>> WE SHOULD GET STARTED. IT'S MY REAL PLEASURE TO INTRODUCE TODAY'S SPEAKERS -- YOU KNOW BACK TO -- EDWIN CHAPMAN -- WE NEVER GET AN OVERLAP, 1992 GET A PH.D. IN THE LAB DEPARTMENT JOINED THE GROUP. IT'S AN IMPRESSIVE PRODUCTIVE SUCCESSFUL POST DOC TRAINING IN YALE. AND HE PRODUCED MORE THAN TEN PAPERS, TEN PUBLICATIONS. AND THE CALCIUM INDEPENDENT INTERACTION [INDISCERNIBLE] SO THAT'S ALL THE RESULTS OF THE STUDIES PRODUCED PROLONGED IMPACT IN THE FIELD FOR MANY YEARS. IN 1996 HE JOINED THE FACULTY AT THE UNIVERSITY OF WISCONSIN HE MOVED UP QUICKLY IN ACADEMIA TO FULL PROFESSOR. THEN HE BECAME INVESTIGATOR. SO DR. CHAPMAN MADE MAJOR CONTRIBUTIONS TO UNDERSTANDING THE MOLECULE INTERDEPENDENCE [INDISCERNIBLE] SO HE IS OBVIOUSLY THE LEADER IN THIS FIELD. EX IT IS VERY IMPORTANT. THE FIRST, HIS LAB I THINK IT'S -- NOT ONLY IS THE CALCIUM SENSOR FOR THE EXOCYTOSIS BUT ALSO FOR THE ENDOCYTOSIS BUT THIS STUDY PROVIDES [INDISCERNIBLE] EXO AND ENDOCYTOSIS [INDISCERNIBLE] THE SECOND STUDY HE DISCOVERED IN HIS LABORATORY IS IDENTIFIED DOC2 WHICH IS CALCIUM SENSOR FOR THE SLOW PROLONGED TRANSMITTER RELEASE [INDISCERNIBLE] SO THIS STUDY PROVIDES MOLECULE -- TO UNDERSTAND SYNAPTIC PROPERTIES. I'M SURE THAT -- INTERESTING STORIES. PLEASE JOIN ME TO WELCOME EDWIN CHAPMAN. [APPLAUSE] >> THANK YOU FOR HOSTING ME. I'VE NEVER BEEN INSIDE THE LABORATORIES OR THE OFFICERS. YOU DO NOT HAVE LAB -- DESPITE RUMORS THAT MIGHT BE TOLD AT NIH. IT MAY BE TIGHT BUT NOT THAT TIGHT. SO BY WAY OF PRODUCTION, I WORKED UP A LITTLE SLIDE HERE TO GIVE PEOPLE A LITTLE BIT OF A SENSE WHAT WE DO IN MY LAB. THIS IS NOT FOR PURPOSES OF THE TALK, THIS IS A SHAMELESS ADVERTISEMENT FOR POST DOCS SO SOME OF THE THINGS WE WORK IN THE LAB HAS SOMETHING TO DO WITH MEMBRANE FUSION. I WILL TALK ABOUT A REDUCED SYSTEM, THE FUSION MACHINERY DIRECTLY -- MEMORY INFUSION REACTION. NO MATTER WHAT REVIEW YOU READ OR WHAT TALK LISTEN TO, NO ONE KNOWS HOW MEMORY IS USED. NO ONE EVEN KNOWS THAT WITH DECIBEL FUSES WITH THE PLASMA MEMBRANE, NO ONE REALLY KNOWS THE STRUCTURE OF THE FUSION FOR THIS INTERMEDIATE CONNECTION OF THE VESSEL. SO WHILE WE DO A LOT OF EXPERIMENTS IN VITRO, WE TRY TO RELATE OUR IN VITRO EXPERIMENTS TO -- EXPERIMENTS. SO THIS INCLUDES A LOT OF EXPERIMENTS TO STUDY A FUSION AND THIS WORK IS DONE IN COLLABORATION WITH MY GOOD FRIEND AND COLLEAGUE MYRA JACKSON. SHE STUDY FUSION -- IN NEURONS. WE ALSO HAVE MOVED UP THE SCALE A LITTLE BIT AND TRIED TO STUDY THE SYNAPTIC TRANSMISSION ITSELF AND THE SYNAPTIC CYCLE AND WE'VE HAD A LOT OF FOCUS ON CALCIUM SENSORS -- I'LL TALK A LITTLE BIT ABOUT THIS TODAY FOR VESICLE REPLENISHMENT -- CHEMICAL GENETICS TO MAKE NEW CALCIUM -- THIS WORK ON EXOCYTOSIS IS ACTUALLY CALLED -- WHICH ARE REGULATORS OF EXOCYTOSIS. THIS WORK ON THE SYNAPTIC -- I'LL SHOW THIS IN MY TALK A LITTLE BIT TODAY, THERE ARE 17 ISOFORMS. WE DISCOVERED ALMOST ALL OF THEM ARE TARGETED TO LARGE DENSE CORE GRANULES THAT SECRETE THINGS LIKE NEURO PEPTIDES AND THINGS LIKE THAT. IN OUR VIEW ONLY TWO SYNAPTIC ISOFORMS -- ANOTHER 15 OR SO REGULATE DIFFERENT MEMBRANE FUSIONS. SO THIS WORK ON NEUROPEPTIDE SECRETION STUDIES ALL KIND OF ASPECTS OF SYNAPTIC PLASTICITY AND ALSO THE SAME WORK ON NEUROPEPTIDES ABOUT NEURO POLARITY. THIS IS KIND OF A WIDE OPEN QUESTION THAT MODERN TOOLS HAVEN'T BEEN APPLIED TO ADDRESS LIKE PHOTO ACTIVATION AND NEUROPROCESSING. THEN FINALLY A PRODUCT OF MINE I'VE BEEN PLAYING WITH SINCE I WAS A POST DOC ARE THE DESTRUCTIVE PROJECTS I SAY DESTRUCTIVE BECAUSE THERE ARE A SET OF TALKENS THAT CAUSE BOTULISM -- SO WE WORK LONG IN THE RECEPTORS HOW THEY TRANSLOCATE ACROSS SYSTEMS. THE TOXINS CAN MOVE AROUND IN A NETWORK OF CONNECTED NEURONS. THEY HOLD INTERESTING PROMISE TO BE TRACERS FOR CONNECTIVE TEAVMENT I'LL TALK MOSTLY ABOUT RECONSTITUTED SYSTEM AND LINK THIS TO CELL-BASED MEASUREMENTS. BEFORE I GET COOKING, SO THIS ISN'T ALL JUST SCIENCE FICTION I ALWAYS LIKE TO BEGIN WITH THIS BEAUTIFUL -- FROM 1981, SIMILAR EM'S FROM -- ITALY. THIS IS THE NEUROMUSCULAR INCLUDE FUNCTION. WHAT IT SHOWS IS PRESYNAPTIC TERMINAL WITH SIGNEE VESSELS AND ORGANELLES. I'VE GOT SOME OF THESE VESICLES ATTACKED -- CALLED DOCKING IT WAS A BLACK BOX UNTIL A FEW YEARS AGO AND THERE'S GOOD PROGRESS ON HOW DOCKING IS MEDIATED ALTHOUGH WE STILL HAVE A LONG WAY TO DO. WHAT THEY DID IN IN THIS EXPERIMENT -- THEY CAPTURED FAMOUS OMEGA SHAPES -- AND IT'S REALLY THIS TRANSITION FROM THE DOCK OF THIS MEMBRANE FUSION -- IT'S AN UNRESOLVED QUESTION AND CAN BECOME A MAJOR QUESTION IN CELL BIOLOGY. I'M GOING TO SHOW A LITTLE BIT, FIRST HALF OF MY TALK IS GOING TO BE A LITTLE BUSINESS OF HISTORICAL SURVEY BECAUSE I'M NOW 50 AND I CAN DO THIS BECAUSE I'VE BEEN DINKING AROUND WITH THIS FOR 20 YEARS. WHEN I JOINED THE LAB BACK IN 19920, THE FIRST REVIEW I READ BY JULIO FERNANDEZ AND JOHNATHAN -- THIS IS A CARTOON I TOOK OUT OF THEIR REVIEW. THERE'S SOME EM EVIDENCE OF THIS -- GIANT SECRETORY GRANULES THAT DURING EXOCYTOSIS ONE IDEA THEY PUT FORWARD 20 YEARS AGO WAS THAT MAYBE THE TARGET MEMBRANE WAS BENT INTO A DIMPLE OR A LITTLE NANO BUMP HERE. THIS IS BEFORE WE KNEW ABOUT SNARES OR SYNAPTIC PROTEINS TO DO THIS SORT OF GENERIC SCAFFOLD. IF WE TUG THE MEMBRANE FORWARD THE PLASMA MEMBRANE THERE IS THIS CONTACT WHICH COULD LEAD TO JOY THAT WOULD POP OPEN TO RESOLVE TO A LIPID LINE FUSION AND IT WOULD DILATE IN THIS WHOLE FUSION. THAT'S OLD STUFF 20 YEARS AGO. I DON'T KNOW IF THIS WOULD BE SENSOR ENOUGH AT THE TIME. IT RAISES THE QUESTION, THIS OLD QUESTION MUST THE TARGET MEMBRANE BE BENT IN ORDER TO ACCELERATE THE RATE OF FUSION. I WANT TO ADDRESS THIS QUESTION FIRST IN THE TALK. THE IDEA THOUGH AND I DON'T WANT TO GO INTO TOO MUCH DETAIL BUT THIS WILL GIVE YOU A COUPLE IDEAS. ONE IS IDEA IF YOU BEND THE TARGET MEMBRANE YOU REDUCE THE