>> GOOD AFTERNOON, EVERYONE. MY
NAME IS CHRIS ALEXANDER THE
ASSOCIATE SCIENTIFIC DIRECTOR
FOR NIHLBI DIR AND -- CHIEF OF
THE LABORATORY STRUCTURAL
CELLOL
BIOLOGY -- TOKYO IN 1999 AND
RECEIVED PHD IN BIOPHYSICS FROM
UNIVERSITY OF TOKYO AND
SOUTHWEST MEDICAL CENTER IN 2005
AND PRIOR TO COMING TO NIH WAS
AN INDEPENDENT GROUP LEADER IN
CHEMISTRY IN GERMANY BEFORE
MOVING HERE TO NIH DURING THE
PANDE
PANDEMIC.
KRIO EM IS A CUTTING EDGE
TECHNIQUE THAT BRIDGES
RESOLUTION GAP BETWEEN LIGHT
MICROSCOPY AND -- LAB USES
INSITUCIO ELECTRO-TOMO GRAPHY
AND CRYSTALO GRAPH IN VITRO
INSTITUTION AND LIGHT MICROSCOPY
AND HOW THEY MAY ACTIVATE
REGENERATE SWITCH AND DR.
MAZUNO'S PRESENT TATATION IS
TIT
TITLED.
>> HELLO, EVERYONE FOR COMING
HERE AND CHEERING ME UP HERE AND
I AM APPOINTED WITH NIAMS AND
TODAY I WANT TO INTRODUCE MY
SCIENCE TO THE COMMUNITY.
TITLE IS INTEGRATIVE STRUCTURE
OF CELL BIOLOGY AND CHRIS
MENTIONED I HAVE A LITTLE OF A
SPECIAL OR UNIQUE BACKGROUND AND
MY LAB WAS FOUNDED IN GERMANY IN
MAX INSTITUTE OF BIOCHEMISTRY IN
MUNICH. THIS IS ACTIVITY
OCTOBER 1ST THAT WAS A RETREAT
AND QUITE A FUN TIME OVER THERE.
BUT I ACTUALLY DID POST GRAD NIH
AND REALLY LIKED THE CULTURE AND
HOW THINGS WORK HERE AND REALLY
DREAMED OF COMING BACK TO NIH.
WE MADE A DECISION TO MOVE TO
NIH IN 2019 AND MOVE WAS TO 2020
WHICH WAS A LITTLE DIFFICULT.
I WILL TELL YOU ABOUT MY
PANDEMIC MOVE LATER.
THIS IS THE LAB IN 2024 AND
RECENT PICTURE AND EVERYTHING IS
SMOOTH AND NICE AND THANKS TO
EVERYBODY WHO HELPED US TO
ESTABLISH OR COMPLETE THE MOVE
THE TECHNOLOGY IS INTEGRATED
BIOLOGY AND QUESTION APPROACH
LOOKING AT THINGS WITH
TECHNOLOGY AND I MYSELF IS
ORIGINALLY KRIO
ELECTROMICROSCOPY SINCE 1999 AND
BACHELORS MATHEMATIC STUDENT I
DID IMAGE ANALYSIS AND YOU CAN
SEE HERE THIS IS A TYPICAL KRIO
EM STRUCTURE THAT YOU CAN GET.
SO, WE STILL WELCOME THIS KIND
OF A STRUCTURE AND IT IS A SUPER
FINE STRUCTURE ANALYSIS AND OVER
TIME MY AIM OF RUNNING A LAB IS
TO [INDISCERNIBLE] PEOPLES
CREATIVITY AND SETTING UP
INFRASTRUCTURE SO PEOPLE CAN
WORK WITH THEIR MIND AND
CREATIVITY.
SO YOU CAN SEE THIS IS A
MOLECULAR ORGANIZATION
OBSERVATION IN A CELLULAR AND
CELL MORPHOLOGY AND MOVING TO
TISSUE ORGANIZATION INVIFO THAT
IS DIFFICULT FOR US AND WE HAVE
COLLABORATION AND THIS IS
BEAUTIFUL AND YOU SEE ELEGANCE
IMAGE AND [INDISCERNIBLE] BY
KEVIN'S LAB AT NIDDK A WONDERFUL
COLLABORATION TO HAVE A REALLY
COMPREHENSIVE UNDERSTANDING WHAT
WE WANT TO SEE.
OKAY. SO TALKING ABOUT IMAGING,
I WANT TO TELL YOU A LITTLE
ABOUT THE TECHNOLOGY DEVELOPMENT
THAT IS HAPPENING IN THE IMAGING
FIELD.
YOU CAN SEE HERE THIS IS THE
SIZE OR THE STRUCTURE RESOLUTION
RANGE THAT YOU CAN ASK BY
MICROSCOPIC TECHNIQUE AND THIS
IS CONTEXT YOU CAN ASK.
IF YOU WANT TO SEE A LARGE
OBJECT, YOU WOULD GO WITH A
CONVENTIONAL ELECTROMICROSCOPE
OR LIGHT MICROSCOPE TO GET AN
OVERVIEW.
HOWEVER, YOU ARE MISSING
MOLECULAR RESOLUTION YOU CAN SEE
HERE IS MISSING AND TO
COMPLEMENT THAT YOU WOULD GO
WITH CRYSTALO GRAPHY OR NMR TO
GIVE ATOMIC RESOLUTION
INFORMATION.
HOWEVER, YOU CAN SEE HERE
IN-BETWEEN THIS IS MISSING UNTIL
2014 WHEN TECHNOLOGICAL
REVOLUTION WE CALL RESOLUTION
HAPPENING IN E M FIELD.
YOU CAN SEE HERE, NOW THE SINGLE
PARTICLE CRYO-EM AND MOLECULE
AND IMAGE ANALYSIS AND GETTING
NEAR ATOMIC RESOLUTION
INFORMATION OR RELATIVELY LARGE
OR LARGER CONTEXT MOLECULE SUCH
AS MICROMOLECULES SUCH AS
COMPLEXES AND FURTHERMORE,
CURRENT REVOLUTION IS HAPPENING
WITH THE INSITU KRIO
ELECTRO-TOMO GRAPHY AND GETTING
CONTEXTUAL VIEW LOOKING AT CELL
OR MOLECULES IN THE CELL TO
HOPEFULLY NEAR OUGHTONOMIC
RESOLUTION THAT IS ONGOING AND
CONCEPTION THING THAT WE ARE
DOING AND WE ARE GETTING SLOWLY
BUT SURELY MOVING TOWARDS THAT
DIRECTION.
AND TALKING ABOUT INTEGRATIVE
STRUCTURE BIOLOGY, WE WANT TO
GIVE ANOTHER DIMENSION AND NOT
ONLY THIS SPECTRUM BUT WE WILL
ADD MORE FACTORS HERE.
FOR EXAMPLE, WE CAN TALK ABOUT
DYNAMICS AND INVIFO IMAGING TO
BE COMBINED WITH THESE STATIC
TECHNOLOGIES OR DISEASE CONTEXT
WE CAN INCORPORATE OR FURTHER
MORE HOW WE CAN LEARN OR APPLY
THE CONTEXT OF WHAT WE HAVE DONE
WITH THE APPLICATION. FOR
EXAMPLE, TRANSLATIONAL ASPECT OF
IT.
SO, WITH THAT SAID I WANT TO
TELL WHAT WE ARE DOING AND BIG
APPROACH QUESTION WE TRY TO
UNDERSTAND IT IS A DECISION THAT
IS A FORMATION THAT IS CONNECTED
TO DECISION MAKING PROCESS.
