>> HEY, EVERYONE.
MY NAME IS YOMI.
I'M A POST-BACC AT THE NIDIC,
IT'S MY PLEASURE TO INTRODUCE
TODAY'S SPEAKER, LING GANG WU,
COMPLETED MD FROM SECOND CAN
COLLEGE FROM SHANGHAI BEFORE
MOVING TO TEXAS AND WAS AT
BAYLOR UNIVERSITY OF MEDICINE,
STUDYING PRESYNAPTIC CALCIUM
CHANNELS AND NEUROTRANSMITTER
RELEASE, WENT ON TO DO TWO
POST-DOC, FIRST AT THE
UNIVERSITY OF COLORADO SCHOOL OF
MEDICINE, WHERE HE STUDIED
KINETICS OF VARIOUS STEPS IN
SYNAPTIC VESICLE RECYCLING.
FIRST ONE IN HEIDELBERG GERMANY,
COMBINED BACKGROUND TO
INVESTIGATE MECHANISMS BEFORE
VESICLE RELEASE AND SHORT-TERM
SYNAPTIC PLASTICITY.
AFTER FOUR YEARS AS AN ASSISTANT
PROFESSOR AT W WASH U, STARTED
HIS OWN LAB AT THE NINDS AND HAS
BEEN HERE FOR 20 YEARS.
ENDOAND EXOCYTOSIS AND UNCOVERED
FUNDAMENTAL MECHANISMS BEHIND
BUDDING AND FUSION AND THEIR
RELATION TO VESICLE RELEASE.
FURTHER HIS LAB USES STATE OF
THE ART IMAGING APPROACHES TO
STUDY THE FUSION PORE, A
STRUCTURE FORMED DURING
EXOCYTOSIS.
JOIN ME IN WELCOMING DR. WU TO
THE FRONT.
THANK YOU.
[APPLAUSE]
>> THANK YOU FOR YOUR GREAT
INTRODUCTION, YOMI.
IT'S MY GREAT PLEASURE AND HONOR
TO BE HERE.
MY FIRST TIME, AS YOMI SAID, IN
THE LAST 20 YEARS OF MY LIFE,
AND AS YOU CAN SEE HERE, IN THIS
CERTIFICATE AWARDED BY OUR
INSTITUTE, 20 YEARS HERE.
A GOOD LIFE HERE AS WELL.
I'M GETTING OLD NOW.
SO I'M GOING TO SHOW YOU IN THIS
20 YEARS, OUR JOURNEY TOWARDS
METHYLIZING THE VESICLE FUSION.
VISUALIZATION, I CAN'T HELP BUT
SHOW YOU SOME OF MY OWN DATD
COLLECTIONS, NOT IN THE LAB, BUT
IN THE NEIGHBORHOOD.
JUST A BIT OF FUN BEFORE WE GET
SERIOUS.
SO HERE WE GO.
I SEE A BIG VESICLE HERE WITH
SOME STRUCTURES THAT I'M SURE
YOU'RE FAMILIAR WITH, AND A
SWOLLEN VESICLE AND A BEAUTIFUL
EVENING ON THE HARBOR HERE, A
DYNAMIC FLOW OF THE MEMBRANE IN
THIS BEAUTIFUL PARK, 20 MINUTES
AWAY FROM HERE.
SO IF YOU'VE NEVER BEEN HERE IN
THIS PARK, I WOULD SUGGEST YOU
TO GO.
BUT NOT RIGHT NOW.
AFTER MY TALK, PLEASE.
YES, MORE OF THE MEMBRANE
DYNAMICS IN THE RESTING
CONDITIONS BEFORE THE SUNRISE,
AND ALSO IMAGES, LANDSCAPES AND
ANIMALS.
HERE ARE THE MOST BEAUTIFUL
PICTURES I'VE EVER SEEN,
BEAUTIFUL MOUNT AN BIRDS
CROSSING THE RIVER BEFORE THE
SUNRISE.
THIS IS THE LAST BOVINE.
WHILE I'M HAVING FUN IMAGING THE
ANIMALS IN MY LAB IS IMAGING
CELLS FROM ANIMALS, PARTICULARLY
BOVINE.
THIS IS A CELL, BOVINE CELLS
CONTAINING MULTIPLE VESICLES
HERE.
THESE VESICLES UNDERGO FUSION
AND BUTTON, SO THAT'S THE -- AND
BUDDING.
THAT'S THE TOPIC OF THE DAY.
AS YOU KNOW, VESICLE FUSION AND
BUDDING IS INVOLVED IN MANY
PROCESS VERY FUNDAMENTAL FOR
EVERY CELL LIFE, FROM THE ESPN
DOUGH PALACE MECHANIC RETICULUM
AND BACK TO THE ENDOSOMES AND
LYSOSOMES.
THEY ARE INVOLVED IN THE GLUCOSE
LEVEL RELATED TO DIABETES, THEY
ARE INVOLVED IN MEDIATING VIRAL
INFECTION INCLUDING THE
CORONAVIRUS INFECTION THAT
CAUSED THE COVID-19 PANDEMIC.
THEY ARE ALSO INVOLVED IN
MEDIATING SYNAPTIC TRANSMISSION
BY EXOCYTOSIS AND ENDOCYTOSIS.
EXOCYTOSIS RELEASES TRANSMITTER
TO ACTIVATE THE TRANGS MITTER
REACCEPT -- TRANSMITTER
RECEPTORS, AND AFTER THE
EXOCYTOSIS, ENDOCYTOSIS,
RECYCLES VESICLE TO SUSTAIN
SYNAPTIC TRANSMISSION.
THIS IS IMPORTANT FOR OUR BRAIN
FUNCTIONS LIKE MY TALK RIGHT
NOW.
THE SYNAPSE IS THE MOST USED FOR
THE STUDY OF FUSION AND BUDDING.
BACK IN 1970S AND 1980S, JIM AND
TOM FROM NIH DISCOVERED THESE
STRUCTURES HERE, THEREFORE THEY
PROPOSED FRR FUSIONS INVOLVED IN
DILATION OF THE FUSION PORE, AND
HERE THE VESICLE IS FLATTENED AT
THE MEMBRANE.
AFTER THIS THEY FOUND THIS
INVOLVING THE ENDOCYTOSIS THAT
REFORMS THE VESICLES FORESEE
CYCLING VESICLES, CLASSICAL
PROCESS, INVOLVING ALMOST THE
REVERSE PROCESS.
AT ABOUT THE SAME TIME,
PERFORMED THE FORM OF EXOCYTOSIS
AND ENDOCYTOSIS BASED ON
OBSERVATION ONLY OF THE PROFILE
THERE.
SO THE FUSION FOR OPTIMUM CLOSE
VERY RAPIDLY, PORE VERY OFTEN
INTERPRETED AS SMALL PORE,
RECYCLES VESICLES REALLY FAST.
SO THAT WAS IN THE 1970S, AND
TWO DECADES PASSED, FUSION HAD
BEEN DEBATED FOR A LONG TIME,
BUT IT WAS FOLLOWED BY
ENDOCYTOSIS HAS BEEN VERY WELL
RESEPTEMBERRED.
AT THE END OF THE 1990S, WE LIKE
TO STUDIED, DETERMINE WHETHER WE
CAN SEE THEM IN THE LIVE CELLS,
AND IF SO, THEIR UNDERLYING
MECHANISMS AND FUNCTIONS.
SO THESE ARE THE THREE QUESTIONS
THAT WE WOULD LIKE TO ADDRESS.