CONTACT AREA SO YOU MINIMIZE THE HYDRATION BARRIERS GETTING THE WATER OUT OF THE WAY. MORE RESENT SINCE LATIONS SAY THESE ARE HIGHLY CURVED AND IF YOU PUT THEM CLOSE TOGETHER LIKE TEN NANOMETERS APART, SOME INTERESTING STUFF HAPPEN. LIPIDS START TO TRANSFER LEADING TO A MEMBRANE INFUSION. THE MOST OBVIOUS ONE IS WHEN YOU BEND VIOLATORS YOU EXPOSE THE [INDISCERNIBLE] MAKES THE MEMBRANE UNSTABLE. THERE'S ALL KIND OF REASONS TO THINK THAT BENDING MIGHT HELP. BUT WHETHER IT HAPPENS OR NOT, IT'S AN OPEN QUESTION. SO BEFORE I CAN ANSWER THAT QUESTION, I GOT TO TO INTRODUCE A COUPLE PLAYERS. THERE'S A LOT OF MOLECULES THAT MEET EXOCYTOSIS. I'LL ONLY TALK ABOUT A COUPLE OF THEM. BY WAY OF INTRODUCTION I WILL STALK ABOUT THE SNARE COMPLEX AND THE -- SO THIS IS THE CORNER OF THE VESICLE AND IT HAS A CALCIUM SENSING PROTEIN CALLED -- THIS IS THE CRYSTAL STRUCTURE FROM BRIAN -- AND THEN THE VESICLE ALSO HAS VESICULAR PROTEIN -- IN BLUE AND SYNAPTO-- FORMS CONTACT WITH TWO OTHER SNARE PROTEINS. TARGET SNARES, T SNARES AND THESE ARE CALLED -- AND THE IDEA IS THAT THERE'S THIS SNARE COMPLEX MIGHT. ONE IDEA IS IT MIGHT START TO BUNDLE FROM THIS END TO THIS END. AS IT ZIPPERS ON DOWN IT IS A CAT LIESED MEMBRANE FUSION. THE OTHER IDEA IS THAT SYNAPTOTAG -- I WANT TO FOCUS ON -- IF WE LOOK AT THE CRYSTAL STRUCTURES -- TWO FLEXIBLE LOOPS SHOWN HERE AND HERE AND -- IF WE JUST -- SORT OF LOOKS LIKE THIS ALTHOUGH BRIAN DID A LOT OF THIS WORK ISN'T ENTIRELY SURE IF THE CALCIUM LINES ARE ALIVE AGAIN. THERE ARE QUESTIONS ABOUT IT, I THINK. NOW I'VE DRAWN A COUPLE ARROWS HERE. THEY ARE POINTING TO GREASY RESIDUES IN THE VERY DISTAL TIPS. IT TURNS OUT THE VERY DISTAL TIPS OF THESE LOOPS LIKE THE -- ALANINE, THE BAY LIEN AND THE -- WHAT WE DISCOVERED, THIS WAS BACK WHEN I WAS A SNOT NOSE ASSISTANT PROFESSOR WE TOOK A C2 DOMAIN -- THE TWO CALCIUM BINDING LOOPS ARE STICKING UP HERE, SHOWN HERE. THERE'S A MINOR LOOP DOWN HERE. THESE ARE THE TWO CRITICAL BINDING LOOPS. WE SCANNED THE SURFACE OF THE C2 DOMAINS AND WE FOUND, WE DISCOVERED YOU CAN PUT THEM IN EVERY SINGLE SURFACE ON A C2 DOMAIN. WHAT WE FOUND IN THE CASE OF SYNAPTOTAGMIN, ONLY THE PROBES PLACED IN THE CALCIUM BINDING LOOPS DIDN'T -- SO IT IS KNOWN THAT -- BINDS -- IN A RESPONSE TO CALCIUM LIKE PROTEIN KINASE -- OR SYNAPTOTAGMIN BUT IT WASN'T KNOWN HOW THESE PROTEINS TOUCH OR ENGAGE THE SURFACES. THAT'S THE FIRST THING WE DID. SO WE DISCOVERED THAT THIS IS THE C2 -- SYNAPTOTAGMIN -- AND THIS INCREASE IN THIS BLUE CHIP -- BECAUSE YOU CAN PUT MEMBRANE IMBEDDED FLORESCENCE IN THE BILAYER AND YOU CAN ACTUALLY MEASURE USING THE -- METHOD, THE DEPARTMENT PENETRATIONS. THOSE ARE THE CARTOONS SHOWING HOW DEEP -- PENETRATES BILAYERS. THE EMPHASIS I WANT TO MAKE ON THIS SLIDE IS NO OTHER PART OF THE -- TOUCHES THE BY LATER. WE MITOCHONDRIA THESE LITTLE CARTOONS ABOUT HOW SYNAPTOTAGMIN IS ON THE VESICLES. I WANT TO EMPHASIZE ONE THING HERE. WE HAVE SHOWN IN A SERIES OF PAPERS THAT I WON'T TAKE YOU THROUGH THAT THE MEMBRANE THAT SYNAPTOTAGMIN PENETRATES IS THE -- THAT'S WHY THE TARGET IS UNDERLINED IN ITALICS. THE REASON WHY IS, ONE REASON IS THE TARGET MEMBRANE HAS -- INDEPENDENT INTERACTION OF THE -- THE OTHER REASON IS THAT THE TARGET MEMBRANE HAS PIP2 -- THAT'S CERTAINLY REQUIRED FOR LARGE -- STILL A LITTLE BIT OF A QUESTION AS TO THE FUNCTION OF -- BUT THERE IS A WEAK CALCIUM INTERDEPENDENT ACTION, NOT ONLY WITH T SNARES BUT ALSO WITH THE PIP2. THE WEAK INTERACTION STEERS THE PROTEIN SO WHEN IT LOADS WITH CALCIUM IT PENETRATES THIS MEMBRANE -- SO WE JUST HAVE THE CARTOON NOW WHERE SYNAPTOTAGMIN IS ON THE VESICLE IN RESPONSE TO THE CALCIUM CAN PENETRATE THE TARGET MEMBRANE. SO WE DO A LITTLE CARTOON, WE WORK OUT THE KINETICS OF THIS. THIS IS A LITTLE CARTOON WHERE WE PUT THE PROBES ON YOUR USING A LITTLE CHEMISTRY TECHNIQUES AND AUTOPSY -- STOPPED SLOW -- IN RESPONSE TO CALCIUM IS THAT -- COMPRISES FOR SPEED AND THAT'S IMPORTANT BECAUSE IF YOU'RE GOING TO NEARBY SYNAPTIC TRANSMISSION BECAUSE ONE NEURON AND ANOTHER NEURON YOU'LL NOTICE THIS RAPID COMPONENT AND IT HAS A VERY FAST RISE, SO YOU WANT A PROTEIN THAT'S GOING TO LOAD CALCIUM AND UNWRAP THE TIME SCALES. I'LL TALK ABOUT THE SECOND COMPONENT OF SYNAPTIC TRANSMISSION A LITTLE BIT LATER WHICH HAS A SLOWER RISE TIME AND PERSISTS. THIS IS CALLED THE -- COMPONENT TRANSMISSION. SO WE'RE LOOKING FOR MAYBE A DIFFERENT CALCIUM AND DIFFERENT -- PROPERTIES FOR THAT COMPONENT. OKAY. NOW, SO HERE'S THE WAY I PUT THIS TOGETHER. IT'S WELL-KNOWN THAT SOME PROTEINS THAT PENETRATE THE MEMBRANE CAN CAUSE THIS SORT OF DISPLAY AND SPREAD OF PHOSPHOLIPIDS. THAT'S JUST SIMPLE GEOMETRY. SO ONE IDEA IS THAT MAYBE SYNAPTOTAGMIN -- THE TARGET MEMBRANE. SO IF SYNAPTOTAGMIN PENETRATION ACTIVEY ABLE TO BEND MEMBRANES DOES IT LOWER THE ENERGY BARRIER FOR FUSION. SO IT IS -- THIS IS FIRST SHOWN BY JOSE -- IN HIS PAPER AND MORE RECENTLY BY HARVEY -- THAT IT WAS THE MEMBRANE FOR THE -- FUSION. I'LL TALK ABOUT THAT IN A MOMENT. SO YOU ALWAYS WORRY ABOUT THIS SORT OF EXPERIMENT BECAUSE LOTS OF PROTEINS WILL CAUSE THIS KIND OF TUBATION -- I LIKE TO SHOW THIS SLIDE. THIS IS A REALLY GOOD TUBELATER. IT HAS NOTHING TO DO WITH THE RNA FUNCTION SO YOU HAVE TO BE CAUTIOUS WHEN DOING THESE KINDS OF EXPERIMENTS. I'LL JUST THROW THAT OUT THERE. SO WE ALSO SEE BY NEGATIVE STAINING BENDING OF MEMBRANES -- AND THERE'S ALWAYS THIS CUT OFF A LITTLE BIT UP HERE BUT THERE'S A LOT OF STUFF HERE. I'M GOING TO POINT OUT A COUPLE FEATURES. ONE IS THAT THIS IS THE C2AB DOMAIN OF SYNAPTOTAGMIN -- SO YOU'LL HEAR IT CALLED C2AB A LOT IN MY TALK. IN CALCIUM -- ONE THING I THING I WANT TO POINT OUT -- THE A DOMAIN IS ACTUALLY A BETTER MEMBRANE PENETRATOR -- A FAILS TO TUBE LATE MEMBRANE. WE'RE TRYING TO FIGURE OUT -- I WANT YOU TO NOTICE THIS PANEL -- SO I