>>> GOOD AFTERNOON, EVERYONE.
GLAD TO SEE EVERYBODY HERE.
WELCOME TO THOSE ON THE WEB.
VERY INTERESTING WEDNESDAY
AFTERNOON LECTURE FROM DAVID
SABTEENY TODAY.
INVOLVING REGULATION OF GROWTH
BY THE mTOR PATHWAY.
ONE OF THOSE PATHWAYS THAT
EVERYBODY SEEMS TO BE WORKING ON
RIGHT NOW.
AND WE HAVE A REMARKABLE
OPPORTUNITY TO HEAR FROM
SOMEBODY WHO REALLY BEGAN MUCH
OF HIS EFFORT AS A GRADUATE
STUDENT AND BUILT MUCH OF THE
FOUNDATION UPON WHICH AT THE
MOMENT WE UNDERSTAND, THIS
CRITICAL PATH WAY THAT REGULATES
CELL GROWTH.
DAVID GOT HIS UNDERGRADUATE
DEGREE AT BROWN, GRADUATING
MAGNA CUM LAUDEY AND THEN WENT
ON TO THE MD-PH.D. PROGRAM AT
JOHN'S HOPKINS WHERE HE BEGAN
THIS EFFORT WITH HIS THESIS
BEING CONTROL OF TRANSLATION BY
A NOVEL RAPAMYCIN SENSITIVE
SIGNALING PATHWAY.
SOUNDS LIKE SOMETHING WE MIGHT
HEAR ABOUT TODAY.
HIS ADVISOR FOR THAT WAS SAL
SCHNEIDER.
SOMEBODY THAT MANY OF US LEARNED
A LOT FROM.
HE WENT ON TO A POSITION AT THE
WHITEHEAD INSTITUTE AS A FELLOW,
A WHAM WHICH PROVIDES
PARTICULARLY CREATIVE AND
INDEPENDENT-MINDED YOUNG
SCIENTISTS AN OPPORTUNITY TO BE
INDEPENDENT AT AN EARLY STAGE
AND A PROGRAM THAT WE HAVE
RECENTLY MODELED A BIT WITH OUR
OWN EARLY INDEPENDENCE PROGRAM
THAT IS BEING SUPPORTED THROUGH
THE COMMON FUND.
HE WAS OBVIOUSLY SUCCESSFUL IN
THAT REGARD, BECAME A MEMBER OF
THE WHITE HEAD IN 2002, AS WELL
AS A PROFESSOR THROUGH THE RANKS
AT MIT WHERE HE IS NOW FULL
PROFESSOR AND MEMBER OF A COKE
CENTER FOR INTEGRATIVE CANCER
RESEARCH AS WELL AS BEING HHMI
INVESTIGATOR AND A MEMBER OF THE
BROAD INSTITUTE.
HIS LAB HAS CONTINUED TO LEAD IN
THIS WHOLE VERY CRITICAL AREA
INVOLVING MECHANISMS OF CELL
GROWTH REGULATION WITH THE MAJOR
FOCUS BEING ON THE TARGET OF
RAPAMYCIN, THE TOR PATHWAY AND
I'M SURE HE WILL BE TELLING US
THE LATEST DEVELOPMENTS IN THAT
REGARD, WITH ALL OF ITS
CONSEQUENCES FOR UNDERSTANDING
BASIC SCIENCE AND FOR
APPLICATIONS TO MANY DIFFERENT
CLINICAL CIRCUMSTANCES.
SO IT'S WONDERFUL TO HAVE DAVID
HERE WITH US THIS AFTERNOON,
PLEASE JOIN ME IN WELCOMING
DAVID SABATINI.
[ APPLAUSE ]
Y.
>> THANK YOU VERY MUCH FOR
ORGANIZING THIS VISIT.
I'M ALWAYS A LITTLE HORSE.
SHOW IF SOMEONE HAS TROUBLE
HEARING ME, PLEASE LET ME KNOW.
IT IS TRUE I HAVE BEEN WORKING
ON THIS PATHWAY WHICH WILL COME
UP PRETTY SOON.
FOR A LONG TIME, SINCE I WAS A
GRADUATE STUDENT, YOU COULD SEE
A GOOD THING, MAYBE IT SHOWS
YOUR PERSEVERANCE AND ALSO MIGHT
SAY IT IS REMARKABLY UNCREATIVE
OF ME.