AT THE END OF THE 19 THE 0S, THE
SUPER MICROSCOPE HAS NOT BEEN
BORN YET, SO WE USE THE
ELECTROSET UP, WE MEASURE THE
CAPACITORS PROPORTION TO
MEMBRANE AREA.
THE MEMBRANE AREAS INCREASE,
THEREFORE CAPACITANCE INCREASES.
SOS DECAY MEANING ENDOCYTOSIS,
WE CAN LOOK AT THE DECAY TIME TO
EXAMINE THE TIME COURSE OF THE
ENDOCYTOSIS.
TO DO THE CAPACITANCE
MEASUREMENTS, WE USE VERY LARGE
PREPARATION IN THE BRAINSTEM,
THE PRESYNAPTIC CELLS AND
POSTSYNAPTIC CELLS.
MY LAB'S PROJECT IS TO ESTABLISH
THE CENTRAL SYNAPSE.
WHAT WE SEE HERE IS THE
CAPACITANCE INCREASE THAT
CORRESPONDS TO THE RECORDED
SIMULTANEOUSLY AT THE SANE
SYNAPSE.
THIS MEANS ARTIFACTS REFLECTS
EXOCYTOSIS.
THEN WE CAN EXAMINE THE TIME
COURSE OF THE ENDOCYTOSIS
BECAUSE INCREASE MEANS
EXOCYTOSIS.
WE SEE THE SLOW ONES AND FAST
ONES TAKING A FEW SECONDS, ONE,
TWO, THREE SECONDS HERE TO HAVE
ENDOCYTOSIS.
AT THE TIME WE COULD INTERPRET
THAT AS THE KISS AND RUN,
BECAUSE IT'S THOUGHT TO BE
REALLY FAST.
ALTERNATIVELY, MOST PEOPLE WOULD
ARGUE THAT THIS COULD STILL BE
VERY FAST TO RUN TRANSITIONS
HERE.
SO WE DON'T KNOW.
SO HERE, IT'S A WHOLE CELL
RECORDING, FROM 10,000 OR 20,000
VESICLE EXOCYTOSIS.
IN ORDER TO SEE THE SINGLE
VESICLE FUSION, WE RECORD THE
MEDIATOR FROM POSTSYNAPTIC CELL,
AND CALL THE POSTSYNAPTIC
CAPACITANCE AND HOPE TO SEE THIS
INCREASE IN DECAY, BUT THE
SIGNAL NOISE IS TOO SMALL TO WE
HAVE TO AVERAGE ABOUT 320,000,
THIS FORMAL POST-DOC, HE'S
PROBABLY HERE, RUNNING ALSO TO
GET TO THIS, WE SEE VERY TINY
SIGNAL, THE CAPACITANCE
INCREASE, 10,000 TIMES LESS THAN
THE SCALE HERE, TO INDICATE
EXO AND ENDOCYTOSIS, BUT THIS IS
TOO LABORIOUS.
IF WE WANT TO CONTINUE FURTHER,
WE HAVE TO RECORD FROM SINGLE
TRACE, AND TO DO THAT, WE HAVE
TO DO THE CELL TEST RECORDINGS
AND ASKING THE VESICLES TO FUSE
ON THE TEST MEMBRANE.
TO DO THAT, WE THEN USE A LARGE
PIPETTE TO PULL AWAY THE
POSTSYNAPTIC CELLS AND THEN USE
A SMALLER PIPETTE TO PASS ON THE
RELEASE PHASE OF THE NERVE
TERMINAL.
UNDER THIS CONDITION WE CAN SEE
SINGLE CHANNEL CALCIUM CODONS IN
THIS CASE AT THE TERMINALS AND
WE CAN ALSO SEE THE SINGLE
VESICLE FUSION THAT'S SHOWN HERE
IN THIS CAPACITANCE INCREASE AND
DECREASE, THAT IS ACCOMPANIED BY
THE FUSION CONDUCTOR INCREASE IN
DECAY.
THIS PROVIDES STRONG EVIDENCE
FOR THE KISS AND RUN, AT THE
SYNAPSE.
AT THE SAME TIME WE ALSO SEE
VERY OFTEN THE CONDUCTANCE
INCREASE TO BEYOND OUR
DETECTION.
THAT'S CONSISTENT.
IF IT'S NOT ACCOMPANIED BY THE
CAPACITANCE DECAY HERE.
AND SOMETIMES, WE ALSO SEE THIS
GIANT VESICLE FUSIONS WHICH
CAUSES THE LAST CAPACITANCE
INCREASE SHOWN HERE, THE SCALE
IS ABOUT 20 TIMES LARGER THAN
HERE, THAT'S BECAUSE OF THE
VESICLE-VESICLE FUSION THAT
CAUSES A COMPOUND VESICLE, AND
THIS UNDERGOES COMPOUND
EXOCYTOSIS.
WE CAN SEE THOSE IN THE ELECTRON
MICROSCOPE.
ACTUALLY, EARLY IN TIME, ALSO
SEES THIS GIANT VESICLE AFTER
THE INTENSIVE STIMULATION.
ALL RIGHT.
SO I SPENT TEN YEARS, MORE THAN
TEN YEARS IN DOING MOST
DIFFICULT PHYSIOLOGICAL
EXPERIMENTS,
ELECTROPHYSIOLOGICAL
EXPERIMENTS, IN MY OPINION.
STILL IT'S AN ESTIMATE BASED ON
THE FUSION CONDUCTANCE WHICH IS
LIMITED TO ABOUT FIVE NANOMETERS
SO STILL THE ASPECT HERE,
ANYTHING BEYOND FIVE NANOMETERS
BELONGS TO SPECULATION.
AT THE TIME THE STEP MICROSCOPE,
ABOUT 60 TO 80 NANOMETERS EVERY
100 SECONDS OR SO, BECOMING
AVAILABLE.
THIS IS STILL INSUFFICIENT TO
RESOLVE THE VESICLE FUSIONS,
SYNAPTIC VESICLE, BECAUSE THE
SYNAPTIC VESICLE IS ABOUT
30 NANOMETERS OR SO.
SO WE HAD TO USE DIFFERENT,
BOVINE CELL, AND THIS IS A
NEUROENDOCRINE CELL SIMILAR TO
THE NEURONS IN MANY PROPERTIES.
MORE IMPORTANTLY, IT HAS VERY
LARGE VESICLES, 300-NANOMETER
VESICLES THAT WE CAN SEE IF THEY
ARE UNDERGOING THE KISS AND RUN
OR THE FULL CLASS FUSIONS.
SO THE CELLS ON THE COVER SLIPS
AND ADD DYE ON 647 OR 532 IN THE
BATH SOLUTION, AND YOU CAN SEE
THE DARK AREA HERE.
THIS IS THE CELL FOOTPRINT, THE
FROZEN SOLUTION, IT'S BETWEEN
THE CELL MEMBRANE AND THE COVER
SLIPS.
WHEN VESICLE FUSION OCCURS, THIS
FLUORESCENT DYE WILL DIFFUSE
INTO THE FUSION SPOTS.
YOU'LL SEE SPOTS.
WE GIVE ONE SECOND
DEPOLARIZATION THAT DECAY IS
INDICATED IN EXOCYTOSIS AND
ENDOCYTOSIS.
I'LL PLAY A VIDEO OF THE PROCESS
TO SEE AFTER THE DEPOLARIZATION
YOU'LL SEE AN APPEARANCE OF THE
SPOTS HERE.
DEPOLARIZATION RIGHT NOW.
SO YOU SEE THE SPOTS COMING
HERE, AND THAT MEANS THAT WE CAN
SEE THE FUSION IN THE BOTTOM OF
THE CELL.