DREW A BOX AROUND IT AND OVER HERE I SAY C2B, C2ABLM. THIS IS A CONSTRUCT WHERE THE C2B DOMAIN CAN'T -- MUTATIONS OF SOME OF THE SPECIFIC ACID RESIDUE -- AND IT WAS FIRST SHOWN -- THIS IS INTERESTING BECAUSE THIS MUTATION KILLS THE FUNCTION OF SYNAPTOTAGMIN IN VIVO. OKAY. SO I JUST WANTED YOU TO MEMORIZE ONE THING. C2B CALCIUM LIGAND DOESN'T FAIL TO BEND MEMORIES. OKAY. NOW HOW DO YOU BEST WHETHER -- THE ONLY WAY TO DO IT THAT WE CAN THINK OF TO DO IT IN VITRO, A LONG TIME AGO BACK IN 2004 WE WORKED HOW THE A SYSTEM WHERE WE TOOK JIM ROTHMAN -- YOU MAKE REEXECUTED D SNARE VESICLE THAT'S WHY IT CALLED DR. THEY HAVE BEEN RECONSTITUTED. YOU MAKE -- KR AND THEY HAVE THE SYNAPTO-- AND SNAP 25. NOW, ON THE D SNARE VESICLE, YOU TAKE PHOSPHO-- AND YOU PUT 1.5% NBDPE AND THE SAME AMOUNT OF RODE MEAN ON THE HEAD GROUP. THEY MAKE A REALLY GOOD THREAT DONOR ACCEPTOR PAIR -- PROTONS WITH THE RODE MEANS. IT'S VERY EASY TO DO, VERY STRAIGHTFORWARD. THE OTHER THING I WANT TO POINT OUT IN THIS SLIDE WE CAN ADD BACK THE CYTOPLASMIC DOMAIN -- AND MAKE THIS REACTION GO IN RESPONSE TO THE CALCIUM SUGAR. THE OTHER THING I SHOULD MENTION HERE IS THAT IT'S BEEN KNOWN SINCE THE VERY FIRST RECONSTITUTED FASHION, YOU CAN SNARE PROTEINS FROM THE ROTHMAN LAB IF THE T SNARE IS NOT -- PRIOR TO RECONSTITUTION, THE ASSAY WON'T WORK. IF YOU PUT -- IN THE BILAYER BECAUSE OF THE TRANSMEMORY DOMAIN AND ADD SNAP 25 -- IT WON'T BIND -- AND IT WON'T -- THAT'S SOMETHING I WILL COME BACK LATER. THAT'S SOMETHING THAT CAN BE EXPLOITED. OKAY. SO I ASKED YOU TO MEMORIZE AND I WROTE THIS CALCIUM LIGAND -- WHICH IS IN RED IT DOESN'T BEND MEMBRANE. WE HAVE KNOWN FOR SOME TIME, WE DID THIS FIRST BACK IN 2005 THAT -- HAS REPEATED THIS A COUPLE YEARS LATER, WE HAVE KNOWN THIS ASSAY IS STILL PRETTY ACTIVE IN THE RECONSTITUTED SNARE CATALYZED FUSION ASSAY. IN THE STANDARD FUSION ASSAY, I SHOULD HAVE MENTIONED THIS BEFORE, IT'S FUSION BETWEEN SMALL -- THERE ARE ABOUT 15 NANOMETERS -- THIS MUTANT STILL WORKS. IT DOESN'T BEND MEMORY BUT IT STILL WORKS. AND HERE'S AN EXAMPLE FROM REINHART'S LAB -- HERE IS WILD TYPE C2AB -- DRIVING MEMBRANE FUSION ON THIS AXIS OVER TIME. AND THEN THE C2B MUTANT IS RIGHT HERE. IT'S THIS BLUE LINE. ACTUALLY IN THEIR HANDS IT WORKS A LITTLE BIT BETTER. IN OUR HANDS IT DOESN'T WORK -- SO THIS KIND OF EXPERIMENT TELLS US THAT THE SUVSUV FUSION ASSAY DOES NOT INVOLVED BENDING OF THE TARGET MEMBRANE. IT DOESN'T BEND MEMORY, IT'S STILL ACTIVE. SO WE ARGUE THAT MEMORY BENDING ACTIVITY HAS NOT BEEN TESTED. THERE'S ONLY ONE STUDY FROM HARVEY MCMAHON'S LAB WHERE THEY CLAIM TO TEST THIS IDEA. WE CLAIM THEY DON'T TEST THIS IDEA BECAUSE THIS ASSAY DOESN'T REQUIRE BENDING. BEFORE I GO ON TO HOW WE THINK WE'VE TESTED IT I WANT TO EXPLAIN WHY THE ASSAY DOESN'T INVOLVE BENDING. THERE'S A BIG SWITCH IN THIS FIELD. IF YOU READ A MEMBRANE TUBULATION -- HAS NOTHING TO DO WITH PHYSIOLOGY BECAUSE THERE ARE NO MEMORIES THAT ARE -- SO WHAT HAPPENS IS THERE'S A REASON THAT WE AND OTHERS USE THEM IS THEY ARE READILY TUBULATED. YOU GET SMALL DIAMETER TUBULE AND GET AGGRESSIVE BEND -- THIS IS THE USUAL STUFF. IF WE BURY THE PS FIRST YOU SEE YOU DON'T GET A LOT OF PUBALS. WHEN YOU GO TO HIGHER PS YOU GO MORE AND MORE TUBULES. THE KEY HERE IS THAT PHYSIOLOGICAL LIPID MIX YOU GET LESS TUBULATION. THE OTHER THING THAT'S IMPORTANT IS THE DIAMETER OF THEM IS VERY DIFFERENT IT'S MUCH LARGER. IF WE MEASURE THE DIAMETER OF THE TUBULES AT 15, 30 AND 45% PS THERE'S AN INVERSE RELATIONSHIP. IF WE GO TO PHYSIOLOGICAL PS CONCENTRATION SORT OF IN THIS RANGE, THE DIAMETER OF THE TUBULES IS ABOUT THE DIAMETER OF A SUV. SO WE THINK WHEN YOU GO TO PHYSIOLOGICAL LIPIDS THE BENDING -- WILL DO IS NO MORE THAN, THE SUV'S ARE ALREADY TOTALLY BENT AT 15 AND 20% PS AND THAT'S WHY THE SUV FUSION ASSAY DOESN'T RELY ON ENDING, THEY'RE ALREADY BENT. HOW DO YOU GET AROUND THEM -- YOU GUYS SEEM TOUGHER THAN MOST CROWDS. I ALWAYS SAY IT'S A STEREO KILLER AND EVERYBODY LAUGHS BUT I'LL SKIP THAT JOKE THIS TIME -- HE'S WORKING ON GED'S AND ANOTHER SET OF PROTEINS CALLED THE -- FAMILY. WHEN HE WAS IN MY LAB HE WAS THE FIRST TO BUILD THE GIANT VESICLES AND NOW WE WORK A LOT ON THESE GIANT -- MEASURE A FEW RECONSTITUTED FUSION PORTS DIRECTLY. THESE ARE KIND OF SOME SORT OF ODD LOOKING ONES WITH SOME INTERESTING STRUCTURES. WE'RE GETTING BETTER AT MAKING THESE. THESE ARE MICRONS INSIDE. THESE ARE FLAT. THIS IS LIKE STUDYING THE EARTH AND IT LOOKS FLAT. THERE ARE MICRONS IN DIAMETER. WHAT COLLIN DID WAS AN OBVIOUS EXPERIMENT. IF YOU TAKE SUV FUSION ASSAYS, THESE A VERY LOW DOSE SO YOU SEE LOTS OF FUNCTION IN THE SUV FUSION ASSAY EVEN. THIS MOLECULE IS MESSED UP -- THERE'S LOSS OF FUNCTION IF YOU GO TO A LOW ENOUGH DOSE. IF YOU INCREASE THE DOSE YOU DON'T SEE ANY DIFFERENCE AT ALL. WHAT'S IMPORTANT IF YOU REPEAT THIS EXPERIMENT -- ALL ALL OF A SUDDEN THIS CALCIUM LIGAND MUTANT IS DEAD AS A DOORKNOB. THE ONLY THING WE CHANGED IS THE STARTING CURVATURE OF THE STARTING MEMBRANE. THIS IS MAYBE SOME OF THE US EVIDENCE THAT SYNAPTOTAGMIN BENDING MIGHT BE IMPORTANT -- I LIKE THIS BETTER BECAUSE THIS IS MORE OF AN INSIDE OUT CELL -- CAN BE AS BIG AS A CELL AND WE ADD THE VESICLES TO THE OUTSIDE. NOW I DIDN'T TAKE THIS FURTHER AND TEST THE IDEA. IS THIS NOT WORKING HERE BECAUSE IT CAN'T BEND THIS RELATIVELY FLAT MEMBRANE. THIS IS ONE OF THOSE EXPERIMENTS YOU GET WHEN YOU'RE ON THE BIKER IN THE SHOWER BUT WE TRIED IT AND IT'S NOT GOING TO WORK. THIS IS THE EXPERIMENT. WE TOOKED THE M BAR DOMAIN, THIS IS A MOLECULE KNOWN TO BEND MEMBRANE HERE'S THE FIGURES FROM -- LOTS OF LABS MAKING PROGRESS ON THIS MOLECULE. IT'S KNOWN THAT THIS M BAR DOMAIN THE CRYSTAL STRUCTURE SHOWN HERE, IT'S KNOWN THAT IT BENDS MEMBRANES. AND IT BENDS MEMBRANES DURING ENDOCYTOSIS TO REFORM A VESICLE. SO