FOR A LONG TIME I FELT THAT AND
TRIED TO STOP WORKING ON THE
MTOR PATHWAY WHEN I MOVED FROM
MY GRADUATE WORK TO THE
WHITEHEAD.
I DIDN'T HAVE BEEN HOW TO DO
ANYTHING ELSE.
SO AT THE END OF THE DAY, I HAD
TO CONTINUE ON THIS SYSTEM.
SO I'LL TELL YOU A LITTLE BIT OF
STORIES AROUND THE TOR PATHWAY,
PARTICULARLY A NEW STORY WHICH
HAS INTERESTING CLINICAL, ONE OF
THE MAJOR REGULATORS OF GROWTH,
SIZE CONTROL.
I THINK ONE OF THE REALLY
INTERESTING ASPECTS OF THIS
PATHWAY, THAT DISTINGUISHES IT
FROM OTHER PATHWAYS THAT
REGULATE GROWTH, IT DOES IT AT
ALL DIFFERENT LYING CALL LEVELS,
SO IT'S A MAJOR CELL SIZE.
ALL BIOLOGICAL LEVELS.
ORGAN SIZE, WHICH I'LL SHARE A
LITTLE BIT WITH YOU, AS WELL AS
BODY SIZE CONTROL WHICH WE ARE
REALLY INTERESTED IN BUT HAVEN'T
DONE ALMOST ANYTHING IN THAT
AREA.
INTERESTING ASPECT OF THE mTOR
PATHWAY, THE FIRST INSIGHTS COME
FROM CHEMICAL BIOLOGY AND THE
STUDY OF A MOLECULE, WHEN I USED
TO FIRST TALK ABOUT THIS, I HAD
TO INTRODUCE, ALTHOUGH RAPAMYCIN
IS A FAMOUS MOLECULE.
THIS IS A BACTERIAL PRODUCT THAT
COMES FROM EASTER ISLAND OFF THE
COAST OF CHILE, IT HAS A NUMBER
OF CLINICAL APPLICATIONS WHICH
ARE SHOWN HERE, PARTICULARLY
LATELY.
A LOT OF INTEREST IN RAPAMYCIN
WHICH ARE ANALOGUES IN THE
TREATMENT OF CANCER AND ALTHOUGH
AT THE BEGINNING, THIS WAS NOT A
VERY PROMISING AVENUE, I THINK
AS WE UNDERSTAND THIS PATHWAY
BETTER, WHAT ARE THE MUTATIONS
THAT LEADS TO ACTIVATION, IT IS
SEEMING TO BE A FRUITFUL
APPROACH.
WHAT TOOK RAPAMYCIN FROM THE
SCIENTISTS AND THE CLINICIANS TO
MORE OF THE PUBLIC SPHERE IS THE
FINDING LISTED HERE AT THE
BOTTOM.
THIS MOLECULE IN A NUMBER OF
DIFFERENT ORGANISMS, MOST
NOTABLY FROM THE PUBLIC'S
ATTENTION IN MIDDLE-AGED MICE
PROLONGS LIFESPAN.
THIS LED HUMAN BEINGS TO START
TAKING RAPAMYCIN LOW DOSE OFF
PRESCRIPTION TO HOPEFULLY
PROLONG THEIR LIFESPAN, IGNORING
THINGS LIKE THIS.
WHETHER I HAVE GIVEN THIS TALK
AT TIMES, I HAVE HAD PEOPLE COME
UP TO ME SAY DAVID, POLICE STOP
LISTING IMMUNOSUPPRESSANT
THERAPY BECAUSE WILL YOU
DISSUADE PEOPLE FROM TAKING
RAPAMYCIN TO IMPROVE THEIR
LIFESPAN.
THAT IS AN ODD THING.
IT'S AN FD Akt APPROVED
IMMUNOSUPPRESSANT.
THIS IS NOT A MOLECULE I WOULD
TAKE FOR RECREATIONAL USES.
I'D LIKE TO USE THIS OLD SLIDE
THAT ILLUSTRATES WHETHER I THINK
THE BIG PICTURE POINTED ABOUT
THIS SYSTEM THAT KEPT OUR
INTEREST FOR SO MANY YEARS.
mTOR IS A LARGE PROTEIN KINE
SPACE RESPONDS TO ANYTHING YOU
GIVE TO THE CELL.