NOW, TO SEE THE MEMBRANE PROFILE
CHANGES, WE OVEREXPRESS THE PS
DOMAIN, AATTACHED -- THIS WAS
DEVELOPED BY THOMAS HERE AT NIH.
IT BINDS TO THE INNER LEAFLET OF
THE PLASMA MEMBRANE.
THEN THE XY PLANE, WHICH MEANS
IT'S PARALLEL TO THE SCREEN
HERE, SO YOU SEE HERE, THE
STRETCH OF THE MEMBRANE AND IF
YOU LABEL THE VESICLE,
NEUROTRANSMITTER, WE SEE THE
SPOTS ABOUT TO DOCK AT THE
PLASMA MEMBRANE.
WHEN THESE VESICLES FUSE, THE
GREEN CAN FUSE INTO THE FUSION
VESICLE AND THEREFORE TO LABEL
THE FUSION VESICLE.
IN OUR EXPERIMENTS, NORMALLY WE
WOULD ADD A DYE OUTSIDE, IN THIS
CASE 532 AND WE HAD THE GREEN
MEMBRANE LABEL, SO HERE IT'S A
STRETCH OF MEMBRANE HERE,
UNDERNEATH THIS MEMBRANE IS A
THIN LAYER OF THE DYE HERE IN
THE XZ PLANE OF THE MICROSCOPE,
AND YOU'LL SEE THE IMAGING UNDER
THE COVER SLIP.
SO DEPOLARIZATION CAUSES EXO AND
ENDOCYTOSIS, AND WE SEE THE
DIFFUSION OF THE GREEN INTO THE
FUSION VESICLES AS WELL AS THE
DYE THAT GETS INTO THE CAVITY OF
THE VESICLES.
SO HERE IS A REALTIME VIDEO OF
THIS PROCESS.
AFTER DEPOLARIZATION, YOU'LL SEE
THIS APPEARANCE HERE, WE SEE
THESE THREE FUSION EVENTS.
SOMETIMES WE SEE THE GREEN
SIGNALS FOLLOWED BY THE RED
SIGNAL AS SHOWN HERE IN THIS
REALTIME VIDEO.
GREEN FIRST.
AND HAD RED COMING LATER.
THAT'S BECAUSE OF THE HEMIFUSION
THAT ALLOWS FOR THE GREEN
SIGNALS, MEMBRANES YOU SHOULD
NEET THE MEMBRANES TO DIFFUSE IN
AND YET BECAUSE THE PORE HAS NOT
BEEN OPENED SO THE DYE CANNOT
PENETRATE INTO THIS HEMIFUSION
VESICLES.
AFTER THE FULL FUSION, THEN THE
DYE CAN PENETRATE, YOU SEE THE
RED DYE APPEAR IN THE VESICLE.
SOMETIMES WE'LL SEE THE GREENER
SIGNALS FIRST AND WITHOUT SEEING
THE RED SIGNALS, AS SHOWN HERE
IN THIS VIDEO.
GREEN SIGNALS FIRST, HEMIFUSION
WITHOUT GOING THROUGH THE FULL
FUSION PROCESS.
SO WITH THIS, THE HEMIFUSION
PATHWAY IN LIVE CELLS.
AFTER THE HEMI TO FULL FUSION,
WE SEE SOMETIMES THE FUSION AS
YOU CAN SEE HERE, THIS EXAMPLE,
AND SOMETIMES THE FUSION IS SO
FAST, FIRST WE'LL SEE THE FUSION
HERE, .1 SECOND AND 26
MILLISECONDS, THAT'S BEFORE THE
DIFFUSION OF THE GREEN PROBE WE
SEE HERE, AND THIS IS THE XY
IMAGES ACROSS THE SCREEN TO SHOW
YOU THAT THE FUSION PORE.
AFTER THE VESICLE FUSION
OPENING, WE NOTICE THAT THESE
VERY LAST PORES CAN CLOSE VERY
QUICKLY, AS SHOWN HERE IN THIS
EXAMPLE.
THIS IS A REALTIME SAMPLE AFTER
DEPOLARIZATION WE SEE THE FUSION
FOR OPEN AND CLOSE AND HAD THIS
PORE IS 200, 300 NANOMETERS OR
SO, WITHIN 100 MILLISECONDS OR
SO.
THIS IS FOLLOWED BY THE
BLEACHING OF THE RED DYE BECAUSE
WE GIVE A STRE STRONG EXCITATION
OF THE RED DYE AND WHEN THE PORE
IS CLOSED, THEN THE DYE CAN ALL
BE BLEACHED BECAUSE THERE'S ONLY
A LIMITED NUMBER OF THE DYE
INSIDE THIS CLOSED CAVITY, SO IT
CAN -- CAN ALL BE CLOSE AND HAD
THIS BLEACHING ALSO CONFIRMS OUR
INDICATION THAT THE PORE IS
CLOSED.
CL WHEN THE PORE IS NOT
CLOSESSED, THEN WE SEE NO
BLEACHING OF THE DYE AS YOU CAN
SEE HERE IN THIS EXAMPLE.
HERE FUSION PORE STAYS LONG
TIME.
NOW WE WOULD LIKE TO KNOW
WHETHER THEY DO THE FOCAL
FUSIONS HERE, AND WE DO NOT SEE
THE FOCALIZED.
INSTEAD IT'S THE SAME FUSIONS AS
SHOWN HERE IN THIS EXAMPLE IS IN
THIS VIDEO.
MEANING THAT THE VESICLE'S BODY
IS SHRINKING INSTEAD OF THE PORE
DILUTION.
AS SHOWN HERE.
HERE IS THE VESICLE FUSION AND
THEN IT SHRINKS IT, AGAIN, NICE
REPEATED AGAIN, SO THE VIDEO
REPEATS AGAIN.
HAD.
I WOULD LIKE TO SHOW YOU ONE
MORE TIME THE SHRINKING OF THE
VESICLES SHOWN HERE.
SO ONCE AGAIN, IT DISERNT
DILATE, INSTEAD IT SHRINKS.
SOMETIMES WE SEE THE ENLARGEMENT
OF THE VESICLES AS SHOWN HERE IN
THIS EXAMPLE AND IN THIS VIDEO.
VESICLES FUSION AND THEN IT
ENLARGES IN ABOUT FIVE TO TEN
SECONDS.
THIS IS REPEATED AGAIN.
SOMETIMES WE SEE THE SEQUENTIAL
COMPOUND FUSION, WHICH MEANS
THAT THE VESICLE FUSES, RECENTLY
FUSED VESICLES, AND IT GENERATES
OR COL LAPS INTO FORMING A LARGE
VESICLE, AS SHOWN HERE IN THIS
EXAMPLE AND ALSO IN THIS
REALTIME VIDEO.
SO VESICLE FUSION OCCURS HERE
AND ON TOP OF THIS FUSION, THEN
THERE WILL BE ANOTHER VESICLE
FUSION.
SO SEQUENTIAL COMPOUND FUSION.
SO I'VE SHOWN YOU DIFFERENT
FORMS OF THE EXOCYTOSIS THAT WE
DID NOT EXPECT FROM LIVE CELLS,
AND NEXT I WILL SHOW YOU THE
ENDOCYTIC MEMBRANE
TRANSFORMATIONS.
THESE ARE SOME OF THE FIRST
VERSIONS OF THE FLAT TO LONG
TRANSITIONS, AND HERE I'LL SHOW
YOU A VIDEO OF THIS PROCESS.