YOU CAN SEE HERE FOR EXAMPLE
THIS IS A PLATELET THAT IS
ACTIVATED IN THE CONTEXT AND THE
WOUND IS DETECTED AND ACTIVATION
WOULD HAPPEN AND CELL SHAPE
CHANGES OR SEE DIVIDING CELL
THAT IS COORDINATING AND SO THAT
THE COMPONENTS ARE DIVIDED
EQUALLY AND IT GETS SHAPE
CHANGES AND DIVISION HAPPENS.
MOST PROMINENT EXAMPLE YOU CAN
SEE WITH CELL SHAPE CHANGE IS
NEURONAL CELL AND NEURON IS MOST
POLARIZED CELL WE KNOW TO HAVE A
METER LONG AXON AND FROM ONE
SIDE TO THE OTHER SIDE OF THE
NEURON FUNCTION IS COMPLETELY
DIFFERENT AND EXAMPLES WE WORK
ON AND NOT ONLY THAT IN HOMEO
STASUS INFORMATION IS DISTURBED
AND PROBLEM CANCER CELL
FORMATION OR SICKLE CELL
FORMATION AND APPLICATION YOU
CAN TRY TO MAKE REGENERATE.
THESE ARE THINGS NEW CHALLENGE
FOR OUR LAB WE CAME FROM BASIC
SCIENCE LAB AND BASIC SCIENCE
INSTITUTE IN GERMANY AND MOVED
TO NIH AND IS OPPORTUNITY TO
ALSO ADDRESS HUMAN HEALTH
PROBLEM.
OKAY. SO NOW HOW WE DO IT, YOU
CAN SEE FROM THE LEFT SIDE TO
THE RIGHT SIDE THAT WE CALL TOP
DOWN, WHICH MEANS WE START TO
LOOK AT LARGE CONTEXT AND TRY TO
REALLY GO USING A MICROSCOPE
INTO THE MOLECULAR VIEW AND
CRYOELECTRON TOMMO GRAPHY
RECONSTRUCTION OF THE POLL HERE
AND ALSO OUT GROSS CON OF THE
NEURON AND TO, FOR EXAMPLE,
SHOWING EXAMPLE OF PLAYERS DOING
THE [INDISCERNIBLE] INFORMATION
AT TIP OF THE GROWTH
[INDISCERNIBLE] OR BOTTOM UP
INSTITUTION MEANS WE CAN COLLECT
PARTICLES OF INTEREST AND TRY TO
UNDERSTAND EACH REGULATION OF
EACH OF THE COMPLEXES OF
MOLECULES AND TRY TO
RECONSTITUTE INTO THE FUNCTIONAL
UNITS SO THINGS START TO MAKE
SENSE FUNCTIONALLY.
SO, NOW, THIS IS AN EXAMPLE OF
BOTTOM UP CONSTITUTION. I WON'T
TALK ABOUT BOTTOM UP
CONSTITUTION TOO MUCH HERE
TODAY.
YOU SEE THESE ARE PROTEINS OR
MOLECULES WE HAVE WORKED ON FOR
THE LAST 10 YEARS AND YOU CAN
SEE THESE HAVE ITS OWN
INTERESTING THINGS GOING ON AND
WHEN WE KNOW WHAT REGULATION OF
PROTEINS ARE WE CAN COMBINE THEM
ALSO.
THIS IS AN EXAMPLE OF THIS IS
INTEGRATING AN IMPORTANT
RECEPTOR PROTEIN DECIDING THE
DECISION OF THE CELL.
YOU SEE THE PLASMA MEMBRANE AND
EXTRA CELLULAR LYINGON THAT IS
ACTIVATING IT FROM INSIDE OF THE
CELL YOU SEE THIS IS TAILING
WHICH IS INTRACELLULAR ACTIVATOR
OF [INDISCERNIBLE] THAT IS
POKING FROM INSIDE OF THE CELL
AND IS ATTACHED BY BOUNDING OF
ACTIN AND MAKING THIS FORMATION
AND WE TRY TO RECONSTITUTE SUCH
STRUCTURE USING IN VITRO BOTTOM
UP RECONSTITUTION.
YOU SEE HERE WE CAN LOCK THE
INTERMEDIATE PROCESS AND TO
REALLY UNDERSTAND MOLECULAR
ORGANIZATION AND MECHANISM OF
HOW ACTIVATION WOULD BE
HAPPENING.
SO THIS IS A STRENGTH OF IN
VITRO RECONSTITUTION WE HAVE
FULL CONTROL OF THE MOLECULE OF
INTEREST.
NOW, NOT ONLY IS THE STRUCTURE
PART, WE CAN ALSO LOOK INTO
DYNAMIC ASPECT OF IT.
THIS EXAMPLE I WANT PT TO SHOW
YOU IS THESE PROTEINS
ENCAPSULATED INSIDE OF THIS
VESICLE THAT IS MIMICKING
INTERMEMBRANE SURFACE OF THE
CELL AND SEE BASICALLY THE GOAL
OF THIS WAS TO ACTIVATE THE
COMPLEX ENOUGH OF ACTIN SO THIS
EXPERIMENT COULD BE PERFORMED ON
PERIPHERY OF THIS.
THIS IS A COMPLETE VIEW OF
CONFIRMATION OF IN VITRO
RECONSTITUTION.
NOW, I'M GOING TO FOCUS MY STORY
TO THE TOP DOWN AND INTRODUCE
MORE DEEP ABOUT THE RESEARCH
THAT IS GOING ON.
SO, BEFORE TELLING YOU ABOUT THE
TOP-DOWN APPROACH I WANT TO
INTRODUCE ABOUT CRYOELECTRO-TOMO
GRAPHY A NEWLY EMERGING
TECHNIQUE I TOLD YOU ABOUT
ALREADY. THIS IS A VIDEO CLIP
THAT INTRODUCE THE TECHNOLOGY
AND THIS IS YOUR CELL OF
INTEREST AND MOLECULE ANYTHING
THAT COULD BE IMBEDDED TO EM
GRADE. SORRY I WILL TAGAIN.
THIS TINY LITTLE THING CAN GO
INSIDE OF A HUGE MICROSCOPE AND
MAKING A [INDISCERNIBLE] SERIES.
IT IS ACUTING INSIDE OF THE
MICROSCOPE AND THIS IMAGE CAN BE
COMBINED IN TWO 3-D BY BACK
PROJECTION AND YOU SEE WE GET
3-D RECONSTRUCTION AND ADVANTAGE
OF CRYO-TOMO GRAPHY IS YOU LOOK
INTO DENSITY DIFFERENCE BETWEEN
PROTEIN AND LIPID TO WATER
DENSITY SO YOU REALLY GET
EVERYTHING THERE.
YOU COULD SEE THIS PRETTY
ENVELOPE BUT NOTHING INSIDE.
OKAY. SO TOP-DOWN
VISUALIZATION, I WANT TO TELL
YOU TWO STORIES THAT ARE GOING
ON IN IN RECENT YEARS AND IN
PRESENCE OF SARS CO-V2 AND THIS
IS IN OUR LAB AND SO FAR A LOT
AND I WILL TELL YOU SOMETHING
ABOUT IT.
SECTION IS NEWEST STORY ABOUT
[INDISCERNIBLE] RESPONSE AND
REGENERATION.
WHAT HAPPENED IS WE WENT TO
GERMANY 2020 AND FEBRUARY WE
PACKED EVERYTHING AND WAS ABOUT
TO SHIP IT.