SO ANY KIND OF NUTRIENT, ANY
KIND OF GROWTH FACTORUX ANY KIND
OF STRESS.
THAT TELLS YOU THE CELL CARES A
LOT ABOUT REGULATING THE
ACTIVITY OF THIS SYSTEM.
IT PRETTY MUCH REGULATES ANY
PROCESS THAT USES OR GENERATES
LARGE AMOUNTS OF ENERGY.
SO FOR EXAMPLE, TRANSLATION,
BIOGENESIS OF RIBOSOMES, LARGE
PERCENT OF THE CELL MASS
ACCOUNTS BY RIBOSOMES.
A NUMBER OF OTHER PROCESSES THAT
ARE NOT ON THIS SLIDE, FOR
EXAMPLE LIPID SYNTHESIS, SO THIS
IS A MAJOR REGULATOR OF ANABOLIC
AND CATABOLIC PROCESSES.
NOW BECAUSE IT SENSES THESE
THINGS, WHICH I THINK IN
AGGREGATE, WE LIKE TO THINK OF
AS EQUIVALENT TO FOOD, SO
GLUCOSE, AMINO ACIDS, GROWTH
FACTORS SUCH AS INSULIN AND IGF
AND THINGS THAT RESPOND TO THE
STATE AND REGULATES THESE
ANABOLIC AND CATABOLIC PROCESSES
AS WE MOVE SOME OF OUR WORK IN
THE IN VIVO DIRECTION, I'LL GIVE
YOU A LITTLE BIT OF A TASTE FOR,
WE ARE INCREASINGLY THINKING OF
THIS SYSTEM AS A MAJOR MODULATOR
OF THE RESPONSE OR AVAILABILITY
OR LACK OF FOOD AND WHETHER THE
ORGANISM IS AN ANABOLIC OR
CATABOLIC STATE.
DEPENDING ON WHERE IN THE LIFE
CYCLE IT IS, IT MAY LEAD TO
CERTAIN KINDS OF RESPONSES NOR A
GROWING ANIMAL TO, ANIMAL THAT
IS SMALLER WHEN IT REACHESSA
ADULTHOOD.
NOW THERE ARE TWO mTOR
PATHWAYS, ONE WE CALL THE mTOR
COMPLEX PATHWAY.
THE BETTER KNOWN PACT WAY --
PATHWAY AND ANOTHER ONE CALLED
THE M TOWER 2 PATHWAY.
IT -- mTOR 2 CAN BE CONSIDERED
PART OF THE PI3 KINASE PATHWAY.
SO IT'S A REGULATOR OF Akt KT
AND HAS IMPORTANT ROLLS IN
TUMORS THAT HAVE HYPERACTIVATION
I'LL
TOLL YOU ABOUT A NEW CONNECTION
TO THIS PATHWAY TO CANCER.
IF WE THINK ABOUT THIS PATHWAY
IN VIVO, WE MANIPULATED THE
ACTIVITY OF THIS SYSTEM.
THIS IS IN A MOUSE LIVER.
A WILDTYPE LIVER AND THIS IS THE
ACTIVITY OF THIS PATHWAY.
HERE WE INHIBITED THE PATHWAY
AND HERE WE ACTIVATED.
WHAT IS MORE INTERESTING TO LOOK
AT IS WHAT IS THE RESPONSE OF
THIS LIVER IN RESPONSE TO
FASTING OR FEEDING?
SO THE LIVER LOSES ABOUT 25% OF
ITS MASS WHEN YOU FAST ANIMAL 24
HOURS B40% IF YOU FAST IT 48
HOURS AND BEYOND THAT THE MOUSE
CAN DIE BY THREE DAY FAST IT IS
NOT LEGAL FOR US TO DO AT MIT
BUT IF IT HAS BEEN KNOWN TO BE
DONE, IT IS NOT GOOD FOR THE
LIFE OF ANIMAL.
WHEN YOU LOOK AT THIS LIVER, YOU
CAN SEE IT NO LONGER SHRINKS IN
RESPONSE TO FASTING SUGGESTING
THE RESPONSE TO THE FASTING,
THIS LIVER IS AS SMALL AS IT IS
GOING TO GET.