THIS IS GOOD BECAUSE IT'S MAYBE ONE PART CURVATURE THAT'S ABOUT THE RIGHT CURVATURE -- YOU CAN'T SEE THIS. WE JUST ASKED IF THE MEMBRANE BENDING THAT DRIVES -- ENDOCYTOSIS -- KIT REST CUE THE MEMBRANE DEFICIENCY IN THIS -- MUTANT FORM OF SYNAPTOTAGMIN. THIS I'M GOING TO BLOW UP FOR YOU AND THE EXPERIMENT IS SHOWN HERE. WE DON'T NEED TO GO THROUGH ALL THE GORY DETAILS BECAUSE I WANT TO COVER MORE TERRITORY TODAY. I WANT TO POINT OUT A COUPLE THINGS. IF YOU DON'T ADD -- NOTHING'S REALLY HAPPENING HERE. IF YOU ADD JUST N BAR, ALONE IT DOESN'T DO ANYTHING TO OUR FUSION ASSAYS -- N BAR ALONE DOESN'T DO MUCH. IF WE PUT IN OUR CALCIUM LIGAND MUTANT MINUS N BAR SO THE RECORDING STARTS HERE WE HAD CALCIUM BOOM WE GET THIS WHIMPY SLOW SLUGGISH FUSION BECAUSE THIS MOLECULE IS MESSED UP AND CAN'T BEND THE GED IN PART AT LEAST. WE TAKE THIS AND DUMP IN N BAR AND LO AND BEHOLD -- THE AMOUNT OF FUSION WE GET WITH JUST WILD TYPE C2 -- IF WE DUMB N BAR ON TOP OF WILD TYPE -- THE KEY POINT IS THAT THIS MESSED UP FUNCTION OF THIS CALCIUM LIGAND MUTANT CAN BE RESCUED BY ADDING -- THIS ASSAY WEIRD, YOU CAN CHANGE THE ORDER OF ADDITION. I JUST WANT TO TELL YOU ONE MORE THING. IN THIS CASE, WE ADDED THE CALCIUM LIGAND MUTANT, THIS SORT OF NOT VERY ACTIVE DOUBLE HERE. WE ADDED IT HERE AND OF COURSE WE ADDED CALCIUM HERE ANDATE STARTS TO COME UP. IN RED WE ALSO, AFTER THE -- GOT N BAR TRIGGED MEMBRANE -- THE FUSION IS TRIGGERED BY N BAR -- AND SO WHAT I LIKE ABOUT THESE EXPERIMENTS IS THEY ASSOCIATE MEMBRANE ACTIVITY -- AND AGGREGATE VESICLES. SO WE JUST ONE LINE OF ARGUMENT. BUT IT'S THE IDEA THAT SYNAPTOTAGMIN ON THE VESICLE, IT KNOWS WHICH MEMBRANE -- THE TARGET MEMBRANE AND IT'S THIS IDEA PROPOSED BY HARVEY MCMAHON THAT MAYBE IT REALLY DOES DRIVE THAT TARGET MEMBRANE ORIGINALLY PROPOSED BY WHOM OH FERNANDEZ 20 YEARS AGO SO MAYBE IT IS THE NEXT STEP -- HERE AT NIH I WANT TO POINT OUT THIS PROJECT ALSO CAME FROM -- WHO WORKS AT NIH -- WAS THE FIRST PERSON, THIS IS INTERESTING TEN YEARS AGO THE FIRST PERSON TO EVER PROPOSE IN LITERATURE THAT ENDOCYTOSIS ARE SIMILAR, IT SOUNDS FUNNY BUT AS FAR AS I KNOW IT'S A TRUE STATEMENT. WE ALWAYS STUDY FUSION IN CONSUMPTION OF VESICLES AND LOTS OF PEOPLE STUDY THE -- FROM THE PERSPECTIVE OF THE LIPID BILAYER THEY'RE SIMILAR INTERMEDIATE STRUCTURES JUST CATALYZE -- IF YOU THINK OF IT IN THOSE TERMS ANYTHING THAT GIVES YOU A STRUCTURE LIKE THIS IS GOING TO HELP YOU RUN THIS REACTION. SO I THINK -- VERY IMPORTANT INTELLECTUAL CONTRIBUTIONS FOR THIS KIND OF PROBLEM. OKAY. SO NOW, TO TAKE THIS FURTHER, YOU KNOW, EVERYTHING I SHOWED YOU SORT OF -- BIOCHEMISTRY. WE NEED TO DO THIS KIND OF EXPERIMENT IN VIVO AND THERE'S NO WAY WE CAN DO THAT SO WE'RE PRESSING IN VITRO EXPERIMENTS A LITTLE FURTHER. WE NEED EMPIRICAL DATA TO START THE STARTING CURVATURE -- THERE'S A LOT OF THEORY AND THE THEORY VARIES BY ORDER OF MAGNITUDE. SO IT'S GREAT TO DO BUT WE NEED EMPIRICAL DATA TO CONSTRAIN OUR THEORY. WE NEED A NEW APPROACH WITH TEMPLATES TO MAKE MEMBRANES WITH A WIDE RANGE OF CURVATURES. THIS IS WORK THAT -- HAS DONE HERE. HE GOT A GREAT BIG FISH IN MADISON. WHAT HE DID WAS YOU CAN BUY THESE BEADS THAT RANGE FROM 150 NANOMETERS TO SEVEN OR 9 -- THEY ARE PERFECTLY ROUND AND YOU CAN COAT THEM WITH THE BILAYERS. THIS IS A TR VESICLE COATED WITH NVDPE AND IF YOU PUT THEM ON A -- I JUST GRABBED THIS, IF YOU PUT THEM ON A -- AND TWIRLD THEM AROUND IT'S KIND OF NEAT BECAUSE BOTH OF US WHO STUDY MEMBRANE FUSIONED -- NOW YOU CAN PUT IT UNDER THE MICROSCOPE -- AND IT'S REALLY GRATIFYING INSTEAD OF HAVING COLORED LIQUID ALL THE TIME. AND SO WHAT HE HAS DONE -- IT'S REALLY HARD TO STUDY -- ON BILAYERS ON GLASS BECAUSE ANY TIME YOU PUT THE BILAYERS ON GLASS, YOU NO LONGER ALL THREE SNARE PROTEINS -- YOU GET THIS ONE. RECENTLY THAT'S BEEN SOLVED AND THE WAY YOU SOLVE THIS IS TO ADD THE MEMBRANES WITH A PE THAT HAS TAG 2000 AND MAKES THE MEMBRANES -- I'M NOT GOING TO SPECULATOR BUT IT NOW RENDERS THE MEMBRANE -- THIS IS PRELIMINARY STUFF, IT'S NOTHING HARD OR NEW, I JUST WANT TO SHOW IT TO YOU BECAUSE IT'S REALLY NEAT -- WE BURN UP THE FOSSIL -- THIS IS A GRAPHIC EXPERIMENT WITH -- FUSED TO THE -- SNAP 25 AND THE -- HETERODIMER AND WE BURNED IT UP AND IT FLOWS RIGHT BACK IN THE SNARES AND MEMORIES ARE FLUID. THEY SEEM TO BE WORKING REASONABLY WELL. AND WE CAN TAKE SMALL SUV'S. THESE ARE REALLY INSIDE OUT CELLS. WE CAN TAKE SMALL SUV'S -- IN THE PLATE READER -- BUT ALSO UNDER A MICROSCOPE THE SINGLE PARTICLE RESOLUTION. IT'S A VERY POWERFUL TECHNIQUE. THIS IS CALCIUM TRIGGERING MEMBRANE FUSION HERE. IF YOU GET RID OF THIS MAP 25 YOU ONLY HAVE -- THERE'S NOT MUCH MEMBRANE FUSION, WE'RE TWEAKING THAT DOWN. I DON'T WANT TO TALK ABOUT THIS AND IT'S TOO CALCULATED AND I'LL GET TO THIS LATER. WE MEASURE FUSION RATE AS A FUNCTION OF THE STARTING DIAMETER AND WE SORT OF HAVE THE DATA. AND THE IDEA IS THAT THERE IS A CURVATURE DEPENDENT RATE OF FUSION WHICH IS EXPECTED. IF WE CLEAN THIS UP AND DO IT QUANTITATIVELY, WE GET GOOD FORMAL EMPIRICAL DATA TO START TO STUDY THIS -- BUT THE QUESTION IS, IF WE ADD BACK THE MEMORY LIKE SYNAPTOTAGMIN TO CHANGE THE RELATIONSHIP BETWEEN STARTING CURVATURE AND THE RATE OF FUSION. I GUESS THE PREDICTION WOULD BE THAT THERE WOULD BE A STEEP RELATIONSHIP -- BUT WITH THE MEMBRANE BENDER IT MIGHT BE -- THAT'S ALL IN VITRO. WE ALWAYS TRY TO RELATE EVERYTHING WE DO IN VITRO TO EXPERIMENTS IN THE NEURONS -- HERE'S ANOTHER FISHING TRIP -- THIS IS AN EXPERIMENT, HE DID THE PHYSIOLOGY -- GRADUATE STUDENT IN THE LAB DID THE MICRO BIOLOGY. AND IN A VERY LONG AND COMPLICATED STORY, I'M NOT GOING TO BORE YOU WITH, IT TURNS OUT, THIS IS THE STRUCTURE -- IT TURNS OUT THAT THE ABILITY OF THE DOMAINS TO PENETRATE DOMAINS IS STRONGLY DEPENDENT ON THE ADJACENT C2 DOMAIN. THIS TURNS