IT COULDN'T BE SHIPPED BECAUSE
OF THE LOCK DOWN AND 7 MONTHS WE
DIDN'T HAVE ANYTHING AND
AFTERWARDS WE HAD EQUIPMENT IN
THE BUILDING AND CONTRACT TO
MOVE SO AND DIFFICULT TIME FOR
US AND FINALLY TRIED TO FETCH
STUDENTS FROM GERMANY TO UP AND
WAS IMPOSSIBLE FOR ARRANGING THE
VISA AND EXCEPT INVESTIGATOR
GAVE VISA TO COVID RESEARCH AND
EVERYTHING WAS SAID FOR US TO
WORK ON COVID RESEARCH AND THIS
IS STUDENT THAT WORKED ON
PROJECT.
OKAY?
NOW, JUST AS A SUMMARY WHAT WE
DIDN'T HAVE IN THE LAB WHEN WE
STARTED ALMOST EVERYTHING AND
BITS AND PIECES OF EVERYTHING
AND PLASMA WAS PACKED AND LOST
PROTEINS WERE LOST AND
EVERYTHING WAS LOST AND WE HAD
PERMISSION TO WORK ON COVID
PROJECT AND THANKS TO DONOR AND
TRIED WE COULD USE AND SPIPROTE
WE COULD BUY AND INSTRUMENT WE
COULD USE INCLUDING CRYO-EM AND
LOOKING WHAT WE COULD CONTRIBUTE
TO THE HEALTH CRISIS, YOU SEE
HERE I DON'T THINK I NEED TO
EXPLAIN MUCH ABOUT COVID AND WHY
IT IS SO DEVASTATING.
ONE THING WE KNOW IS ACTIVATION
OF THE PLATELET COULD BE A BIG
PROBLEM AND COVID WHEN IS VERY
SEVERE YOU GET CLOTTING OF THE
BLOOD AND WE THOUGHT WE COULD
WORK ON THE PLATELET ON THE
SPECIAL BACKGROUND OF THE
[INDISCERNIBLE] AND KNOW WHAT IS
HAPPENING WITH THE RECEPTOR AND
WHAT IS HAPPENING WITH THE
ACTIVATION PRO S PROCESS AND
DECIDED TO WORK ON THAT AND
SPIKE PROTEIN STRUCTURE AND
NOTICE IT QUICKLY AND COULD GET
NICE STRUCTURE AND GOOD QUALITY
CONTROL FOR US.
THEN WE LEARNED HOW TO PURIFY OR
EXTRACT THE PLATELET OUT OF THE
BLOOD AND MIX IT TOGETHER AND
THAT IS AN S PROTEIN ON THE
PLATELET.
NOW YOU CAN SEE HERE THIS IS A
LIGHT MICROSCOPIC ANALYSIS OF
THE PLATELET OF THE CONTROL AND
YOU SEE MORE OF THESE LEADERSHIP
ARE NOT MUCH CHANGING AND
HAPPILY AROUND AND SEE ACTIVATED
ON THE SURFACE AND IN THE
PRESENCE OF THE COVID SPIKE
PROTEIN YOU CAN SEE LOTS OF THE
ACTIVATION ACTUALLY ARE GOING
ON.
SO THIS YOU CAN SEE VISUALLY
ALREADY THAT ACTIVATION WAS
HAPPENING IN THE PRESENCE OF THE
S PROTEIN AND MORE
INTERESTINGLY, WE COULD SEE A
LOT OF SPIKE -- A LOT OF
PLATELETS THAT GET ATTACHED TO
ONE SIDE OF THE ECM AND START TO
PROVIDE MICROACTIVATED AND START
TO GET STRETCHED THAT YOU CAN
SEE HERE.
THIS WAS QUITE INTERESTING
MORPHOLOGY THAT WE SAW.
WE QUANTIFIED IT AS YOU CAN SEE
IN THE PRESENCE OF THE SPIKE WE
REALLY SEE A LOT MORE OF THIS
STRETCHED MORPHOLOGY.
THIS WAS ALSO DOSE-DEPENDENT
MEANING THERE IS DIRECT
CORRELATION BETWEEN PRESENCE OF
SARS CO-INDIVIDUAL FORMATION ON
THE PLATELET.
WE DECIDED TO HAVE A LOOK AT
WHAT IS GOING ON THERE USINGCIO
EM AND HERE WE PREPARE A
PLATELET MIXING WITH SPIKE
PROTEIN TO MAKE CRYO-EM GREAT
[INDISCERNIBLE] AND HAVE A LOOK
AT TOMMO GRAPHY AND YOU SEE HERE
MEDIUM SNAPSHOT TO VISUALIZE
OVERVIEW OF THE PLATELET AND YOU
CAN SEE HERE THIS IS A COLLAGEN
FIBER AND THIS IS ACTUALLY THE
DISTORTED PLATELET THIS IS A
CONTROL AND SEE THE EFFECT OF IT
AND THERE IS DIRECT
VISUALIZATION WE CAN DO.
HERE YOU CAN SEE THIS IS THE
AREA WE COULD COLLECT
TOMOGRAPHY.
SO THIS IS ONE EXAMPLE OF
HUNDREDS OF TOMMO GRAMS THAT WE
TAKE AND YOU SEE HERE THIS IS
THE PLATELET AND FEEDO PODIUM
HAPPENING WITH ACTIVE STRESS
FIBER. YOU SEE HERE THE DIRECT
ATTACHMENT OF THE SPIKE PROTEIN
ON THE SURFACE OF THE MEMBRANE.
WE CAN REALLY SEE QUITE A BIT OF
THE S PROTEIN ATTACHED ON THE
MEMBRANE AND IT IS CAUSING THE
DEFORMATION.
NOW, YOU MIGHT WONDER IF THIS IS
A [INDISCERNIBLE] AND SURFACE
RECEPTORS ARE MOSTLY INTEGRATED
ON THE PLATELET AND LOOKING AT
CONTROL, YOU DON'T SEE ANY OR
NOT MUCH OF THE PROTRUSION OUT
OF THE MEMBRANE SURFACE AND MUST
BE THE PROTEIN THAT IS ATTACHED
FROM THE OUTSIDE.
SO TO VALIDATE THAT, WHAT WE DO
IS CALLED SUB-TOMO GRAM
AVERAGING AND COLLECT DENSITY
AND 1,284 PARTICLES AND DID
[INDISCERNIBLE] OF IT.
IF YOU DO SOMETHING SIMILAR,
LARGER AMOUNT ON TO-DO SINGLE
PARTICLE WAY YOU WOULD GET
ATOMIC STRUCTURE THAT I HAVE
SHOWN YOU ALREADY.
WE HAVE TOMOGRAPHY AND
RESOLUTION THAT IS A LITTLE
COMPROMISED BUT GOOD ENOUGH TO
IDENTIFY WHAT IT IS.
SO AFTER RECONSTRUCTION, YOU CAN
SEE HERE THIS IS THE DENSITY
THAT WE GOT.
SO THIS SHOWS THE VERY TYPICAL
TIME ARRANGEMENT OF THE SPIKE
PROTEIN AND HERE ALSO YOU CAN
SEE THAT OUR TOPOGRAPHIC AFTER
OF ATOMIC STRUCTURE -- THE
STRUCTURE ON THE S PROTEIN AND
DOING MORE OF ANALYSIS WE COULD
ALSO REALIZE SOME OF THE
POPULATION OF THE SPIKE PROTEIN
HAVE OPEN CONFIRMATION AND ONE
OF THE RBD DOMAINS RECREPTOR
BINDING DOMAIN IS STICKING UP
AND LIFTING UP AND ATTACHING TO
THE SURFACE OF THE PLACELET.