FLAT MEMBRANES AND AFTER THE
DEPOLARIZATION INDICATED HERE AS
A GRAY TRIANGLE HERE, THEN THE
MEMBRANE BECOMES CURVED AND
EVENTUALLY FORMS A VESICLE
WITHIN ABOUT 10, 20 SECONDS.
SO I'LL SHOW YOU THIS PROCESS.
SO NOW YOU SEE SOME SPIKES
COMING HERE.
IT FORCES ON THE SIDE THAT
FORCES THE VESICLE TO BECOME A
VESICLE.
THIS IS FOLLOWED BY BLEACHING OF
THE RED DYE, CONFIRMING THAT THE
PORE IS CLOSED.
SO THIS PROCESS CAN BE DIVIDED
INTO THREE MULTIPLE STEPS.
ONE IS TO THE LUMBAR
TRANSITIONS, SECOND IS LUMBAR TO
OMEGA TRANSITIONS, HERE IS THE
FROM THE TIME IT LAWM BAR
TRANSITIONS SHOWN FLAT TO LAMBDA
TRANSITIONS, NOTICE SOME SPIKES
PULL THE MEMBRANE FORWARD TO
FORM THIS LAMBDA SHAPED
MEMBRANE.
AFTER DEPOLARIZATION WE SEE.
LET ME SEE IF I CAN PLAY IT
AGAIN.
OKAY.
SO YOU SEE THE SPIKES COMING
HERE.
THIS MEMBRANE PROFILE DOES NOT
PROCEED INTO BECOMING AN OMEGA
SHAPED PROFILE.
SO IT STOPS HERE.
AND BECAUSE OF THIS REASON, YOU
SEE THE PREFORMED IF LAMBDA
EXPHAIPPED OMEGA SHAPED
PROFILE -- SHAPED AND THE OMEGA
SHAPED PROFILE CONDITIONS, THEY
ARE PREFORMED.
ELECTRON MICROSCOPES.
THESE STRUCTURES CAN FURTHER
BECOME A VESICLE AFTER THE
DEPOLARIZATION AS SHOWN IN THIS
EXAMPLE HERE.
THIS IS LAMBDA SHAPE GROWS UP TO
DEPOLARIZATION AND THEN BECOMES
AN OMEGA SHAPE.
HERE SHOWS YOU AN EXAMPLE OF
THIS PROCESS.
SO THIS IS LAMBDA AND FORCES ARE
PUSHING HERE, SO IT BECOMES
OMEGA SHAPE.
THE SMALLER ONES ARE GENERALLY
SMALLER OMEGA PROFILES.
SO THIS SHOWS YOU A PREFORMED
OMEGA SHAPED MEMBRANE CAN CAUSE
THE PORES UP TO DEPOLARIZATION
SHOWN HERE IN THIS EXAMPLE.
SO HERE CLOSES THE PORES REALLY
QUICKLY, FOLLOWED BY THE DYE.
SO THESE ARE THE PREFORMED OMEGA
CLOSURES.
I HAVE SHOWN YOU DIFFERENT
TRANSITIONS FROM ARE FLATD TO
LAMBDA TO OMEGA TO O'S AND
DIFFERENT LAMBDA SHAPES CAN
GENERATE DIFFERENT SIZE OF THE
VESICLES.
ALL RIGHT.
SO I HAVE SHOWN YOU THE LIVE
CELL OBSERVATIONS OF THE FUSION
AND BUDDING, I ALSO INCLUDED THE
COMPOUND FUSION AS SYNAPSE HERE.
NEXT, I WOULD LIKE TO TALK ABOUT
THE MECHANISM, THE MOLECULAR
MECHANICS UNDERLYING THIS
DIFFERENT TRANSITION THAT WE
HAVE STUDIED SO FAR.
THE FIRST IS ENDOCYTOSIS.
FLAT TO LAMBDA TRANSITION, THE
ACTION, BECAUSE IF YOU BLOCK THE
ACTIN, WE SEE THE REDUCTION OR
THE FLAT TO ROUND TRANSITIONS
THEY MATT CLI.
AND WITH K44A WHICH IS NEGATIVE,
IT BLOCKS THE FLAT TRANSITION.
IF YOU LABEL THE ACT INWITH --
ACTIN, YOU SEE THE ACTINS THAT
APPEARS TO PULL THE MEMBRANE
INWARD, IN THIS EXAMPLE.
AS YOU CAN SEE HERE IN THIS
REALTIME.
SO HERE IS ACTEDIN FILAMENTS
HERE AND IT'S VERY THIN RIGHT
NOW, AND AFTER DEPOLARIZATION IT
BECOMES STRONGER AND STRONGER
AND FORMS FILAMENT THAT APPEARS
TO PULL THE MEMBRANE INWARD.
SO IT EVENTUALLY FORMS A
FILAMENT THAT PULLS THE
MEMBRANES INWARD HERE.
WE ALSO LABEL IN RED AND YOU CAN
SEE THE DYNAMIN IN THE BASE OF
THE LAMBDA ANDS HERE IS THE
DYNAMIN IN RED RAE CRUTD TO THIS
GROWING PROFILE -- RECRUITED TO
THIS GROWING PROFILE.
SO WITH THESE RESULTS, WE
PROPOSE THAT THE ACTIN AND
DYNAMIN FORMS BUNDLES TO PULL
THE MEMBRANES INWARD AND FORM
THIS.
SO NEXT QUESTION IS HOW THIS
LAMBDA SHAPE IS CONVERTED INTO
THE OMEGA SHAPE.
WE NOTICE THAT THE DYNAMIN FORMS
THE BASE OF THE LAMBDA SHAPE AND
ALSO THE BASE OF THIS OMEGA
SHAPE.
AS YOU CAN SEE HERE, AND IS THE
XY IMAGES OF THE DYNAMIN
SURROUNDING THE POLAR REGIONS.
SO IT APPEARS THE DYNAMIN
CONSTRICTS THE BASE OF THE
LAMBDA AND THEREFORE TO
GENERATED AN OMEGA BASE.
THIS WILL SHOW YOU A VIDEO OF
THE PROCESS.
DYNAMIN IS IN RED AND
SURROUNDING ALWAYS ON THE BASE
AND THE SIDE OF THE LAMBDA AND
WHY THE LAMBDA IS GOING TO
BECOME OMEGA SHAPE.
SO HERE IS THE DYNAMIN IN RED
THAT SURROUNDS THE BASE AND
BECOMES OMEGA BASE.
IT STOPS HERE SOMEHOW.
SO WE'LL PLAY AGAIN.
SO DYNAMIN IS SURROUNDING AND
APPEARS TO CONSTRICT THE
MEMBRANES.
IF IT BLOCKS WE SEE THE LAMBDA
TO OMEGA TRANSITIONS REDUCTIONS
DRAMATICALLY.
THIS MEANS THE DYNAMIN IS ABLE
TO CONSTRICTED THIS VERY LAST
LAMBDA SHAPE WHICH IS ABOUT 800,
SOMETIMES 800 NANOMETERS.
WHAT WE KNOW ABOUT DYNAMIN IS IT
CAN CONSTRICT ABOUT 10-NANOMETER
PORES.
WHY THIS IS A SURPRISE TO US.
IN CORD WITH NIDHI AND JENNY
HINSHAW, THEY PUT DYNAMIN ALONG
IN VITRO AND CAN CONSTRICT
LYSOSOMES TO BECOMES VERY SMALL,
FROM HUNDREDS OF NANOMETERS TO
ABOUT TENS OF NANOMETERS.