OUT TO BE REALLY WEIRD STUFF. THE B DOMAIN IS NOT A VERY GOOD MEMBRANE PEN TRAITOR ON ITS OWN SO IF YOU FUSE AN A DOMAIN NEXT TIGHT THE B DOMAIN PENETRATES MEMORIES LIKE GANGBUSTERS. HERE'S THE EDWARDNESS -- THE A DOMAIN DOES NOT EVEN BIND CALCIUM OR MEMBRANE. WHEN YOU FUSE THAT DEAD DOMAIN NEXT TO THIS B DOMAIN THE B DOMAIN STILL BECOMES -- THE FUNCTION IS ACTUALLY THE C2 DOMAINS THAT I CAN'T BEGIN TO UNDERSTAND -- WE HAVEN'T DONE THAT YET. THE REASON I'M TELLING YOU THAT INFORMATION IS BECAUSE IF YOU, WHAT IT MEANS IS IF YOU SPLIT THE LINK BETWEEN A AND B AND MAKE THEM FAR APART THE A DOMAIN CAN NO LONGER ACTIVATE THE B DOMAIN AND VICE VERSA. INSTEAD OF JUST SEVERING IT WE PUT DIFFERENT LINKERS IN HERE -- WE PUT IN PROTEIN RODS -- WE PUT IN A COUPLE DOZEN LINKER LEADS. WE LOOKED AT THE ABILITY OF THE C2B DOMAIN -- WE LOOK AT THE ABILITY -- THIS IS THIS SPECTRUM, WE MIXED THE PROTEIN WITH LYSOSOME WE ADD CALCIUM THE LOOPS PENETRATE AND BOOM WE GET THE BIG FLUORESCENT FOR THIS SET OF LINKERS AND FOR THIS SET OF LINKERS. AND THEN WE ALSO MEASURED SYNAPTIC TRANSMISSION. THESE ARE EPS -- SO I'M GOING TO SHOW YOU A GRAPH WHICH IS -- I'M GOING TO PLOT AN IN VITRO FLORESCENCE CHANGE ON THIS AXIS, I'M GOING TO PLOT THE AMPLITUDE OF THE RESCUE OF THE -- HERE IS A WILD TYPE, HERE IS A SYNAPTIC -- THE COMPONENT IS GONE, THAT'S OLD HAT AND YOU GET A LOT OF -- SO THERE'S STILL PLENTY OF RELEASE IN THESE NEURONS. YOU HAVE ANY WILD TYPE SYNAPTIC -- AND LO AND BEHOLD YOU GET NORMAL RESCUE AND YOU PUT IN THESE VARIOUS -- WHEN YOU MEASURE THESE CURRENT AND YOU PLOT THE AMPLITUDE OF THE RESCUE AND THIS FLORESCENCE CHANGE -- 65.66. THERE'S A PRETTY SEVERE CORRELATION. I SHOULDN'T SAY TOO MUCH THINGS BECAUSE I PLOTTED THINGS THAT SHOULD NEVER WE PLOTTED TOGETHER. I POINT THIS OUT TO YOU BECAUSE OF ALL THE THINGS WE KNOW TO MEASURE IN TEST TUBES NOTHING CORRELATES EXCEPT FOR MEMBRANE CORRELATION. NOTHING WE MEASURE CORRELATES WITH RESCUE. I'M BINDING THE IDEA THAT SYNAPTIC -- PART OF PENETRATING MEMBRANES. OKAY. I WANT TO SHOW YOU SOMETHING, I'M GOING TO SKIP OVER THIS PART PRETTY QUICKLY. I DON'T WANT TO LEAVE YOU WITH THE IDEA THAT SYNAPTOTAGMIN ONLY ACTS ON THE -- BUT ACTS ON SNARE PROTEINS. THIS IS A VERY OLD SLIDE THAT WARD TUCKER -- USING THE SUV FUSION ASSAY. I SHOULD THANK THOMPSON -- WHO WAS THE GUY IN THE LAB THAT FIRST RECONSTITUTED SNARES -- MOUNT SIGH INDEMNIFY AT THE TIME, REALLY NICE GUY. SO EVEN IN THE VERY FIRST PAPER-- PLEASE DIRECT YOUR EYES TO THIS PANEL HERE, THIS LOG INFORMATION HERE, THE AMOUNT OF ACTIVITY IN THE FUSION ASSAY DEPENDED, SO WE DID FUSION ASSAYS WITH 30 COPIES OF T SNARES FOR T SNARE VESICLES OR 80 COPIES OR TO COPIES OF THE T SNARE. THE AMOUNT OF SYNAPTOTAGMIN WE NEEDED SCALED WITH THE AMOUNT OF -- SO THE AMOUNT OF -- WAS NEEDED EVEN BACK IN THIS FIRST PAPER WITH THE AMOUNT OF T SNARES. EVERY PAPER WE EVER DONE, I'LL JUST SKIP THE REST OF THIS BUT WE'VE KEPTLY SEEN INDICATIONS OF SYNAPTOTAGMIN DOESN'T GO THROUGH MEMORIES BUT ALSO GO THROUGH SNARE PROTEINS. I WANT TO SHOW YOU SOMETHING REAL BRIEFLY HERE THAT I THINK IS REALLY INTERESTING. SO WE DON'T HAVE TO WORRY ABOUT THIS. I JUST WANT TO POINT OUT THAT THE ASSAY HERE, SO YOU CAN MIX PROTEINS AND LYSOSOMES IN THE CENTRIFUGE TUBE AND SPIN THEM. THE LYSOSOMES -- THE PROTEINS WILL COME WITH THEM AND YOU CAN RUN GELS ON THEM. IF YOU DIRECT YOUR ATTENTION TO THIS PANEL THERE'S SOMETHING VERY INTERESTING THAT HAPPENS. THIS EXPERIMENT, WE RECONSTITUTED SOME -- ONLY INTO VESICLES AND WE'VE ADDED SNAP 25. THERE ARE T SNARES. WE'VE ADDED CYTOPLASMIC DOMAIN -- AND WE ADDED THE CYTOPLASMIC DOMAIN IN CALCIUM AND ASKS WHAT HAPPENED. TO GIVE IT AWAY, WHAT HAPPENS IS I'VE ALREADY ALLUDED TO THE FACT THAT -- ALREADY SHOWN THAT SNAP 25 WILL NOT BIND TO MEMBRANE SYNAPTOTAGMIN -- ANY APPRECIABLE DEGREE. WE DON'T KNOW WHAT THAT IS, IT'S JUST AN OBSERVATION. NOW THE EXCEPTION TO THIS IS IF WE ADD SYNAPTOTAGMIN AND CALCIUM, THE SYNAPTOTAGMIN WILL FOLD THE SNAP 25 -- AND THAT WILL THEN RECONSTITUTE SYNAPTO-- SO HERE'S A VERY SIMPLE GEL THAT SHOWS YOU SYNAPTOTAGMIN AND CALCIUM CAN RESEMBLE SNARE PROTEINS. WE FLOAT THE -- VESICLES AND THIS IS ALL SYNTAXEN. THE SNAP 25 BAND IS DOWN HERE AND YOU CAN SEE THERE'S A LITTLE BIT OF SNAP 25 BOUND IN SYNTAXEN BUT NOT MUCH IS HAPPENING HERE. WE ADD CALCIUM NOTHING HAPPENS. WE ADD TAGMIN NOTHING HAPPENS AND WE ADD -- AND BOOM SOME THINGS HAPPEN -- YOU SEE HERE THERE'S A LOT OF SNAP 25 SIGNAL AND DOWN HERE AT THIS LOWLY SET OF DOMAINS -- IT'S ALSO CALLED AND YOU SEE A LITTLE BIT OF BINDING HERE AND BOOM IT POPS IT. SO YOU CAN TAKE THESE, I'M NOT SHOWING IT HERE BUT YOU CAN TAKE THIS SAMPLE, ADD ETTA, GET RID OF THE TAG MIN WILL POP RIGHT BACK UP AGAIN AND THE SNAP 25 AND THE SYNAPTO-- WILL STAY DOWN. IF YOU USE STAMP NO TAG MINUTE TO DRIVE THE ASSEMBLY -- WE'VE SHOWN, I THINK I TOOK THE SLIDE OUT BUT THE SNARE COMPLEX CAN NOW DRIVE MEMBRANE FUSION. THERE'S NO QUESTION IN TEST TUBES SYNAPTOTAGMIN CAN DRIVE FULLLY TRANSITION OF SNARES. THE REASON I'M SHOWING YOU THIS, I'M GOING TO SKIP AHEAD BECAUSE I'M TAKING TOO LONG IS TO MORPH FROM -- SO WE'RE VERY INTERESTED IN THE IDEA THAT DOCKING OR PRIMING, AND I JUST SKIPPED THOSE SLIDES, MIGHT INVOLVE PARTIAL DIFFERING OF SNARE COMPLEXES. AND IN RESPONSE TO CALCIUM -- MAYBE THE SNARE COMPLEX FINISHED ZIPPERING AHEAD. -- SO THE QUESTION IS WHAT ARE THE CHANGES FROM THE SNARE COMPLEX DURING MEMBRANE FUSION AND HOW DOES STAMP NO TAG MINUTE REGULATE THOSE STRUCTURE CHANGES. I WANT TO BE CLEAR THAT SYNAPTOBRAGGEN IS -- SHOWN BY -- WHEN IT'S SOLUTIONS IT HAS A RANDOM STRUCTURE. WHEN IT IS IN THE SNARE COMPLEX IT SNAPS IN AND OUT OF THE HELIX. WE FOUND JUSTIN'S PAPER FROM -- LAB WHERE HE PUTS TWO HISTAMINES ON ADJACENT TERMS -- WHEN PROTEIN SNAPS -- REGISTER