SO, HAVING THIS RECONSTRUCTION
AND ANALYSIS DONE, WHAT WE CAN
DO IS SOMETHING CALLED BACK
PROTEIN WE CAN PUT COORDINATE
BACK OF THE ANALYSIS.
YOU SEE HERE THIS IS AN S
PROTEIN DIRECTLY ATTACHING TO
THE MEMBRANE AND EACH OF THEM WE
CAN REALLY UNDERSTAND HOW THIS
PROTEIN IS ATTACHING TO THE
PLATELET SURFACE AND TO THE
INFORMATION WE GOT FROM THAT,
YOU CAN SEE THE CARVED PLATELET
AND SPIKE PROTEIN ATTACHED TO
IT.
OBVIOUSLY, IT LOOKS LIKE WHEN
CURVATURE OF THE MEMBRANE IS
HIGHER THAN YOU SEE MORE OF THE
SPIKE PROTEIN WHICH WE
QUANTIFIED AND AVERAGE COULD BE
QUANTIFIED AND YOU CAN ALSO
CORRELATE THAT TO THE DENSITY OF
THE SPIKE PROTEIN.
THIS INDICATES THAT MORE OF THE
CURVED PART IS PROTEIN TO ATTACH
AND IT ALSO INDICATES THAT THE
POED YA INFORMATION HIGH
CURVATURE S BINDING TO THE
PLATELET CAUSES SUPERCURVATURE
HAVING ACTIVATION OF THE -- FOR
THE PLATELET. NEXT INFORMATION
THAT WE COULD GET AS YOU CAN SEE
HERE, ORIENTATION OF THE SPIKE
ON THE MEMBRANE SEEMS TO DIFFER
FROM EACH OTHER AND COULD TAKE
STATISTICS OF IT AND WE CAN SEE
THAT A SPIKE PROTEIN IS NOT
REALLY BINDING ON MEMBRANE IN A
ROBUST WAY BUT WOBBLES AROUND
AND NOT A SURPRISE AND THIS IS A
TRIMER AND THERE ARE
OPPORTUNITIES FOR THE SPIKE
PROTEIN TO BE BOUND TO THE
MEMBRANE SURFACE AND WE CAN
VISUALIZE THAT BY DIRECTLY
LOOKING AT SPIKE PROTEIN
ATTACHING ON THE MEMBRANE
SURFACE THAT WE THINK IS THE WAY
OF MAXIMIZING THE SPIKE PROTEIN
TO RECOGNIZE THE SURFACE OF THE
PLATELET.
SOMETHING SIMILAR WAS OBSERVED.
BOTTOM PART OF THE SPIKE COMING
TO THE CORE OF THE VIRUS.
YOU SEE THEY ARE WOBBLING AROUND
AND WE HAVE [INDISCERNIBLE] OF
THE SPIKE PROTEIN THAT WOULD
MAXIMIZE FOR THAT ATTACHMENT OF
THE VIRUS TO THE HOST.
SO NOW WHAT KIND OF A RECEPTOR
IS THERE TO RECOGNIZE THE SPIKE
PROTEIN AND WE HAVE A GOOD
HUNCH. SO MANY OF THE PRO E-
TEENS ARE BINDING ON THE
MEMBRANE AND WHAT WOULD BE
MOSTLY EXPRESSED AND THAT WOULD
BE INTEGRATING.
ALSO, INTERESTINGLY, YOU CAN SEE
THIS RGD PEPTIDE OR SEQUENCE
THAT IS INCORPORATED INTO SARS
CO-V2 OR [INDISCERNIBLE] AS
CORONAVIRUS DOESN'T HAVE LGD
THAT IS A FAMOUS STRETCH OF THE
ACID KNOWN AS [INDISCERNIBLE].
WE ALSO SYMPTOMS OF THE
STRUCTURE OF THE INTEGRIN OPEN
FORM OF PRESENCE OF FIBERMECTIN
YOU SEE RGD THAT IS STICKING TO
HEAD OF INTEGRIN THAT IS
CONTRIBUTING TO ACTIVATION OF
THE INTEGRIN AND SOMETHING
SIMILAR MUST BE HAPPENING FOR
INTEGRIN OF THE PLATELET AND USE
THIS PURIFIED VERSION OF THE
INTEGRIN ALPHA 3 BETA 3 TO BE 3
BETA 3 AND ALPHA 5 BETA TESTING
INTERACTION.
YOU SEE HERE 5 BETA 1 DO HAVE
AFFINITY TO THE SPIKE PROTEIN
AND JUST AS A NOTE TO BE BETA 3
IS THE MOST MAJOR INTEGRIN THAT
IS EXPRESSED ON THE PLATELET.
THAT ALSO EXPLAINS WHY THIS IS
STOCHASTIC.
IF THIS PROTEIN, S PROTEIN LOCKS
ON TO BETA 3 IT WILL BE A BIG
MUCH, MUCH BIGGER PROBLEM.
ALL BLOOD GETS CLOTTED.
THIS IS THE -- THE MECHANISM ON
THAT WE DERIVED FROM THE
OBSERVATION THAT SPIKE PROTEIN
IS RECOGNIZING ON TO INTEGRIN
SURFACE OF THE PLATELET IS
STOCHASTIC INFORMATION -- IT
COULD GO TO IRREVERSIBLE FORM OF
THE PLATELET AND COULD GO BACK
AND ONE PART IS ATTACHED TO ACM
AND THIS IS A STUDY TO FINISH IT
AND ARE HAPPY TO DO SOMETHING
FOR AT LEAST THE PUBLIC HEALTH
CRI
CRISIS.
WE WILL GO TO LONGSTANDING
PROJECT ABOUT THE AXIUM OR THE
NEURON STARTED SOME YEARS AGO
ALREADY BACK IN GERMANY AND PHD
STUDENT STARTED TO MAKE THE
PIPELINE THAT WAS ALREADY SIX OR
SEVEN YEARS AGO.
WE WERE QUITE PIONEERING THE
CURVATURE OF THE NEURON TO EM
GRADE.
YOU SEE HERE THIS IS MOST THAT
WE GET EMBRYO AND THE PRIMARY
CULTURE GOING TO EM GRADE AND WE
GO TO CORRELATE AND CRYO-EM AND
HAVE A LOOK AND GET A DEEPER
VIEW.
HAVING THIS DONE, THIS IS THE
FIRST TIME THAT WE OBSERVED HOW
ORGANIZATIONAL ORCHESTRATION OF
THE MOLECULE AT AXIUM BRANCHING
POINT AND THIS IS FIRST TAKE
HOME STUDY AND MESSAGE WAS
INTERESTING THAT [INDISCERNIBLE]
WAS CONCENTRATED AT AXIUM BRANCH
POINT AND WHERE IT GENERATED
FROM AND LOTS OF PRODUCTIONS OF
THE MATERIAL HAVE TO HAPPEN.
THIS WAS OUR KIND OF SHOWCASE
STUDY THAT IS PASSED ALREADY.
I WON'T TALK ABOUT IT TODAY.
TODAY, I WANT TO FOCUS ON THE
STORY THAT IS RECENT ABOUT
AXONAL GENERATION IN PRESENCE OF
DRUG EPOBY AND PROJECT
ORCHESTRATED AT NIH AND
[INDISCERNIBLE] WHO IS A MEDICAL
DOCTOR HAVING NO EXPERIENCE
BEFORE COMING HERE AND HAD VERY
GOOD VIEW AND GUTS AND FOCUS AND
REALLY MANAGED TO HAVE THE
STORYLINE GO FORWARD.