AND HERE IT SHOWS THE
RECONSTRUCTION OF THE DYNAMIN
THAT APPEAR TO SURROUND HELICES,
PROPOSING THAT THE DYNAMIN FORMS
HELICES AROUND THE BASE OF THE
LAMBDA AND CONSTRICTION OF THE
OMEGA PROFILES.
AFTER THAT DYNAMIN WILL CONTINUE
ON CONSTRICTED AND FORCE THE
PORE TO BECOME A VESICLE AS
SHOWN THIS EXAMPLE HERE WITH
DYNAMIN IN RED SURROUNDING THE
PORE REGION AND COME TOGETHER TO
FORCE THE MEMBRANE TO CLOSE THE
PORE, SHOWN HERE IS A REALTIME
VIDEO.
DYNAMIN IS IN RED AND SURROUNDS
THE BASE PORE REGIONS.
MY FILE I GUESS IS TOO LARGE.
SO WE'LL PLAY IT AGAIN.
SO THESE TWO COME TOGETHER, AND
THESE PORES ALSO COME TOGETHER,
SO IT CONSTRICTS.
OKAY.
IF WE BLOCK DIE NAM MINIMUM WITH
THESE TWO -- DYNAMI234 WITH
THESE TWO RESULTS, WE PROPOSE
THAT ACTIN 1K3 DYNAMIN IS
INVOLVED MR. IN CONSTRICTION TO
PULL THE VESICLES WITH.
SO ONLY TWO PORES SUFFICIENT TO
GENERATED VESICLE FROM FLAT
MEMBRANE AND OUR COOPERATOR DID
THE MATHEMATICAL MODELING TO
SHOW THAT ONLY TWO FOLDS ARE
SUFFICIENT TO GENERATE VESICLE,
SO THIS HERE, THE VIDEO SHOWING
A PULLING FORCE HERE, AND LAMBDA
CONSTRICTION FORCE WILL GENERATE
OMEGA SHAPE, SO A VIDEO.
SO NOW IT'S PULLING AND THEN
THEY WILL APPLY CONSTRICTION AND
THEN IT BECOMES OMEGA SHAPE.
SO THIS ACTIN IS INVOLVED IN
CONVERTING THE FAT MEMBRANE INTO
A ROUND -- THE FLAT MEMBRANE
INTO A ROUND VESICLE.
I'VE SHOWN YOU THAT THE
PREFORMED OMEGA CAN BE CLOSED
AFTER DEPOLARIZATION, THIS
ACTUALLY PROVIDES US WITH A
GREAT OPPORTUNITY TO SCREEN THE
FISHER MECHANIC -- THE MISSION
MECHANISMS BECAUSE WE DON'T NEED
TO STUDY HOW THE OMEGA PORE IS
FORMED, THIS IS BYPASSING THAT
PROCESS.
THIS SHOWS YOU SOME OF AGAIN
THESE PREFORMED OMEGA PORE,
WAITING FOR THE DEPOLARIZATION
TO CLOSE THE PORES.
WE SCREEN THE PROTEINS AND WHAT
WE FOUND IS REALLY SURPRISING,
INVOLVED IN CLOSING THE PORE,
VERY WELL KNOWN TO COAT THE
VESICLES, HERE IS AN EXAMPLE 10E
VESICLES, COATED ON THE BODY
HERE.
CLATHRIN.
WHAT IS NOT KNOWN IS IF IT
MEDIATES FISSION.
WE KNOCK DOWN CLATHRIN, AND WITH
THE POLYMERIZATION, WE SEE THE
DRAMATIC DECREASE OF THE OMEGA
PREFORM CLOSURE.
SO CARBON IS -- SO CLATHRIN IS
INVOLVED 2349 FITIONZ PROCESS.
-- SO CLATHRIN IS INVOLVED THIS
IN THE FISSION PROCESS.
SO CLATHRIN IS INVOLVED IN THE
FUSION PORE CLOSURE.
THIS MEANS THAT CLATHRIN COULD
ANTAGONIZE THE FUSION PORE
EXPANSION.
IF THAT'S THE CASE, THEN WHEN
YOU BLOCK CLATHRIN, THE FUSION
PORE EXPANSION WILL BE FASTER,
AND WE DO SEE THAT THE KNOCKDOWN
OF CLATHRIN WILL GENERATE A
FASTER RELEASE.
HERE WE LABEL THE VESICLES WITH
NEUROTRANSMITTER INSIDE VERY
WELL KEHL WITH THIS 511 -- THE
FFN 511 AND AFTER DEPOLARIZATION
CAN BE RELEASED.
SO HERE IS AN EXAMPLE I'LL SHOW
YOU AND KNOCKDOWNS OF CLATHRIN,
IT SPEEDS UP THE VESICLE AND
RELEASE OF TRANSMITTEDDER.
THE QUESTION IS HOW CLATHRIN
CLOSE THE PORE.
SURPRISING RESULTS.
WE LABEL WITH COLORS HERE AND
YOU CAN SEE THE CLATHRIN
SURROUNDS THE BASE OF THE OMEGA
SHAPED PROFILES AND HERE IS A 3D
IMAGING OF CLATHRIN IN MAGENTA
TO SHOW YOU CLATHRIN SURROUNDS
THE BASE OF THE OMEGA PROFILES.
HERE IS THE XY IMAGES ACROSS THE
PORE REGION, MORE EXAMPLES OF
CLATHRIN SURROUNDING THE GREEN
PORE.
SOMETIMES THE GREEN PORE IS TOO
SMALL TO BE RESOLVED BUT WE
STILL SEE THE CLATHRINS.
THIS SHOWS RECENT IMAGING TO SEE
THE INDIVIDUAL CLATHRIN
MOLECULES HERE SURROUNDING THE
REGIONS HERE AS GREEN CONFOCAL
IMAGES, YOU CAN SEE SOMETIMES
THEY ARE VERY CLOSE TO EACH
OTHER, AS IF CLATHRIN IS
POSITIVELY LIMB RIETZED IN
THE --S POLYMERIZED IN THE PORE
REGIONS HERE.
AND WE SEE, THE POLYMERIZED
CLATHRIN AT THE BASE HERE, AND
IT APPEARS TO BE IN THE PORE
REGIONS.
SO IT COULD BE THAT PRELIMINARY
ZIKS OF CLAT -- POLYMERIZATION
OF CLATHRIN.
HERE POLYMERIZATION IS BROUGHT
HERE, AS SHOWN HERE AND UNDER
THESE CONDITIONS, THE FUSION
CLOSURE AND THE PREFORMED OMEGA
PORE CLOSURE IS DECREASED.
SO IT MEANS THAT CLATHRIN
POLYMERIZATION IS REQUIRED TO
MEDIATE THE PORE CLOSURE.
THE MATHEMATICAL MODEL, IT WAS
PRELIMINARY ZIEZED AROUND THE
BASE REGION AND FOUND THE
PROCESS CAN JN RATE CURVE OF
FORCE TO CONSTRICT THE OH
MECHANIC GA PORES, AND PORE IS
UNDERGOING FISSION.
SO WITH THIS WE CONCLUDE THAT
CLATHRIN IS A NEW CLASS OF THE
FISSION ARE, BOLD HYPOTHESIS,
CAN CLOSE THE FISSION PORE AS
WELL AS THE FUSION PORE.
OKAY.