CAN BIND THE TRANMISSION METAL AND THAT THEN CAN RECEIVE ENERGY FROM A THREAT DONOR UP STREAM AND YOU GET -- IT'S THE SAME SORT OF TRICK, TWO HISTAMINES BIND THE METAL -- SO THESE ARE VERY PRELIMINARY DATA THAT I'M GOING TO SHOW YOU NOW. SO HERE'S THE IDEA. SYNAPTOBREAK IS A RANDOM COIL -- THIS IS THE PRIMITIVE THING -- AS I SPEAK HERE SO THE IDEA IS WE DUMP IN THE CYTOPLASMIC DOMAIN THAT WILL FORM -- IT WILL SNAP -- AS WE ZOOM IN, THE HISTAMINES WILL BIND TO NICKEL. WE CAN'T USE COPPER BECAUSE COPPER BINDS TO SYNAPTOTAGMIN AND MESSES IT UP. YOU GET QUENCHING OF THE MBPR. HERE IS A NICKEL TITRATION. YOU CAN JUST SEE -- YOU GET SOME QUENCHING OR SOMETHING TAKING PLACE HERE. BUT HERE IS, OVER HERE WE'VE TAKEN -- AND ADDED THESE CDP SNARES. SUPPOSEDLY DRIVE THE ASSEMBLY SNARE COMPLEXES AND WE TITRATED THE NICKEL AND LO AND BEHOLD WE GET THIS -- BETWEEN THE TRANSITION METAL AND THE BINDING. WE FIXED THIS AT TEN MICRO MOLAR NICKEL. WE REPEATED THE EXPERIMENT NOW AT -- WHERE THE THING WE VARIED WAS THE AMOUNT OF CYTOPLASMIC DOMAIN THAT THE SYNAPTOTAGMIN HAD. HERE IN THE SOLUTION BASED AWE SAY AS WE ADD MORE AND OR -- CHANGES THE SIGNAL. THIS WHOLE PROMISE TO USE JUSTIN ' S TECHNIQUE -- PROBABLY EVERYBODY KNOWS THIS BECAUSE HE WORKS HERE -- YOU CAN PRESSURE PRECISE CHANGE AND WE HOPE IF WE MOVE THESE PROBES FROM ONE END TO THE OTHER DURING THE DOCKING OF THE VESICLES WE MIGHT GET EVEN TRP TERMINAL THREATS -- UNTIL AFTER THE CALCIUM TRIGGER. THE REASON I'M SO INTERESTED IN THIS, I MIGHT AS WELL SHOW THIS REAL QUICK, IS THAT -- RECENTLY GOT MEMBRANES IMBEDDED IN -- RESUBSTITUTED SYSTEM AND THERE WAS SOME WEIRDNESS. TO GET IT TO WORK YOU MUST HAVE PIB2 IN THE TARGET MEMBRANE NOT IN THE VESICLE MEMBRANE. AND SO IF THERE'S NO PIT2 IN THE TARGET MEMBRANE CALCIUM DOESN'T WORK IN THE SYSTEM AT ALL. AS WE TITRATE -- MEMBRANE FUSION. AND THE OTHER THING I WANT TO POINT OUT HERE THAT WAS VERY INTERESTING PIECE OF STRANGENESS IS DOWN HERE. IF WE DO NOT PREINCUBATE THE B AND T SNARE VESSELS WITH TAG MIN PRIOR TO THE CALCIUM TRIGGER THERE IS NO CALCIUM TRIGGERED FUSION. THAT'S SHOWN DOWN HERE. YOU CAN'T SEE THIS HERE. I APOLOGIZE WE BUNCHED IT UP -- CALCIUM HAS NO EFFECT. IF WE GO OUT HERE SAY TO THIS PURPLE TRACE FOR 60 MINUTES, BOOM YOU GET THIS ROBUST AND VERY FAST CALCIUM EFFECT. NOW THE POINT HERE IS THIS -- IF YOU LOOK AT THE EXTENT OF THE FUSION VERSUS THE RATE OF FUSION, THE HALF MAXIMAL TIME FOR PREINCUBATION IS THREE TO NINE MINUTES. BUT THE DOCKING, RECONSTITUTE THE DOCKING PARTICLE HERE AND I CAN TALK ABOUT THAT IN THE Q&A BECAUSE I'M RUNNING OUT OF TIME. THE DOCUMENT TAKES PLACE BETWEEN THE -- IS VERY FAST AND IT'S SHOWN HERE. THIS IS A LIGHT SCATTER ASIAN SAY. WE'VE MIXED SYNAPTOTAGMIN B VESSELS WITH -- AND IT'S ALMOST AS ANALYSIS AS WE CAN MEASURE IT. WHEN WE MIX THEM TOGETHER BOOM EVERYTHING AGGREGATES AND WE ASK CALCIUM WITH NO FURTHER AGGREGATION BECAUSE EVERYTHING'S ALREADY DOCKED. IN CONTRAST IF WE HAD THE CYTOPLASMA -- IT'S NOT ANCHORED TO ONE AND BIND TO THE OTHER. WE DISCOVERED RECENTLY IS THAT SOME COPIES OF SYNAPTOTAGMIN -- DOMAIN BIND TO ONE VESICLE AND ANOTHER SET OF COPIES BIND TO OF THE VESICLE -- BUT THE MAIN POINT HERE IS THE DOCKING STUFF IS VERY FAST. SO WE HAVE AN INTERESTING SYSTEM TO PROBE WITH THESE PROBES BECAUSE DOCKING IS COMPLETE IN LESS THAN A MINUTE. THE PRIMING PART IS 3-9 MINUTES. WHAT'S HAPPENING DURING THE DOCKING STEP IS THAT N TERMINAL THREAT. AND THEN THE FUSION ASSOCIATION WITH CTMO THREAT. OKAY. SO I'VE GOT TWO MINUTES LEFT. I'LL SHOW YOU JUST ONE OR TWO MORE QUICK SLIDES. THAT'S SYNAPTOTAGMIN 1 -- IT'S MEANT FOR SPEED -- A LOT OF PEOPLE SAID YOU MIGHT THIS HAVE WRONG -- ALL YOU KNOW IS YOU NEED IT. BUT THERE ARE OTHER INTERPRETATIONS ABOUT KNOCKOUT EXPERIMENTS. THERE'S A VERY IMPORTANT PAPER THAT I LOVE. THIS IS THE FIRST PAPER TO EVER DO -- IN A SMALL CENTRAL NEURON. WHAT THIS GROUP DID, THIS IS -- THIS PAPER CAME OUT TWO YEARS AGO. LIKE ANY POST DOCTORAL STUDENTS OWE IT TO YOURSELVES TO -- WHAT THEY DID HERE IS THEY GREW HIPPO CAMPO NEURONS, WE DO THIS ALL THE TIME -- SO ONE NEURON AND THE NEURON'S -- AND SO THEY FORMED SNAP PARTICULARS ON THEMSELVES, THEY FORMED -- HIPPO CAMPO NEURONS ALWAYS GOT TO FORM -- SNAP SIST -- ON THEMSELVES. THEY COULD LOAD THE NEURON WITH CASE CALCIUM, THEY COULD DO -- AND GET A TYPE LIKE INCREASE THROUGHOUT THE ENTIRE NEURON. THEN THEY CAN END THE CALCIUM CONCENTRATION IN LARGE STRUCTURES LIKE DENDRITES -- THE TERMINALS ARE TOO SMALL TO DO THIS KIND OF MEASUREMENT ACCURATELY. THAT'S WHAT THEY DID. THIS IS THE FIRST EVER EXPERIMENT DOING THIS. HERE'S WHAT THEY SHOWED. I DON'T WANT TO GO THROUGH TOO MUCH DETAIL. THIS FLASH RIGHT HERE IS THE FLASH AND THEN THE CONTROL, THIS IS THE WILD TYPE HIPPO CAMPO NEURON -- THE CALCIUM'S BEEN DELIVERED AND TAKES A WHILE TO CLEAR. THEY AREN'T THAT INTERESTING HERE. NOW THAT THEY TOOK A SYNAPTIC -- KNOCKOUT NEURON AND REPEATED THIS EXPERIMENT. THEY SAW SOMETHING REALLY INTERESTING. SO IN THE -- KNOCKOUT NEURON THEY DO THE FLASH AND THE RISE TIME IS DESPERATELY SLOWER -- IT MUST BE SLOWER TRANSFUSION THAN SYNAPTOTAGMIN 1. THE REASON THAT'S A BIG DEAL TO A LOT OF US IS THERE'S ANOTHER POSSIBILITY AND THAT IS FUSION WILL BE JUST AS FAST IN THE ABSENCE OF TAG MIN THE ONLY THING YOU'VE DONE IS MOVED VESICLES AWAY FROM CALCIUM CHANNELS. SO THIS IS THE FIRST REALLY EXPERIMENT IN A SMALL CENTRAL SYNAPSES TO RULE OUT. THIS IS NOT THE LACK OF GETTING A BIG FAST EPST IN HIPPO CAMPO NEURONS IS NOT BECAUSE THE VESSELS ARE FURTHER AWAY FROM CALCIUM CHANNELS IT'S BECAUSE THE OTHER SENSORS FUNCTION MORE SLOWLY. WE WONDER IF THE OTHER SENSOR WAS A TAG MIN AND TO MAKE A REALLY LONG STORY SHORT WE DID ON -- SCENE THIS IS SOMETHING YOU'RE ALL FAMILIAR WITH. WE PUT A PH SENSITIVE -- ON THE PROTEIN -- WHEN THEY FUSE THE GFP LIGHTS UP AND THE ENDOCYTOSIS GOES BACK OUT. TO MAKE A REALLY LONG STORY SHORT, THE ONLY TAG MIN THAT GO TO SYNAPTIC VESICLES IN OUR HANDS AND I KNOW PEOPLE AREN'T GOING TO AGREE WITH THIS IS SYNAPTOTAGMIN 1 AND 2. A 5, 7 AND 17 -- LOOKS LIKE A LARGE -- 3 AND 11 GAVE US RESPONSES IN DENDRITES AND ONLY IN DENDRITES. 