AND ALSO IN VERY, VERY GREAT
TEAM WHO FINISH UP THE STUDY AND
KENNY HAD TO LEAVE TO GO BACK TO
MEDICAL STUDY HE IS A NEUROCELL
BIOLOGIST.
JUST HAVE A LOOK AT THEIR
EFFORT.
SO, WIDER [INDISCERNIBLE] IN
REGENERATION IS IMPORTANT.
IT IS BECAUSE IN THE CONTEXT OF
THE HEALTH, IT IS SOMETHING TO
DO WITH THE BRAIN INJURES.
TRAUMATIC BRAIN INJURY.
WHAT IS HAPPENING IN YOUR BRAIN
IS IN AXON. THIS IS A PROBLEM
CENTRAL NEURAL SYSTEM BRAIN
DOESN'T WANT TO REGENERATE AND
DEEP PART OF THE BRAIN THAT WE
WORK ON AND WHAT HAPPENS YOU GET
UNCONSCIOUS AND HAVE COGNITIVE
COGNITION PROBLEM OR EVENTUALLY
YOU DIE.
THIS IS SOMETHING THAT WE WANT
TO ADDRESS AND ALSO POSSIBILITY
OF THERAPEUTIC OPTIONS.
MOLECULARLY OR CELL BIOLOGICALLY
WE SEE INJURED AXON THAT IS
FOLLOWING.
YOU SEE HERE THAT THIS AXON IS
LITERALLY CUT OFF AND MEANSNENTS
COMPONENTS ARE LEAKED OUT AND
[INDISCERNIBLE] WHICH IS
DISRUPTED AND FIRST REACTION OF
THE AXON IS TO CLOSE AND TO
PROTECT SURROUNDING TISSUE AND
CALCIUM GOES IN AND
[INDISCERNIBLE] MICROTUBULE SO
IT SHRINKS AND WAY THAT
DEGENERATION OF THE NEURON IS
HAPPENING.
NOW, THIS IS A COLLABORATOR FROM
DATA FROM 2015 AND STUDY WAS
DONE IN PRESENCE OF DRUG EPOB
WHICH IS INJECTED TO MOST THAT
HAS SPINAL CORD GIRAFFE 8 WEEKS
MOUSE CAN WORK NICELY AND SHOWS
THIS DRUG IS WORKING.
WHAT IS DRUG DOING? IT IS
[INDISCERNIBLE] THAT IS A DRUG
THAT WAS ORIGINALLY DEVELOPED AS
ANTICANCER DRUG.
IDEA IS THAT IT IS RECOGNIZING
MICROTUBULE TO STABILIZE
MICROTUBULE SO IT ARRESTS CELL
CYCLE AND CANCER CAN'T PROPAGATE
ANYMORE AND ORIGINAL IDEA AND
REPURPOSING MOUSE, SOMEHOW MOUSE
IS FIXED WE DON'T KNOW
MOLECULARLY WHAT IS HAPPENING
AND SET UP PIPELINE SO MODIFIED
OR ADVANCED VERSION COMPARED TO
OUR SHOWCASE STUDY.
YOU SEE HERE WE PUT MORE LIGHT
MICROSCOPIC EMPHASIS ON STUDY TO
HAVE A LOOK WHAT IS GOING ON AND
WE HAVE THIS EM GRADE PUTTING
NEUROTISSUE HERE TO LET AXON TO
GROW.
ONE BIG THING WE DO IS AXOTOMY
AND WE CAN IMPLEMENT TO TOP OF
AXON ON EM GRADES I WILL SHOW
YOU LATER TO OBSERVE WHAT IS THE
RESPONSE ALSO ON CRYO-EM.
SO, I SHOW YOU HOW WE ARE DOING.
THIS IS THE SCOMPLANT AND
NEURONS COMING OUT FROM THE
TISSUE AND THEY ARE AXONS THAT
ARE USEFUL FOR US.
WE HAVE GOOD CONTROL OF IT AND
WE CAN IMPLEMENT THAT ON TO EM
GRADE YOU CAN SEE HERE.
THIS IS A TISSUE AND AXONS AND
WE CAN GROW AXONS UP TO SOME
DAYS AND AROUND 8 DAYS IT
DOESN'T GROW ANYMORE AND IS TIME
WE CAN TEST REGENERATION.
SO, THIS IS THE LIGHT MICROSCOPE
STUDY TO SHOW YOU WHAT IS
HAPPENING AND SO IN CELLULAR
LEVEL WE CAN SEE ALSO I DON'T
KNOW IF YOU NOTICE THAT CUT AXON
WAS REGENERATED IN PRESENCE OF
EPOB. IN PRESENCE OF IT IT
SHRINKS TO MAKE IT EASIER LIKE
THIS IT GROWS BACK THIS EPOB AND
SHRINKS EPOB AND WE CAN
REPRODUCE WHAT STUDIES SHOWS IN
CELLS THAT IS QUITE NICE AND WE
CAN ALSO QUANTIFY THAT.
NOW LET'S HAVE A LOOK AT SINGLE
AXON CAREFULLY AND YOU SEE HERE
THIS IS THERE. IT CAN BE QUICK.
IT JUMPED AND IT IS STARTING TO
REGENERATE ALREADY.
SO LET'S JUST MAKE A SNAPSHOT OF
IT AROUND 50 MINUTES OR ONE HOUR
YOU ACTUALLY DON'T SEE THE
TYPICAL GROWTH OF THE GROWTH
CON.
I DO ACTUALLY SEE THE GROWTH
CON.
WE KNOW IT BETWEEN ZERO AND 50
MINUTES OF REGENERATION IT IS
REGENERATING WITHOUT FAMOUS
GROWTH CON.
WHAT DOES IT MEAN?
WE WONDER HOW THIS CAN
REGENERATE WITHOUT IMPORTANT
GROWTH CON FORMATION.
YOU CAN ALSO SEE IT HERE IN THE
PRESENCE OF THE EPOB AFTER ONE
HOUR.
YOU GET IT REGENERATED BUT DON'T
SEE GROWTH COM.
THIS IS HYPOTHESIZED THIS IS A
MICROTUBULE DOING THE PUSH TO
REGENERATE REGENERATING AXON AND
THIS IS IT COMPROMISED OR NOT?
HOW IS MEMBRANE LOOKING LIKE?
WITH THAT WE ACTUALLY ENDED UP
MEASURING MEMBRANE TENSION USING
MEMBRANE TENSION DIKON FLIPPER
AND TIP OF REGENERATION AXON
INDICATING THIS IS VERY TENSE
AND MEMBRANE IS TENSE AND NOT
NORMAL MEMBRANE.
SORRY.
IN THE MEANTIME AFTER SOME OF US
AFTER FOUR HOURS YOU SEE TENSION
TO BE NORMALIZED AND THIS MEANS
AT BEGINNING THERE IS A VERY,
VERY FORCED PUSH FOR THE AXON TO
REGENERATE AND AT SOME POINT
GROWTH CON COMES BACK AND IS
NORMAL REGENERATION AND LET'S
HAVE A LOOK AT THAT ON CRYO-EM.
YOU CAN SEE HERE THIS IS THE
NEURON.
THIS IS THE EM GRADE.