SPEAKING OF THE FUSION PORE, WE
WOULD LIKE TO KNOW THE
MECHANISMS UNDERLYING THE FUSION
PORE DYNAMICS AND BECAUSE OF THE
LIMIT OF TIME, I WILL NOT BE
ABLE TO GO THROUGH ALL THESE
DETAILS, BUT THE BOTTOM LINE
HERE IS THAT THE FUSION PORE
DYNAMICS IS DETERMINED BETWEEN
THE COMPETITION BETWEEN TWO
FORCES, SOMEONE ON THE
RIGHT-HAND SIDE, FUSION PORE
EXTENSIONS, SNARE, ACTIN,
PROMOTES PORE OPEN.
CONSTRICTION, CLATHRIN AND
DYNAMIN WE UNDERSTAND HERE.
AND WHETHER IT STOPS HERE OR
FUSION PORE CLOSURE.
NEXT WE WOULD LIKE TO KNOW WHY.
WE OBSERVE AND COLLABORATED WITH
COMPUTER SIMULATION AND FOWFNED
POSITIVE DIFFERENCE BETWEEN THE
INTRACELLULAR AND EXTRACELLULAR,
SO THIS PRESSURE DIFFERENCE CAN
SQUEEZE THE MEMBRANE TO MAKE THE
VESICLE MEMBRANE CRUMBLE AND
RELEASE THE TENSION.
HIGHER TENSION AT THE PLASMA
MEMBRANE CAN RELIEVE THE LOAD
TENSION OF THE VESICLE MEMBRANE,
CAUSING THE VESICLES TO SHRINK
AND THIS IS THE SIMULATION
RESULT THAT THEY'RE SHRINKING
INSTEAD OF COLLAPSE INTO THE
MEMBRANE.
SO THE MIDDLE PREDICTED THAT IF
YOU DECREASE THE DRIVING SOURCE
BY APPLYING HYPERTONIC SOLUTION,
THEN YOU SEE REDUCTION OF THE
FUSION, AND WE SEE
EXPERIMENTALLY A DRAMATIC
DECREASE AFTER WE APPLY THE
HYPERTONIC SOLUTION.
SO WHEN WE APPLY F-ACTIN, IT
REDUCES THE TENSION HERE AND
BLOCKS THE FUSION AS WE SEE
HERE.
SO THAT'S AGAIN CONSISTENT WITH
THE MODEL'S PREDICTION, AND WE
INCREASE THE DRIVING FORCE BY
APPLYING HYPOTONIC, LAYER
SOLUTION, THEN IT WILL RESCUE
POSSIBLY AT LEAST THE INHIBITION
OF THE FUSION AS SHOWN HERE IN
THIS BLUE BAR HERE.
SO WE CONCLUDE THE TENSION AND
ABILITYIN ARE INVOLVED IN
SHRINKING THE VESICLE.
SO THIS IS THE MECHANISMS
BIOMECHANICS THAT I'VE STUDIED
SO FAR, SHOWN HERE.
IN THE NEXT FEW MINUTES I WOULD
LIKE TO TALK ABOUT SOME
FUNCTIONS OF THESE
TRANSFORMATIONS THAT WE HAVE NOT
BEEN ABLE TO RECOGNIZE BEFORE.
THE FIRST ONE IS THE HEMIFUSION
WHICH CAN ACTUALLY GENERATE --
IT CAN CONTROL RELEASE ASSEMBLY.
ARE SO IT COULD CAUSE RELEASE
FAILURE.
HEMIFUSION TO FULL FUSION IS
DELAYED AS SHOWN HERE, IT
GENERATES ASYNCHRONOUS RELEASE
WHICH HAS NOT BEEN RECOGNIZED
BEFORE AS MECHANISM.
THE OTHER WAY TO GENERATE
ASYNCHRONOUS RELEASE IS BY THE
SEQUENTIAL COMPOUND FUSION, AND
HERE WE'LL SHOW YOU AN EXAMPLE
OF THIS FUSION, VESICLES WITH
THE PHOSPHONEUROTRANSMITTER,
FOLLOWED BY THIS VESICLE RELEASE
AND THIS WILL FORM ASYNCHRONOUS
VESICLES, THIS IS THE GREEN
PROBE THAT WE'LL LABEL THE
MEMBRANES, I'LL SHOW YOU SEVERAL
TIMES HERE.
VERY FAST.
RELEASE AND REPEAT AGAIN.
NO, IT DOES NOT GO.
ONE MORE TIME.
SO NOW IT REPEATS.
OKAY.
SO THIS IS A I SEQUENTIAL COMPOD
FUSION THAT CAN GENERATE SAING
NUS RELEASE.
OF COURSE WHEN THE FUSION PORE
IS NOT RESERVABLE, IT WILL VERY
OFTEN ACCOMPANIED BY THE SLOW
RELEASE SHOWN HERE AND WE CAN
SEE THE FUSION PORE, THEN
RELEASE OF THIS FORCED
NEUROTRANSMITTER WOULD BE MUCH
FASTER.
SO THE FUSION PORE CAN SHOW THE
RATE OF THE RELEASE.
AND ALSO OUR RESULT DEFINED KISS
AND RUN.
KISS AND RUN IS ALWAYS DEFINED
AS VERY FAST FUSION FOR OPENING
AND CLOSING WITHIN THE PORE AND
NOW IT CAN BE ACTUALLY FAST OR
SLOW, IT CAN BE NARROW OR LARGE,
IT ALSO CAN FORM LARGE VESICLES
OR SMALL VESICLES.
IF THE VESICLE ARE LARGE, THEY
CAN CAUSE THE PORE TO FORM LARGE
VESICLES.
IF SHRINK RS CAN CAUSE THE PORE
TO FORM SMALL VESICLES AND
THEREFORE TO CONTROL THE VESICLE
SIZE.
OBSERVATION ALSO SHOWS THE FOCAL
FUSION AS THE DOMINANT
MECHANISMS IN THE FUSION.
ALSO, I WOULD LIKE TO TALK ABOUT
THE ENDOCYTOSIS.
THE ENDOCYTOSIS WE SEE HERE FROM
WHOLE CELL POINT OF VIEW, MOST
PEOPLE WOULD INTERPRET THAT AS
THE FLAT TO O TRANSITIONS AS WE
SEE HERE.
WHAT WE SEE HERE AGAIN IS REALLY
A SURPRISE TOWSES, THAT THE FLAT
TO O TRANSITION IS MINIMAL FROM
AFTER THE DEPOLARIZATION SH
THESE TWO FORMS OF MECHANISMS
ARE THE DOMINANT ONE TO FORM
VESICLES RATHER THAN THE FLAT TO
O TRANSITIONS.
BECAUSE THIS IS A SURPRISE, I
WOULD LIKE TO RECONFIRM THIS BY
RECONSTRUCTING THE POPULATION
EXO AND DOANCYTOSIS FROM
INDIVIDUAL ENDOCYTOSIS HERE.
THIS SHOWS YOU THE CELLS
RECORDING, WHOLE CELL RECORDINGS
HERE OF THE CAPACITANCE DECAYS,
FAST DECAYS, AND EXO HYPHEN
DOUGH FROM EACH INDIVIDUAL --
EXO AND ENDO, EACH OF THE CELLS
WE SEE THE FUSION EVENTS.
IF THERE'S A FUSION EVENT, WE
GIVE UP STEPS.
IF FUSION CLOSURE, WE GIVE DOWN
STEP.
IF NO FUSION CLOASH -- WE SEE
TONS OF THEM IN ONE CELL AND WE
ADD THEM HERE, THIS SHOWS THE
NUMBER OF EVENTS, FUSION OPENING
AND CLOSURES.
SO WE CAN MIMIC THE CAPACITANCE
INCREASE HERE.
