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>> 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