THIS IS THOUGHT, EACH OF THE
THOUGHT HAS DIAMETER OF A
MICROMETER THAT WE TYPICALLY USE
FOR THE IMAGE ANALYSIS OR
OBSERVATION OF THE AREA AND THIS
INSITU WE DON'T USUALLY CARE
ABOUT THE HOLE BUT SEE WHAT WE
SEE.
NOW THAT WE PERFORMED AXE ON MY
ON TO EM GRADE YOU SEE HERE WE
ARE CLEANING DEBRIS SO WE DON'T
GET CONFUSED.
NOW LET'S JUST HAVE A LOOK AT
WHAT IS HAPPENING THERE.
SO TO CONFOUND REGENERATION WHAT
WOULD HAPPEN ON EM GRADE YOU SEE
COLORED AXON THAT GOT
REGENERATED AND WE CAN REALLY
FOLLOW REGENERATION AND HAVING A
LOOK AFTER ONE HOUR.
SO AFTER ONE HOUR HOW IT LOOKS
IS LIKE THIS.
THIS IS A TISSUE THAT IS THE
EXPLANT AXONS.
YOU SEE TWO AXONS WE DID
PERFORMANCE OF AXONOMY AND
REGENERATION YOU SEE HERE OF
STUFF HAPPENING AND LOOKING A
LITTLE MORE, WITH THIS I'M
PLEASED TO NOT GET ALERTED.
WE DO NOT HAVE THE MEMBRANE
DENSITY WHICH WE DON'T. IT IS
DUE TO TENSION.
SO, DURING THE FREEZING PROCESS,
MEMBRANE DID NOT SURVIVE.
WHAT WAS ADVANTAGE IN
[INDISCERNIBLE] AND WE GET A
VERY GOOD SIGNAL RATIO TO ALLOW
US TO DO HIGH RESOLUTION
ANALYSIS.
LET'S HAVE A LOOK.
YOU CAN SEE HERE THIS IS NORMAL
AXON AND YOU SEE THIS CUT SIDE
AND SEE CRAZY AMOUNT OF THE
REGENERATION TO BE STARTING TO
HAPPEN.
YOU SEE LOTS OF MICROTUBULE AND
VESICLES TRANSPORTED.
WE DON'T KNOW MUCH ABOUT BASIC
TRANSPORT ISSUE YET. WE ARE
DOING IT RIGHT NOW AND KNOW LOTS
OF STUFF IS TRANSPORTED ALONG
MICROTUBULE THAT IS SHOOTED OUT
AND THIS LENGTHWISE IS AROUND 20
MICROMETERS QUITE A LOT OF
ELONGATION OF CELL THAT IS
HAPPENING WITHIN ONE HOUR.
NOW, IN ORDER TO UNDERSTAND HOW
AND WHAT IS THE MOLECULAR BASIS
OF THIS SUPER SHOOTING OUT
EVENT, WE DECIDED TO DO LARGE
SCALE AXOTOMY. THAT MEANS YOU
HAVE LOTS OF AXONS HERE AND WE
CAN ACTUALLY CUT THAT AND WE CAN
OBSERVE THE ENTIRE AREA HERE AS
THINKING THAT THE TENSION OF THE
MEMBRANE IS ALL APPLIED SO THAT
IF YOU DO CRYO-EM ALL OVER HERE
DON'T HAVE THE MEMBRANELESS LESS
COMPONENTS AND WE CAN DO
ANALYSIS AND WE COLLECTED DATA
OF 2000 IMAGES AND HAVE A LOOK
AT IT AND DO SPS [INDISCERNIBLE]
THAT GIVES HIGH RESOLUTION
INFORMATION AND WE CAN DO
TOMOGRAPHY TO GET CONTEXT AND
FOR TODAY'S TALK I WILL TALK
ABOUT SPA VERSION OF IT.
BY COLLECTING SO MANY DATA, WE
COULD GET 3-D RECONSTRUCTION OF
THE MICROTUBULE AT REGENERATING
AXON. YOU SEE HERE THIS IS
FILAMENT THAT IS PROTEIN THAT IS
IMPORTANT FOR MICROTUBULE IF YOU
[INDISCERNIBLE] IT WILL BE AND
IS NOT REALLY CONVENIENT FOR
CELLS.
YOU CAN SEE [INDISCERNIBLE] THAT
IS A PROTEIN THAT IS MAKING
[INDISCERNIBLE] OF MICROTUBULE
TO BE CERTAIN PROTEIN ALL OF
THEM 1 THOUSAND 6 HUNDRED 63
MICROTUBULES THAT WE OBSERVED.
WE CAN SEE AND GO ANALYSIS OF
DIRECTIONALITY TO SHOW THEY ARE
ALL HAVING SAME DIRECTIONALITY
WHICH IS AN IMPORTANT IDENTITY
FOR THE AXON.
DENDRITE MICROTUBULES ARE
SWAPPED AROUND.
SO, SORRY.
SO THIS REALLY INDICATES THAT
MICROTUBULES ARE READY TO BECOME
THE REAR AXON THAT WE ARE
REGENERATING.
THIS RESOLUTION, WE CAN ALSO SEE
AN INTERDIMER DISTANCES 83.3
[INDISCERNIBLE]IN SITU AND IN
VITRO WE CAN SEE IT AND CONTROL
IT IN STABLE CONDITION WE KNOW
IT HAS 84 [INDISCERNIBLE] AND
MICROTUBULE WHEN IT IS NOT
STABLE IS 81.8 ANGSTROM THIS
INDICATES MICROTUBULES AT
REGENERATING SITE OF THE AXON IS
A STABILIZED FORM OF THE AXON OR
MICROTUBULES.
WE WONDER THAT THERE MUST BE
EPOB THAT IS ATTACHED TO
STABILIZE MICROTUBULE AND DIDN'T
SEE IT WHEN WE HAVE HIGH
THRESHOLD OF THE DENSITY AND
WHEN WE HAVE -- WE CHANGE
THRESHOLD WE START TO SEE EPOB.
THIS IS FIRST TIME WE CAN REALLY
OBSERVE THE DRUG THAT IS ACTING
ON TO THE MICROTUBULE IN
REGENERATING CELL AND WE CAN SAY
THAT DRUG WE ADDED IS
PHARMACEUTICALLY RELEVANT
CONCENTRATION THAT IS ONLY ONE
NANOMOLE AR OR MICROTUBULES
DENSITY IS IN MICROMOLARS
THOUSANDS OF MAGNITUDE LOWER
AMOUNT OF THE DRUG THAT IS ADDED
COMPARED TO TUBULIN AND THIS
DENSITY HERE AND CONCENTRATION
OF THE DRUG IS HAPPENING AT
REGENERATING SITE OF THE AXON.
OKAY. NOW WE ARE AWARE WHERE IS
MICROTUBULE COMING FROM? WE
HAVE THREE HYPOTHESIS TO ASK AND
GOING ONE-BY-ONE AND FIRST
HYPOTHESIS THAT TUBULIN IS
SYNTHESIZED AT REGENERATING SITE
AND WE THOUGHT SO BECAUSE WE
SOMETHING LIKE THAT ACTING AND
THIS IS AXON AND AXON BRANCH AND
YOU SEE RIBOSOMES HERE AND SEE
ACTIN THAT IS COMING OUT FROM
THE RIBOSOME.
WE HAVE REGENERATION.
SORRY AXON THAT IS BRANCHING
GENERATION WE WOULD SEE SINNIS
OF ACTIN AND REGENERATION SIDE
AND DON'T SEE MUCH INTERACTION
WE HAVE NOT SEEN IT SO FAR AND
STILL LOOKING AROUND AND HAVEN'T
FOUND IT SO FAR AND IS NOT A
PRIME MECHANISM TO PRODUCE
TUBING COMING TO REGENERATION OF
THE AXON.