WE GIVE A DOWN STEP HERE AND WE
ADD ALL OF THEM TOGETHER, WE SEE
SOME FIVE TO TEN EVENTS HERE,
REFLECTING THE PREFORMED OMEGA
PROFILE CLOSING EVENTS.
THEN WE ADD ALL THIS TOGETHER
AND WE GET THE RED THAT SHOWS
RESEMBLES CAPACITANCE RECORDED,
AND WE SEE THE FUSION CLOSURE
AND PREFORMED OMEGA CLOSURE IS
RESPONSIBLE FOR THE SMALL
ENDOCYTOSIS, FAST AND
COMPENSATORY ENDOCYTOSIS.
RATHER THAN THE FLAT TO ROUND.
SO FLAT TO ROUND IS NOT THE
PRIMARY MECHANISM, RATHER IT'S
THE TWO HERE, FUSION CLOSURES
AND OMEGA.
SO I'VE SHOWN YOU VERY QUICKLY
20 YEARS LAB IN VISUALIZING THE
FUSION AND BUDDING
TRANSFORMATIONS MECHANISMS AND
FUNCTIONS WE HAVE STUDIED SO
FAR, AND THIS IS QUITE DIFFERENT
FROM THE CLASSICAL MODEL THAT WE
STARTED WITH.
FINALLY, I WOULD LIKE TO THANK
MANY, MANY PEOPLE INVOLVED IN
THIS WORK.
[READING NAIMEDZ]
INVOLVED IN ESTABLISHING THE
WHOLE CELL AND CELL, MANY PEOPLE
HERE, VERY IMPORTANT
CONTRIBUTIONS TO OUR MANY YEARS
OF STUDIES HERE.
[READING NAMES]
I WANT TO SINCERELY THANK.
AND MANY OTHERS ARE INVOLVED IN
THE IMAGING EXPERIMENTS.
PROVIDED MANY OF THE VIDEOS, AND
THESE ARE MY LAB MEMBERS.
I WOULD ALSO LIKE TO THANK
CAROLYN SMITH FOR HER SUPPORT
AND MANY OF THE COLLABORATIONS I
MENTIONED IN MY TALK.
ALSO HUGE THANKS TO NINDS FOR
REALLY STRONG SUPPORT.
I ALMOST CANNOT IMAGINE ANY
OTHER PLACE THAT WILL ALLOW ME
TO TRANSFORM ELECTROPHYSIOLOGY
INTO AN IMAGING IN THE FRONT.
SO IMPORTANT, HUGE THINGS TO
NINDS, AND ALSO FINALLY THANK
YOU VERY MUCH FOR YOUR ALL
ATTENTION.
[APPLAUSE]
>> HI.
CAN YOU HEAR ME?
>> YES.
>> I HAVE PERHAPS A NAIVE
QUESTION, BECAUSE I KNOW NOTHING
ABOUT VESICLE FUSION, BUT WHAT
IS THE MECHANISM FOR THE
COMPOUND VESICLE FUSION?
BECAUSE I CAN UNDERSTAND LIKE
THE NORMAL VESICLE FUSION
INVOLVES, THE FORCE REQUIRED BY
THE SNARE COMPLEX AND CALCIUM.
>> YEAH.
THAT'S A VERY GOOD QUESTION.
I ACTUALLY DO NOT KNOW.
BUT WHAT WE KNOW IS THAT WHEN WE
KNOCK OUT TECH 1 -- SNARE 2 I'M
SORRY, WE SEE A DRAMATIC
REDUCTION OF THE COMPOUND
FUSIONS.
SO IT MEANS THAT IT WASN'T
REQUIRED CALCIUM SENSORS, AND
YOU'RE ASKING WHETHER THE
SNARES, I BELIEVE IT'S INVOLVED,
AND VESICLE HAS NOT BEEN KNOWN
TO CONTAIN SYNTACTIC SNARES BUT
I THINK SOME OF THEM SHOULD BE
THERE, OTHERWISE -- I CANNOT
IMAGINE WITHOUT SNARE THEY WOULD
FUSE.
>> A RELATED QUESTION ABOUT THE
PULLING FROM THE LAMBDA TO OMEGA
TRANSITIONS, I'M ASSUMING THE
ACTIN FILAMENT WOULD HAVE TO LET
GO OF THE VESICLE, OR DOES THAT
STAY ATTACHED AND THE PULLING
FORCE JUST CONTINUES?
12K3W4R50 IT'S A MODEL BECAUSE
WE SEE THE ACTIN FILAMENTS
THERE, AND IT APPEARS TO PULL
MEMBRANES, BUT WE DON'T KNOW
WHERE THE ACTIN HOLDS.
MAIB THE MICROTUBULE OR
SOMETHING AND HOW THE ACTIN
FILAMENT WOULD PULL THE
MEMBRANE, WE DO NOT KNOW AT THIS
POINT.
SO IF ANYONE HAS GOOD IDEAS, I
WOULD LOVE TO TAKE IT.
>> THANK YOU.
>> AGAIN.
THANKS FOR THAT.
THAT'S AN AMAZING ARC OF
ACHIEVEMENT OVER A LONG TIME.
IT'S JUST AMAZING TO SEE.
I THINK WE TAKE FOR GRANTED
PRETTY IMAGING PICTURES THESE
DAYS, BUT THE ACHIEVEMENT IS
AMAZING.
>> THANK YOU.
>> AS YOU ALLUDED TO, THE
ELECTROPHYSIOLOGY WASN'T SO EASY
EITHER.
IT'S VERY CLEVER HOW YOU TOOK
ADVANTAGE OF THE BLEACHING OF
THE RED DYE, AND TO SHOW THAT
THE VESICLE HAD CLOSED OFF.
IT GOT ME THINKING ABOUT THE
OTHER IMAGING THAT YOU WERE
DOING.
IS THERE ANY OTHER BLEACHING
GOING ON?
I MEAN, YOU HAVE THE DYNAMIN
WHICH IS IN THE SAME SORT OF
AREA, THE SPECTRUM, PRESUMABLY
FORMING A STRUCTURE AROUND, BUT
THAT DOESN'T BLEACH?
>> WE HAVE TO USE A VERY DIM
LIGHT SO THERE'S A BALANCE.
IF IT'S A STRONG LIGHT, THEN
VISION IS HIGHER BUT THEN YOU
BLEACH BEFORE YOU CAN SEE IT.
SO WE USE VERY STRONG INCREASE
OUR POWER TO 80% TO BLEACH THEM
VERY QUICKLY.
OTHERWISE, WE HAVE TO -- THE
DYNAMIN, IT'S MUCH LESS
BLEACHING, SO THEY ARE STILL
THERE AND SHOULD BE FINE.
YEAH.
>> SO JUST RELATED TO THAT, AND
I'M SORRY, YOUR TRICK THAT WAS
MAKING ME THINK ABOUT THIS.
BUT WHEN YOU SHOWED THAT THE
VESICLE KIND OF DISAPPEARS, IS
IT POSSIBLE THAT YOU COULD BE
RESTRICTING THE FLOW OF THE
LABEL OF IP3 OR WHATEVER IT IS
TO THAT AREA AND IT'S JUST
BLEACHING, OR --
>> IS IT POSSIBLE?
SO WHEN THE DYE IS BLEACHING, WE
THINK IT'S BECAUSE IT'S CLOTHED
SO WE COULD ALSO -- SO YOUR
QUESTION IS, IS IT POSSIBLE THA-
>> I WAS WAITING TO SEE A
VESICLE THAT HAD BEEN FULLY
PINCHED OFF AND I JUST DON'T
REMEMBER SEEING THAT.