IN CONTRAST, THE LIVER THAT IS
IN ACTIVATIVE PATHWAY IS
RESISTANT TO THESE EFFECTS
ALTHOUGH NOT COMPLETELY.
WHEN WE THINK ABOUT THE TYPES OF
SIGNALS THEY NEED TO RESPOND TO
MAKE THE DECISION TO GROW OR
DE-GROW, WHICH IS WHAT IS
HAPPENING IN THIS SITUATION, AND
THE RE-FEEDING SITUATION IS THE
GROWING, WHAT WE LIKE TO THINK
ABOUT IS THE TWO CLASSES OF
SIGNALS THAT MATTER.
THERE ARE THE LOCAL NUTRIENT
LEVELS.
NO CELL SHOULD TRY TO GROW IN
THE ABSENCE OF THE BUILDING
BLOCK FOR MASS AND TO GENERATE
ENERGY.
BUT ALSO IN A MULTICELLULAR
ORGANISM, ALL THE HUMORAL CREW
THAT IS TELL THE CELL THROUGHOUT
THE BODY THAT THE ORGANISM IS IN
THE FED STATE.
SO A PATHWAY LIKE THE mTOR
PATHWAY NEEDS TO INTEGRATE THESE
KINDS OF SIGNALS TO MAKE THE
GROW/NO GROW DECISION.
WE HAVE BECOME PARTICULARLY
INTERESTED IN WHAT I WOULD SAY
IS THE SIGNAL INTEGRATION
PROBLEM FOR THIS PATHWAY, WHICH
IS PARTICULARLY ACUTE FOR THE
mTOR PATHWAY BECAUSE IT SENSES
SO MANY DIFFERENT THINGS.
SO WE KNOW A FAIR AMOUNT AT THE
MOLECULAR LEVEL HOW THIS
HAPPENS.
AND A LOT OF IT HAPPENS THROUGH
A TUMOR SUPPRESSOR CALLED TSC,
MUTATED IN THE DISEASE CALLED
TUMOR SCLEROSIS COMPLEX.
MANY SIGNALS FEEDING THROUGH
OTHER GENES MUTATED IN CANCER
TALK TO TSC AND THEN NEGATIVELY
REGULATES A SMALL PROTEIN CALLED
REB AND WHEN IT IS LOADED, IT
ACTIVATES mTOR 1 KINASE
ACTIVITY AND DRIVE GROWTH.
IN OUR HANDS, RHEW IS ESSENTIAL.
GENETICALLY IT'S EQUIVALENT OF
DELETING THIS COMPLEX IN OUR
HANDS.
NOW THE SIGNAL THAT IS NOT
ILLUSTRATED HERE AND WE HAVE
BECOME PARTICULARLY INTERESTED
IN AGAIN IN THE LAST FEW YEARS,
ARE AMINO ACIDS.
THIS IS ONE OF THE ANCESTRAL
SIGNALS AND IS QUITE A BIT OF
EVIDENCE IT DOESN'T GO THROUGH
THIS PATH WAY.
INSTEAD, IT APPEARS THAT AMINO
ACIDS SIGNAL A DIFFERENT CLASS
OF SMALL PROTEINS, WHICH OPERATE
AS HETERODIMERS.
SO THESE ARE QUITE INTERESTING
PROTEINS.
WHEN WE FIRST STARTED STUDYING
THEY WERE POORLY STUDIED.
THEY ARE PART OF THE RAS FAMILY.
ALL YOU CAN SEE IS THE GTP
BINDING SITE.
THEY HAVE A GTP Akt SE DOMAIN
AND A C-TERMINAL DOMAIN.
WE HAVE 4 OF THEM.
BUT THE PART AURAS EXPECT OF
THESE THAT IS INTERESTING IS
THAT THEY COME IN TWO FLAVORS.
Akt AND B FLAVOR THAT ARE
PRACTICALLY IDENTICAL.
YOU ADD AMINO ACIDS AND THIS
CHANGES AND WE ARE UNDERSTANDING
HOW THIS MIGHT HAPPEN AND NOW
THEY INTERACT VERY WELL WITH
MTOR 1.
HERE IS DATA SHOWING THAT.