SO, NEXT HYPOTHESIS THAT WE
WONDER, SORRY.
I SHOWED THIS ONE FIRST.
THIS IS [INDISCERNIBLE] OF
TUBULING AND REGENERATION THAT
IS HAPPENING AND CLUSTER OF
TUBULING OR MICROTUBULE IS
SHATTERED TO TIP OF REGENERATING
AXON AND MEANS THERE IS SOME
SORT OF A GENERATION OF THE
TUBULIN OR MICROTUBE YULE AND
PRECUT SIDE AND IT GETS
SHATTERED IS IT MICROTUBULE OR
TUBULIN? IF MICROTUBULE WE KNOW
NORMAL MICROTUBULE IN NEURON
CONTAINS MIPS THAT ARE USUALLY
THERE WHEN MICROTUBULES ARE
MATURE AND LOOKING AT
REGENERATING AXON MICROTUBULES
HAVE MUCH LESS OF MIPS INSIDE
AND MEANING THEY ARE YOUNG
MICROTUBULES NOT MATURED YET TO
CONTAIN INTERLUMIN PARTICLES WE
CAN MEASURE IT AND YOU SEE HERE
MICROTUBULES ARE MUCH LESS
CROWDED COMPARED TO THE NORMAL
CONTROL.
THIS INDICATES THAT MICROTUBULES
ARE NOT CARRIED FROM THE SOMA OR
UPSTREAM OF THE AXON BUT IS
POLYMERIZED AT THE REGENERATING
SITE AND EXTREME EXAMPLE, YOU
CAN SEE HERE THEY ARE TIP OF
REGENERATING SITE AND
MICROTUBULES DON'T HAVE MIPS.
THEY ARE FRESHLY MADE
MICROTUBULES AT THE TIP.
NOW, HAVING THAT IN OUR MIND WE
LOOK INTO POLYMERIZATION PROCESS
OF MICROTUBULE AND TRIED TO FIND
IT.
HERE IS THE BASIC INFORMATION
THAT IS SHOWN ALREADY AND SHOWN
WHEN MICROTUBULE IS POLYMERIZED
AND SEE IN THE BACKGROUND
OLIGGANDS IN MICROTUBULE AND IN
THIS PRIOR CRYO-EM STUDY IN
VITRO YOU SEE THIS SPIRAL LIKE
OLIGGAND THAT GETS ATTACHED TO
THE MICROTUBULE AND STRAIGHTENED
INTO THIS AND IT GETS
INCORPORATED AND WE LOOK INTO
THAT IN OUR OWN TOMMO GRAMS OF
REGENERATING AXON.
YOU WOULD SEE HERE THERE IS A
BIG CLUSTER OF OLIGGANDS THAT
ARE LOOKING SIMILAR TO WHAT IS
SHOWN AS IN VITRO TUBULE
OLIGGANDS HERE IS SNAPSHOT YOU
CAN SEE MORE.
WE MEASURE DIAMETER OR CURVATURE
OF OLIGOMER THAT IS SHOWN IN
VITRO AND WE DO SEGMENTATION TO
SEE AND IT SHOWS SPIRAL
FORMATION AND DETECT SIZE OF
MONOMER THAT IS INCORPORATED
INTO SPIRALS.
YOU SEE HERE THIS STAND OUT
NANOMETER OR.5 NANOMETER THAT
FITS IF IN UNIT THAT YOU SEE IN
MICROTUBULE IN TOMMO GRAM AND
TOMOGRAPHY RESOLUTION IS GOOD
ENOUGH TO DISTINGUISH SIZE OF
ACTING MONOMER AND TUBULIN
MONOMER AND YOU CAN SEE ALL
TOGETHER THIS IS TUBULIN
MONOMER.
TO GET FINAL CONFIRMATION OR
FINAL VALIDATION OF IT YOU SEE
OLIGOMERS OF TUBULIN WE ATTACH
TO END OF MICROTUBULE THAT ARE
POLYMERIZING TO CAPTURE SNAPSHOT
OF THE MICROTUBULES
POLYMERIZINGIN SEATU INTO
REGENERATING AXON AND THIS IS A
SIDENOTE WE HAVE BEEN FOLLOWING
UP FOR SOME TIME.
YOU SEE HERE THIS IS A
MICROTUBULE THAT GETS BRANCHED.
HERE YOU SEE THIS SHARING
MICROTUBE YULE BRANCH.
WHY WE CARE ABOUT THAT IS
BECAUSE WE -- SOME YEARS AGO WE
PUBLISHED A PAPER SHOWING THAT
MICROTUBE YULE CAN BRANCH OUT IN
VITRO AND IN PRESENCE OF THE
PROTEIN THAT IS ABUNDANT IN
NEURON.
WE LEFT IT AS AN OPEN QUESTION
IF THIS WOULD BE SOMETHING
RELEVANTIN SITU OR IN VIVO AND
DON'T THINK IT IS MAJOR
MECHANISM FOR THIS OR REMODELING
AND WONDERING IF YOU WOULD CATCH
IT AND SINCE WE ARE VERY HAPPY
WE CAUGHT IT WITH REGENERATING
AXON.
YUP. CONCLUSION OF THIS PART OF
THE STUDY IS IN THIS TUBULE
OLIGOMERS TRANSPORT IT IN AXON
AND REGENERATION SIDE.
NOW, I HAVE SHOWN UP TO ONE HOUR
AND 24 HOURS WHAT IS GOING ON
YOU SEE HERE THIS IS A CUT SIDE
AND NEURON MANAGED TO REGENERATE
QUITE WELL.
WE CAN ALSO TAKE TOPOGRAPHIC
SNAPSHOTS OF CERTAIN AREAS ALONG
REGENERATING AXON THAT SEEM TO
BE OKAY.
I MEAN, HEALTHY ENOUGH.
IT ALSO HAS THE GROWTH CONE
CAPPED ALREADY AND CAME BACK
NICELY.
NOW, CONCLUSION, AFTER ONE HOUR
OF THE AXOTOMY YOU SEE NO
TYPICAL GROWTH CONE I SHOW
MICROTUBE YULE GROW RAPIDLY THAT
IS ASSOCIATED WITH INCREASED
MEMBRANE TENSION AND INCREASE
REGENERATING SIDE AND TUBULE
OLIGOMERS UP TO 6 HOURS AND NEW
MEMBRANE AND LIPID THAT IS
STARTING WITH AXON GROWTH SEEING
BY CHANGE OF THE TENSION OF THE
MEMBRANE.
YEAH. THEN FINALLY SOME HOURS
AFTER WE SEE RECOVERY OF GROWTH
CONE THAT IS IMPORTANT THING FOR
THE AXON GUIDANCE ALSO THAT IS
GUIDED MOVEMENT OF THE GROWTH
AXON REGENERATING AXON AND
ECONVENIENT TULELY THIS COULD BE
CONNECTED HOPEFULLY INTO NEURAL
NETWORK AGAIN AND THIS IS A
FINAL NOTE OF MY TALK.
I SHOWED YOU ABOUT THE
RESOLUTION REVOLUTION OF WHAT IS
HAPPENING IN THE EM FIELD AND
IMAGING FIELD AND ALSO SOME OF
THE TWISTS AND ADDITIONAL
FACTORS AND DIMENSION THAT WOULD
BE GIVING US A LOT MORE OF
COMPREHENSIVE VIEW OF WHAT WE
ARE TRYING TO ADDRESS.