I WAS WONDERING WHETHER THAT
WOULD BLEACH VERY QUICKLY, AND
IF SO, COULD A SIMILAR THING
EXPLAIN YOUR DISAPPEARING --
>> YEAH, VERY GOOD POINT.
SO WHEN WE SEE THE KISS AND
RUNS, WE ALWAYS ASSUME THAT THE
VESICLE IS DIGGING AWAY AND WE
TRY VERY HARD AND WE DON'T SEE
THEM, THEY ALL STAYED THERE,
SOME STRUCTURE STILL ATTACHED.
WHAT WE SEE IS THE DYE IS
BLEACHING -- THE VESICLE
MEMBRANE IS STILL THERE AND DYE
IS BLEACHING.
SO THE VESICLE STAYS THERE.
I HAVEN'T SEEN ONE IMAGE TO --
SO HERE IS IMAGING, I THINK THIS
IS THE CLATHRIN HERE AND
UNDERNEATH HERE, AND AFTER
DEPOLARIZING, WE DON'T SEE THEM
FOR THE LARGE VESICLE.
LARGE VESICLES, THEY STAY THERE
FOR SOME TIME.
OCCASIONALLY WE SEE THAT DATA
BUT VERY FEW, AND I DON'T KNOW
WHY.
>> (AWAY FROM MICROPHONE) AROUND
THE VESICLE?
>> THAT SUGGESTED CLATHRIN, THIS
ONE, YEAH, COULD BE CLATHRIN,
THIS BINDS TO CLATHRIN AS WELL
AS THE LIPID PITP, AND THEN IT
DISAPPEARS.
>> SO MUCH FOR YOUR BEAUTIFUL
TALK.
-- THANK YOU SO MUCH FOR YOUR
BEAUTIFUL TALK.
I WAS THINKING ABOUT THE
CLATHRIN YOU SHOWED BENEATH THE
VESICLE.
THIS IS A NEUROSCIENCE SEMINAR
SERIES, SO DO YOU THINK WE'RE
JUST MISS GOING IN NEURONS AND
THAT IT'S ALSO PRESENT AT THE
PORE IN NEURONS, SYNAPTIC AS
WELL?
>> VERY GOOD POINTED.
THE QUESTION IS WHETHER THE
CLATHRIN FOR THE SMALL VESICLE
RS THERE ALSO IN THE BASE OR NOT
1234 I DO NOT KNOW.
BUT THE SIMULATION A LONG TIME
AGO IN 2003, 20 YEARS AGO,
SHOWING THAT CLATHRIN ON THIS
COATED AREA CAN ALSO CONSTRICT
THE MEMBRANE, SO THEY DON'T NEED
TO BE ON THE BASE.
IF THEY ARE ONLY AT THE HEAD
THEY CAN ALSO CONSIDER THE PORE,
SO ALSO IT'S SHOWN HERE THAT TH,
SHOWING THE PIT CAN ALSO BE --
THE CLATHRIN CAN BLOCK THE VERY
WELL KEHL PINCH SO I THINK IT'S
ALSO INVOLVED IN THE SMALL
VESICLES PINCH PROCESS.
>> HI LING GANG.
THANKS FOR THE GREAT
PRESENTATION.
I THINK YOU BROKE THE WORLD
RECORD ON AVERAGING.
I'VE NEVER SEEN A SIGNAL WITH
300,000 AVERAGING, SO THANK YOU
SO MUCH FOR THE BEAUTIFUL WORK.
I HAVE A COUPLE QUESTIONS.
ONE IS THAT SOME OF THIS
MOVEMENT -- HOW DO YOU DEAL WITH
MOVEMENT IN THE SORT OF XY
DIRECTION?
YOU KNOW, DO THINGS BASICALLY
STAY THE SAME?
1K3 THEN THE SECOND THING IS, I
THOUGHT THAT THE MODEL WITH THE
OSMOLARITY WAS
SUPER-INTERESTING.
SO DOES THAT MEAN THAT INSIDE
THE VESICLE YOU PRODUCE LIKE A
HYPOTONIC -- HOW IS THAT REALLY
WORKING?
SO YOU HAVE SOMETHING THAT'S
HYPOTONIC INSIDE SO THAT THE
SOLUTION IS MORE LIKELY TO
LEAVE?
THE CONTROL FOR SORT OF MAKING
THE VESICLE SMALLER USING THE
OSMOLARITY INSIDE THE VESICLE,
INSIDE THE CELL, INSIDE THE
VESICLE, I'M ASSUMING 1234.
>> IT'S SOLUTIONS.
SO IT'S THE SECOND QUESTION.
SO WE APPLY THE DIFFERENT OSMO
SOLUTION OUTSIDE THE CELL.
WE HAVE THE IMAGES I DID NOT
SHOW YOU, BUT THE CELLS CRUMBLE
AND TENSION IS LOSE, WE ALSO
MEASURED THE TENSION AFTER THAT
AND WE SEE THE TENSION REDUCED.
THEN WE APPLY THE HYPOTONIC,
WHERE IT GETS INTO THE CELL AND
WE SEE THE CELL SWELLING AND THE
TENSION IS HIGHER.
WE MEASURED THE TENSION WITH
PIPETTE ASPIRATION AND SO I
THINK IT SHOULD BE RIGHT, WE
HAVE EVIDENCE TO SUPPORT.
>> SO YOU THINK THAT'S A NORMAL
MECHANISM BY WHICH HOW THE
VESICLE GETS SMALLER?
>> MY VESICLE GETS SMALLER.
IT CHANGE THE TENSIONS.
SO THE TENSION AT THE MEMBRANE,
PLASMA MEMBRANE IS HIGH.
NOW, IF YOU REDUCE THE MEMBRANE
TENSIONS, THEN THIS LOWER
MEMBRANE TENSION, THE -- THE
VESICLE MEMBRANE, TO LOWER THIS
VESICLE MEMBRANE, YOU NEED
HIGHER MEMBER TENSION, SO
THERE'S A DIFFERENT TENSION AT
THE PLASMA MEMBRANE AND THE
FUSION VESICLE AND THAT PROVIDES
THE DRIVING FORCE TO LOWER THE
MEMBRANE TENSION, LIKE WATER
FORCING INTO THE FOLD, ACCORDING
TO THE SIMULATION.
I'M JUST TRANSLATING MY
COLLABORATORS.
AND FOR THE FIRST QUESTION --
>> (AWAY FROM MICROPHONE).
>> MY FIRST TWO PAPERS, I GOT
THIS QUESTION, AND WE HAD TO
SPEND HALF A YEAR TO ONE YEAR TO
ADDRESS THAT PARTICULAR
QUESTION.
THE QUESTION IS WHETHER WHAT WE
SEE IS SIMPLY THE MOVEMENT AWAY.
IT'S NOT BECAUSE XY AND XZ AND I
DO NOT HAVE THE DATA HERE TO
SHOW YOU, BUT WHAT WE'RE TRYING
TO DO IS DO THE 3D'S, INSTEAD OF
SINGLE, SO EVEN THOUGH THEY ARE
MOVING, WE WILL KNOW.
THE ANSWER IS THEY ARE NOT THE
MOVEMENTS.
BUT BECAUSE THEY CAN BE BLOCKED.
IF THEY ARE MOVEMENT, IT CANNOT
BE BLOCKED BY ANYTHING.
AND THEN OTHER 3D MEASUREMENTS.
THANK YOU VERY MUCH.
[APPLAUSE]