4, 6, 9 AND 12 GAVE US RESPONSES IN BOTH COMPARTMENTS. THESE GUYS AREN'T EXPRESSED AND IT WAS THIS SCREEN THAT DROVE US TO LOOK AT OTHER PROTEIN FAMILIES FOR THE OTHER SLOW CALCIUM SENSOR AND THE SCREENS ARE ALL OF THEM. ONE OF THE ONES WE LAND ON ARE THOSE DUCK 2. THIS IS JOHN -- WORK ALONG WITH -- HOOKED UP WITH FRED GAUGE AND SO WHAT JOHN DID FIRST HE DID THE IN VITRO STUFF AND THEN -- I'LL JUST SAY THIS REAL QUICK. IF YOU DO A SPOT FLOW EXPERIMENT -- OR DOCK 2 BIND TO A VESICLE AND WATCH IT BIND, THIS IS THE SPOT FLOW EXPERIMENT. THE TAG MIN BINDS SO FAST IT'S HARD TO MEASURE. AND THE DOCK -- BINDS MUCH MORE SLOWLY. WE WORKED OUT ALL OF THE -- FOR THIS. THE SECOND THING I WANT TO POINT OUT IF YOU BUILD A TAG MIN OR DOCK 2 -- AND ADD CHELATOR -- FROM THE VESICLES, TAG MIN IS SO FAST IT'S HARD TO MEASURE. SO THE TIME SCALE HERE IS .2 SECONDS. THIS IS A FULL SECOND OUT HERE. AND DOCK 2 IS JUST SLOW. THE TIME COURSE OF DOCK 2 LETTING GO OF CALCIUM VESICLES -- WHAT THAT MEANS, I DON'T KNOW. SO COMPARED TO SYNCHRONICITY -- SLOW ONSET IN RESPONSE TO CALCIUM AND IT'S LONG LASTING -- THAN ANTICIPATED. IF WE DO THIS BIOPHYSICS, AND THIS IS PRETTY REDUKION -- REDUCTION STUFF -- WE WENT AHEAD AND DID EXPERIMENTS. HERE'S AN EXPERIMENT. HERE'S AN EPSC FROM SYNAPTOTAGMIN 1 -- THERE'S NOT A LOT OF -- YOU GET RID OF A -- ALTHOUGH SOME STILL PRODUCE. WE GET THE SAME RESULTS EVEN IN KNOCKOUT MOUSE. IN OUR HANDS ONLY DOCK 2 ALPHA -- WE CAN'T DETECT DOCK 2 BETA -- TO RESCUE THE -- FOR A NORMAL SYSTEM FOR A SYNAPTIC EFFECT. IF YOU LOOK AT WILD TYPE NEURONS, IF YOU GET RID OF DOCK 2 AS SHOWN IN THE RED TRACE HERE, YOU INCREASE THE KINETICS OF THE DECAY OF THE DPSC AND THAT IS COPIED PERFECTLY BY -- AND GET RID OF -- RELEASE. AND THE OTHER THING THAT'S KIND OF NEAT ABOUT THIS IS IF YOU OVER EXPRESS DOCK 2, YOU GET INCREASES, THERE'S TWO COMPONENTS OF TRANSMISSION. JUST DO DOUBLE -- AND WHAT YOU SEE IS THIS IS THE FAST COMPONENT'S NOT AFFECTED BUT THE SLOW COMPONENT -- OVER EXPRESS DOCK 2 BETA WE CAN INCREASE THE RELEASES. SO THE NICE THEN ABOUT THIS IS BY UP REGULATING WE GET MORE -- SO NOW WE'RE IN A POSITION TO TWEAK THE -- RELEASE AND WHAT WE'RE DOING IS LOOKING AT A RELAPSE REVERBERATIONS -- WHICH IS IN -- IT WILL SIT THERE AND OSCILLATE FOR LIKE TEN SECONDS. WE'RE USING THIS AS A WAY TO FINE TUNE THE RELEASE TO UNDERSTAND THE RULES ABOUT -- REVERBATION FOR ON-LINE MEMORY -- WE END UP WITH A MODEL THAT DRIVES FAST CHEMISTRY AND MAYBE DRIVES MEMBRANE FUSION. DOCK 2, WE DON'T KNOW THE CALCIUM -- FOR SLOW RELEASE. THEY HAVEN'T SHOWN THAT. IT'S REQUIRED FOR SLOW RELEASE AND IT HAS ABOUT -- PROPERTIES CONSISTENT WITH THE CALCIUM SO THE LAST THING I'M GOING TO SHOW IS AN EXPERIMENT THAT -- DID, ARE WE'VE BEEN WANTING TO DO THIS FOREVER. SO WE STARTED MAKING DOCK 2 -- CHIMERAS -- I WANT TO POINT OUT IF WE TAKE THE DOCK 2 -- DOMAIN DA AND FUSE IT TO THE SYNAPTOTAGMIN -- WE GET THIS BLACK COVER AND IF WE DO THIS -- WE GET THE INTERMEDIATE GENETICS. IF WE INCLUDING THE CHIMERAS INTO THE SLOW SLUGGISH -- IRRELEVANT MEMBRANE FUSION ASSAY. HERE'S THE DOCK 2 AND THE CHIMERAS AGAIN HAS INTERMEDIATE GENETICS. IF WE SHOOT THIS CHIMERAS INTO A -- THESE ARE EPSC'S HERE. THESE ARE NOT SCALED, NORMALIZED. SO IT RECOVERS A CURRENT IN THE KNOCKOUT BUT DOESN'T RECOVER ALL THAT WELL. THE DOCK 2A SYNAPTOTAGMIN -- IF WE NORMALIZE THE PRAISES THOUGH YOU CAN SEE THE -- IS INTERMEDIATE. SO WE MIGHT BE ABLE TO -- TO TUNE THE CONNECTIVE SYNAPTOTRANSMISSION BY MAKING THE -- CHIMERAS WE BUILT A COUPLE DOZEN MOLECULES, WE SCREEN A BUNCH OF THEM BUT WORK IS PROCEEDING. IT'S ALWAYS BEEN A FANTASY IF THERE REALLY ARE TWO COMPONENTS OF TRANSMISSION -- THOSE ARE BIG F'S WHEN I CAME TO THE FIELD WHEN IT DOES RAISE THE POSSIBILITY YOU COULD MAKE DESIGNER MOLECULES TO MAKE SYNAPSES AT ANY SPEED YOU WANT. THAT'S SOMETHING WE REALLY WANT TO DO. AND THAT'S SOMETHING THAT I WOULD REALLY LOVE TO GET -- JOIN THE LAB TO DO. SO SORRY I WENT A LITTLE BIT OVER. HERE'S THE GROUP AND SHEILA JUST LEFT AND SO WE'RE JUST IN THE MIDST OF INTERVIEWING PEOPLE. SO LET US KNOW IF YOU'RE INTERESTED IN DOING THIS WORK. THANKS FOR YOUR ATTENTION. [APPLAUSE] I WILL ANSWER QUESTIONS IF THERE ARE ANY. >> SO YOUR BAR DOMAIN BENDING ASSAY'S REALLY NEED TO LOOK AT THE ROLE OF BENDING. HAVE YOU STARTED TO LOOK AT EFFECTS OF LIPID COMPOSITION ON THAT AND CAN YOU MODULATE THE ABILITY TO BEND THE MEMBRANE BY DOING THAT? >> A GREAT QUESTION. YES AND YES. I GOT TO BE LAILG CAREFUL OR I'LL GET CONFUSED. SO IT TURNS OUT THAT I SHOWED YOU THAT THE DIAMETER OF THE TUBULE DEPENDS ON THE -- PAS DOES A HAVE SHAPE -- IT HAS A LITTLE BIT OF THIS SHAPE. SO IF YOU AGGREGATE PS TOGETHER, JUST THAT ALONE WILL GIVE YOU -- NOW EVERYONE TELLS ME ALL THE CURVATURE JOCKEYS WHO I CAREFULLY TRY TO PAY ATTENTION TO BECAUSE I HAVE A LOT OF QUESTIONS ABOUT THE WHOLE THING. THEY DON'T THINK IT'S THAT INTERESTING BECAUSE PS ISN'T VERY, YOU KNOW -- SHAPED LIKE THIS. BUT QC OUR RESIDENT CHEMISTRY GENIUS IN MADISON -- DID A SIMULATION AND THE SHAPE OF PS IS ENOUGH THAT AG GATING PS ALONE IS ENOUGH TO GIVE YOU NARROW TUBULES. SO WE HAVE A CONUNDRUM. THE AMOUNT OF PS DETERMINES THE DIAMETER OF THE TUBULES AND THAT MIGHT BE PART OF THE REASON WHY. BUT THE DIAMETER OF THE TUBULES DOES NOT DEPEND ON THE AMOUNT OF SYNAPTOTAGMIN. YOU EITHER GET BENDING OR YOU DON'T. AND SO WITH QC HAS COME UP THROUGH A SERIES OF MEETINGS WE CAME UP WITH A COMPLICATED MAYBE