SO MINUS US PLUS AMINO ACIDS, IF
WE MAKE A MUTANT OF RAG B IN
THIS CASE, THAT IS
CONSTITUTIVELY LOCKED TO GTP, SO
EQUIVALENT TO A RAS ACTIVATING
ALLELE, YOU HAVE A CONSTITUTIVE
INTERACTION, MOREOVER, IN CELLS
EXPRESSING THIS PROTEIN, THE
PATHWAY NO LONGER CARES ABOUT
AMINO ACIDS.
WHAT I MEAN IS THAT IF YOU
REMOVE AMINO ACIDS THE PATHWAY
DOESN'T HAVE BEEN.
IT'S CONSTITUTIVE T LOST THE
CAPACITY TO SENSE THE ABSENCE OF
AMINO ACIDS.
WE ARE WORRIED ABOUT THOSE
EXPERIMENTS SO WE KNOCKED IN
THIS MUTANT ALLELE NOT IN THE
RAG B LOCUS WHICH IN THE MOUSE
IS POORLY EXPRESSED AND HERE ARE
THE CONTROLS MINUS OR PLUS AMINO
ACIDS.
THIS IS ONE OF OUR FAVORITE
OUTPUTS FOR mTOR 1 ACTIVITY.
YOU CAN SEE NICE REGULATION N
CONTRAST, THE MESS WITH THE
ACTIVATED TWO COPIES OF THE
PROTEIN, YOU CAN SEE NO
REGULATION WHATSOEVER.
EACH THOUGH THE PROTEIN IS NOT
WELL EXPRESSED AND THIS IS NOT A
MOUSE ENGINEERING ISSUE.
IT IS A PROTEIN STABILITY
PROBLEM.
IT SEEMS LIKE THE ACTIVE PROTEIN
IS TURNED OVER MORE QUICKLY.
I'LL GETBA CAN TO THESE MIGHT U.
MICE LATER ON.
THEY ARE NECESSARY AND
SUFFICIENT TO MEDIATE SO YOU
DELETE THEM.
THERE IS NO AMINO ACID
SIGNALING.
AND THIS IS NOW SHOWN IN A
NUMBER OF DIFFERENT ORGANISMS.
THE PART OF THIS STORY WHERE I
ARGUE IT BECAME MORE INTERESTING
AND LESS LIKE A CONVENTIONAL
SIGNALING STORY, IS THE ANSWER
TO THIS QUESTION.
HOW DO THEY WORK?
BECAUSE THE FIRST ASSUMPTION WE
MADE IS THAT THESE WERE
ACTIVATORS OF mTOR ONE KINASE
ACTIVITY LIKE REV AND WE SPENT A
LONG TIME TRYING TO PROVE THIS
AND WE HAD TO CONCLUDE THIS WAS
NOT THE CASE.
THE FIRST WILL YOU WE HAD AS TO
HOW THIS WAS HAPPENING WERE
MOVIES SUCH AS THIS.
THIS IS MT TORE C1.
NO AMINO ACIDS -- mTORC1.
THERE IS A DRAMATIC CHANGE IN
LOCALIZATION.
YOU ADD AMINO ACIDS AND WITHIN
MINUTES IT BECOMES PUNKITATE.
YOU CAN DO MINUS OR PLUS AMINO
ACIDS ALL YOU LIKE.
THERE ARE FOR SMALL PUNK TA AND
THEN THEY BECOME LARGER.
AND THEN IT TOOK US A LONG TIME
TO FIGURE OUT WHAT THESE ARE.
BUT THEY ARE LYSOSOMES.
WHAT IS HAPPENING IS THAT MT --
mTOR 1 COMES OFF THE SURFACE
WHEN YOU REMOVE THEM.
THIS PROCESS IS CONTROLLED BY
THE RAG GTPPASES.
THE CELLSY SHOWED YOU, mTORC1
NO LONGER CARES ABOUT AMINO
ACIDS.
AND THE RAGS, I HAVEN'T SHOWN
YOU THIS, ARE ALSO AT THE
LYSOSOMAL SURFACE.
DATA LED TO A MODEL WHICH I'M
ILLUSTRATING HERE, IN THE
ABSENCE OF AMINO ACIDS, WE
CONTINUING IS A MEMBRANE
COMPARTMENT BUT HAVEN'T BEEN
ABLE TO DEFINE WHAT THAT IS.
