANK YOU FOR COMING