TESTABLE MODEL TO FIGURE THIS OUT. AND WE'RE IN THE MIDST OF DOING THAT. SO THERE IS QUITE A BIT OF WORK. NOW -- WAS THE GUY WHO PIONEERED THIS STUFF WITH CONE SHAPED LIPIDS AND HE DID VERY CLEVER EXPERIMENTS OF LIPIDS WHETHER THEY'RE SHAPED THIS WAY OR THIS WAY WHETHER YOU ADD THEM TO THE OUTSIDE OF THE FUSION REACTION OR THE INSIDE OF THE FUSION REACTION YOU HAVE DIFFERENT RESULTS -- THOSE EXPERIMENTS ON FUSION -- A LOT OF THE WORK WAS RUN BY ALL FUSION PROTEINS. WHEN -- JACKSON REPEATED THOSE EXPERIMENTS IN -- HE GOT THE OPPOSITE RESULTS -- SO MY CRAW CONSTRUCTED A VERY COMPLICATED THEORETICAL FRAMEWORK TO TRY TO INTERPRET THAT FROM THE PERSPECTIVE OF A -- PROTEIN LINE FUSION WORK -- SO AGAIN, THIS IS ALL INTERESTING STUFF BUT DIRECT MEASUREMENTS ARE LACKING. WE'RE VERY INTERESTED. I DON'T WANT TO GIVE TOO LONG OF AN ANSWER BUT A BRIEF INTERESTING DETAIL. IF YOU SPLIT SYNAPTOTAGMIN IN A AND B -- BUT THEN YOU ADD UP -- WHICH IS ONE OF THE MAJOR PHOSPHOLIPIDS THEMSELVES, THE C2B DOMAIN WORKS GREAT. YES, I MEAN WE'RE A LITTLE LATE IN COMING TO THIS BUT MORE AND MORE WE'RE GET AN APPRECIATION FOR THE ROLE OF LIPIDS. THE CLEANER AND MORE DEFINE YOUR SYSTEM IS -- THE MORE WRONG ANSWERS YOU'RE GOING TO GET. WITH PHOSPHOLIPIDS THE DIRTIER THE BETTER. >> TWO RELATED QUESTIONS. DO YOU THINK THE POWER RELATIONSHIP HAVE ANY RELATIONSHIP TO THE TOTAL -- THE SECOND QUESTION [INDISCERNIBLE] IS REQUIRED. >> YES. SO I HAVE NO ANSWER FOR EITHER QUESTION. BUT I THOUGHT ABOUT THOSE QUESTIONS EVERYONE KNOWS IN GENERAL AGREEMENT -- AT THE LOWER LIMIT. AND THEN WHEN THE STRUCTURE STUDIES OF TAG MIN 1 INDICATED THERE WERE -- MAYBE ON C2A AND -- THERE WAS FIVE CALCIUM IONS AND WE HAVE A MAY DAY WITH MAP, RIGHT. THERE'S BEEN A LOT OF MUTAGENESIS AND I COULD EASILY SPEND A WEEK SEMINAR SHOWING HOW CONFUSING MUTAGENESIS IS. SO LET ME JUST GIVE YOU THE PAIRED DOWN VERSION. THERE ARE MANY PAPERS THAT HAVE MUTATED THE CRAP OUT OF CT5 AND ALMOST NOTHING HAPPENS. THEN MUTATED THE CALCIUM LIGAND -- C2A DOES NOT. THEN -- THAT'S BEING DEBATED. WHEN WE GET TO THAT POINT WE GET A COLLABORATION WITH -- AND SHE DID SOMETHING THAT NONE OF US REALLY BOTHERED TO DO AND THAT IS IF YOU HAVE A -- REST DO WITH D EVERYONE GOES TO, RIGHT OR THEY GO TO ALAN. THAT'S WHAT EVERYONE DOES. NOREEN WENT FROM D TO E -- AND WHEN SHE DID THAT IN THE C2A DOMAIN, SHE SHOULD NOT HAVE DONE ANYTHING BECAUSE A IS NOT IMPORTANT AND D TO E IS CONSERVATIVE, RIGHT. LIKE 80% OF FUNCTION OF THE PROTEIN. MOREOVER, THAT B TO E BY -- IN MY LAB LOOKS LIKE IT MESSED UP THE OTHER CALCIUM BINDING SITES IN SYNAPTOTAGMIN. THEN WE GO TO -- THE GUY WHO DID THE FIRST CRYSTAL STRUCTURES AND I INTERACTED WITH BRIAN I TRY TO UNDERSTAND THIS. WE'VE COME TO THE CONCLUSION WE DON'T REALLY KNOW HOW SYNAPTOTAGMIN BINDS TO CALCIUM -- IT'S NOT GOING TO BE A CASE WHERE YOU CAN CHANGE THE HILL SLOPE IN A DISCRETE WAY -- ANY OF THE CALCIUM IONS YOU KILL FUNCTION OF THE PROTEIN. IF YOU -- A DOMAIN IT DEPENDS ENTIRELY ON WHAT DOMAIN YOU MAKE, WHAT THE SUBSTITUTION IS WHETHER IT'S A LOSS OF FUNCTION OR GAIN OF FUNCTION OF THE SAME RESIDUE. SO IT'S REALLY GOING TO BE A MESS. THE GENERAL QUESTION IS I THINK IT'S UNLIKELY THAT THE SMOKE IS GOING TO MAP ON TO TAG MIN. IT'S GOING TO BE THE NUMBER OF TAG MIN RATHER THAN -- THAT ARGUMENT, THAT WAS THE FIRST QUESTION SO I DON'T HAVE A GOOD ANSWER FOR YOU. WHAT WAS THE SECOND QUESTION? >> [INDISCERNIBLE] >> UNKNOWN. SO THE ONLY THING I HAVE TO ADD THERE IS THAT REINHART -- DID THE PROTEOME AND THE SYNAPTIC VESSEL YOU HAVE ON AVERAGE 15 COPIES OF SYNAPTOTAGMIN. AND IF YOU LET THE SYNAPTOTAGMIN REACH OUT ABOUT THE LENGTH OF THE CYTOPLASMIN DOMAIN -- VESICLE. AND THEN IF YOU LOOK AT THE REALLY BEAUTIFUL FLASH -- EXPERIMENT IN -- THAT WAS PIONEERED BY -- LOTS OF PEOPLE DOING THIS BEAUTIFUL WORK. WHAT THEY DID IS THEY DID -- AND THEN THEY DID -- POST SYNAPTIC -- WITH A STANDARD CURVE AND SAID HERE'S HOW MANY VESSELS COME OUT IN RESPONSE TO THIS MUCH CALCIUM SO YOU GOT A STANDARD CURVE. AFTER THEY'VE ALL DONE THEY HIT IT WITH AN ACTION POTENTIAL AND GET A SYNAPTIC RESPONSE. HOW MUCH CALCIUM IS THAT THAT. THAT WAS A BIT OF A SHARK. THE NUMBERS EVER CHANGING. I DON'T KNOW WHAT THE NUMBERS ARE THIS HE CAN WITH -- AT THE SENSORY DRIVE -- SO THE QUESTION THEN BECOMES WHAT'S THE -- TAG MINUTE IS YOUR REAL QUESTION, RIGHT. IF YOU HAVE 20 MICRO MOLAR IN THIS SPACE, SO THIS AMOUNT OF TIME, THIS PROTEIN -- HOW DOES IT WORK. AND THAT'S ALL IN THE REALM OF -- IT GETS EVEN MORE COMPLICATED BECAUSE IF THE -- IS OVER HERE, WE HAVE AN -- TROPHIC SYSTEM, THE TAG MIN IS ON THIS SIDE AND YOU GET OCCUPIED -- WHICH MAKES THE THING GET REALLY MUCH MORE COMPLICATED. SO I GUESS WHAT I WOULD SAY IS IT LOOKS LIKE THE CALCIUM SENSOR HAS PRETTY MINIMAL OCCUPANCY UNDER NORMAL CONDITIONS. I THINK 10 OR 20 MICRO -- OCCUPY A SMALL NUMBER OF BINDING SITES AT ANY GIVEN TIME. SO THE SYSTEM IS OPERATING WELL AT THE BOTTOM OF THE DYNAMIC RANGE. I DON'T REALLY NECESSARILY KNOW WHY THAT WOULD BE BUT THAT'S WHAT IT LOOKS LIKE. IF YOU LOOK AT THE MODELS FROM SNARE AND -- AND THOSE GUYS, THEY WILL SAY THE CALCIUM CENTER HAS THE INFINITY UP AROUND 70 MICROMETERS BUT 10 OR 20 MICROMETERS. >> [INDISCERNIBLE] >> SO THE -- TELLS US IN THE ABSENCE OF TAG MIN ONE -- SLOWER FUSION. SO THAT ARGUES TO ME THAT THE SECOND SENSOR OR SENSORS, THERE'S MORE THAN ONE HAS TO HAVE SLOWER VESICLE PROPERTIES I WAS SO RELIEVED TO SEE THAT IN THE -- PAPER. SO THE IDEA WOULD BE, THEN, THAT WHEN THE CALCIUM LEVEL RISE, TAG MIN DOES THE CHEMISTRY FIRST -- TAG MIN COMMITTEELY RELAXED -- GIVEN BY THE RESIDUAL CALCIUM AT DOCK 2 THE RESPONSE IS A LITTLE MORE SLOWLY AND STAYS RESPONDING TO. SOD IDEA -- THERE IS EVIDENT FROM THAT -- ALTHOUGH WE HAVE SOME QUESTIONS ABOUT THAT. THANK YOU FOR YOUR ATTENTION.