THIS WOULD BE NEW KIND OF BIG
PICTURE STUDY THAT WE TRIED TO
PASS THROUGH.
THAT IS THE DIRECTION THAT WE
HAVE GOING.
OKAY. SO WE START TO TRY TO
FINISH MY TALK AND I WANT TO
START BY ACKNOWLEDGING MY
COLLEAGUES AND STUDENTS AND
POST-DOCS FROM INSTITUTE OF
BIOCHEMISTRY AND THESE ARE
PEOPLE THAT STARTED UP TOGETHER
AND THEY DECIDED TO REMAIN IN
GERMANY AND MOVE ON ALREADY AND
THESE ARE THE PEOPLE THAT
ACTUALLY BRAVELY CAME WITH ME
FROM NIH AND THAT WAS REALLY
NICE.
WE COULD HAVE A KIND OF PRAITION
THAT WAS CONTINUING.
THESE ARE THE GRANTS I HAVE BEEN
GETTING IN GERMANY.
THIS IS QUITE GENEROUS SUPPORT I
WAS GETTING AND BEING A
NON-EUROPEAN BUT THEY SUPPORTED
ME.
THAT WAS VERY NICE.
HERE IS THE CURRENT NIH LAB.
SO WE ARE HAVING A VERY GOOD
TIME NOW AND TRYING TO MAKE SIN
EAR
EARTHY AND GOOD TEAM. I'M HAPPY
WITH MY NEIGHBOR.
WE HAVE A GOOD TIME TOGETHER AND
CO-FACILITY PARTICULARLY
MICROSCOPE CO-FACILITY AND THEY
ARE LIKE OUR EVERY DAY
DESTINATION TO GO IF YOU DON'T
FIND POSTER WE GO THERE TO FIND
THEM USUALLY AND FINALLY
COLLABORATORS AND YOU CAN SEE WE
HAVE LOTS OF COLLABORATION GOING
ON.
SO SOME OF THE COLLABORATORS AND
NEW COLLABORATORS AND WE HAVE
THEIR HELP AND THEIR INSIGHT AND
DIFFERENT PERSPECTIVE AND
ADDRESS WHAT WE START OUT TO
ACHIEVE AND THAT IS MY TALK AND
THANKS SO MUCH FOR YOUR
ATTENTION.
>> AUDIENCE: [APPLAUSE].
>> WE WILL NOW OPEN UP TO
QUESTIONS.
IF ANYONE WOULD LIKE TO ANSWER
A QUESTION IF YOU COULD GO TO
THE MIC.
ONLINE ALSO IF YOU COULD USE THE
LIVE FEEDBACK I WILL PASS
QUESTIONS ON TO DR. MAZUNO.
>> OKAY.
>> VERY NICE OVERVIEW FROM
[INDISCERNIBLE] MY QUESTION IS
ABOUT EPLE B THE DRUG YOU SHOW
AND IN STRUCTURE YOU SHOW IT IS
BIND DIRECTLY TO TUBULING ON
TUBULING FILAMENT FORM AND ON
FUNCTION YOU MENTIONED IT
ACTUALLY HELPED TRANSPORTING OF
TUBULIN OLICCO MERS AND THAT
DOESN'T SEEM TO BE EXACTLY IN
THE STRUCTURE THAT DOESN'T
EXPLAIN THE FUNCTION.
>> RIGHT. RIGHT. WHAT WE THINK
IS HAPPENING IS THAT IN THE
PRESENCE OF EPOB AND THEN WHEN
THE POLYMER -- DEPOLYMERIZATION
OF THE MICROTUBULE TO BE
HAPPENING AND IN VITRO PEOPLE
HAVE SHOWN ALREADY THAT IT
BECOMES PIECES OF OLIGOMERS AND
WHAT WE THINK IS HAPPENING IS
THAT MAYBE IN E POB AND
MICROTUBULES THAT TRY TO DO
DYNAMIC INSTABILITY AND TRY TO
DO DEPOLYMERIZATION HERE AND
THERE AND PASSIONATELY
DEPOLYMERIZE MICROTUBULE
PRODUCING OLIGOMERS AND WHAT WE
ARE SEEING AS DOT FORMING WHEN
FORMING EPOB.
>> RIGHT. THEN YOU AT THE
BEGINNING YOU MENTIONED EPOB WAS
DEVELOPED AS AN ANTICANCER DRUG.
>> RIGHT.
>> HOW DID IT WORK AS ANTICANCER
DRUG IN TERMS OF
DEPOLYMERIZATION OF MICROTUBULE
OR MOVING IT.
>> YEAH. SO I THINK THE DESIGN
IS THAT IT ARREST THE -- SO
MICROTUBULE NEED TO REMODEL WHEN
CELL DIVISION HAPPENED BUT EPOB
STABILIZED ENOUGH SO IT DOESN'T
DEPOLYMERIZE COMPLETING AND CELL
CYCLE CAN'T MOVE TO THE NEXT
STAGE IS WHAT IS HAPPENING OR IS
THE DESIGN.
>> OKAY. TO STABILIZE.
>> YEAH, YEAH.
>> THANKS.
>> THAT WAS BEAUTIFUL. I HAD A
QUESTION ABOUT YOUR SPIKE
INDUCED MEMBRANE DEFORMATION AND
NOW WITH YOUR SYSTEM DO YOU HAVE
ANY INTENTION OF LOOKING AT
DIFFERENT AT LIKE FOR EXAMPLE
DIFFERENT RESIDUES OR SEGMENTS
OF SPIKE AND HOW THEY CONTRIBUTE
TO THE MEMBRANE DEFORMATION.
>> TO BE HONEST, WE HAVE MOVED
ON FROM SPIKE SO.
>> NO PROBLEM.
>> SORRY IT WAS A GOOD CLOSURE.
>> HI. I'M INTERESTED IN THE
SPIKE PROTEIN BINDING. YOU SAID
IT IS NOT REALLY CLEAR IF IT IS
BINDING TO THE CURVATURE OR
INDUCING THE CURVATURE.
DO YOU HAVE ANY SENSE WHICH IS
MORE DOMINANT?
>> I HONESTLY THINK IT IS
INDUCED INDIRECTLY INDUCING THE
CURVATURE.
SO IT WOULD BIND. IN MY MIND
WHAT I THINK IS IT WOULD BIND
STOKE AFTICALLY AND SOMETIMES IT
BINDS TO LOCK INTO INTEG GRIN SO
INTEGRIN GETS ACTIVATED AND DOT
INDUCES CURVATURE MAKING
[INDISCERNIBLE] TO BE FORMED.
SPIKE ITSELF DOES NOT CAUSE
CURVATURE FORMATION AND
ACTIVATING INTEG GRIN IT INDUCES
CURVATURE FORMATION.
>> MIGHT THERE BE A CERTAIN
AMOUNT OF FLUCTUATION NORMALLY
AND WHEN IT HITS A CURVE THING
SPIKE BINDS IT AND LOCKS IT IN
OR SOMETHING LIKE THAT.
>> YEAH INTEGRIN ENHANCES IT.
>> THANK YOU.
>> YEAH.
>> ANY OTHER QUESTIONS?
OKAY. I WOULD LIKE TO THANK DR.
MAZUNO FOR YOUR WONDERFUL
PRESENTATION AND FOR EVERYONE
COMING TO ATTEND TODAY.
>> THANK YOU.
>> AUDIENCE: [APPLAUSE]