THE RAGS ARE ON THE LYSOSOMAL
SURFACE IN THIS NUCLEOTIDE STATE
AND WE HAVE QUITE GOOD EVIDENCE
THIS IS THE CASE FROM OUR LAB
BUT THERE WILL BE BETTER
EVIDENCE COMING OUT SUGGESTING
THIS IS THE CASE.
WHEN YOU ADD AMINO ACIDS, THE
NUCLEOTIDE STATE NOW FLIPS AND
mTORC1 DOCKS HERE AND IT CAN
INTERACT WITH THIS ACTIVATOR,
RHEB.
YOU NEED AMINO ACIDS TO GET TO
THE RIGHT PLACE AND YOU NEED ALL
THESE OTHER SIGNALS TO LOAD RHEB
WITH GTP.
WE HAVE DONE A LOT TO PROVE THIS
MODEL IN PUBLISHED DATA.
BUT THE KEY TOOLS WE USED IS A
MUTANT OF mTORC1 WE CAN PUT ON
THE SURFACE OF THE LYSOSOME IN
THE ABSENCE OF THE RAGS.
IF YOU DO THAT, YOU OBTAIN CELLS
THAT AGAIN HAVE CONSTITUTIVE
SIGNALING AND DON'T CARE ABOUT
AMINO ACIDS AND NO LONGER CARE
ABOUT THE RAGS.
YOU CAN DELETE THEM AND THEY
DON'T CARE AT ALL.
BUT THEY CARE ABOUT RHEB.
SO WE HAVE A LOT OF CONFIDENCE
THAT IN A BIG PICTURE POINT OF
VIEW, THIS MODEL IS CORRECT.
SO SOME OF THE QUESTIONS WE ARE
PARTICULARLY INTERESTED IN IS
THIS PATHWAY DEREGULATING
CANCER.
THAT'S WHAT I'LL SPEND THE LAST
HALF OF THE TALK TELLING YOU
ABOUT.
REALLY THE BIGGEST QUESTION IN
THIS FIELD IS WHAT AND WHERE IS
THE AMINO ACID SENSOR.
I'LL TELL YOU WE DON'T KNOW WHAT
THE AMINO ACID SENSOR IS.
IT'S NOT THE RAGS.
IT'S NOT ANY OF THE PROTEINS
THAT WE SEEM TO HAVE IDENTIFIED.
WE ALL STARTED TO BECOME
CONVINCED ON THE WHERE
COMPONENT.
AND THAT IS BECAUSE OVER AT LAST
FEW YEARS WE IDENTIFIED ANOTHER
COMPLEX WHICH IS ALSO ON THE
LYSOSOMAL SURFACE, A COMPLEX WE
CALL THE RAG LATER.
THIS COMPLEX SEVERS TWO
FUNCTIONS HERE.
IT TEATHERS THE RAGS TO THE
SURFACE OF THE LYSOSOME AND ACTS
AS A GEF FOR THE RAG A AND B
VERSIONS OF THE RAGS.
SO, THIS AT THE END WAS POINTING
TO SOMETHING THAT WE COULDN'T
IGNORE ANYMORE.
AND THAT IS THAT SO MANY OF
THESE COMPONENTS ARE ON THE
LYSOSOMAL SURFACE, RAILINGS, RAG
LATER AND RHEB AND mTOR 1.
NONE OF THESE PROTEINS, DESPITE
THE WAY THEY ARE DRAWN ARE
TRANSMEMBRANE PROTEINS.
SO EVOLUTION EITHER PICKED THE
LYSOSOME TO BUILD THESE
STRUCTURES BECAUSE THEY NEEDED
SOMEWHERE TO PUT THEM, OR THERE
IS SOMETHING SPECIAL ABOUT
LYSOSOMES AND AMINO ACID
METABOLISM AND WE FAVOR THAT
LATTER INTERPRETATION.
NOW I THINK IN RETROSPECT LIKE
MANY THINGS IN SCIENCE, WE
SHOULD HAVE BEEN ABLE TO GUESS
THIS.
BECAUSE LYSOSOMES IS THE PAYOFF
STEP FOR AUTOPHAGY.
mTORC1 IS THE MAJOR REGULATOR.
SO YOU WANT TO BE RIGHT THERE
WHEN YOU'RE PRODUCING MORE AMINO
ACIDS TO TURN OFF AUTOPHAGY.
IN MANY ORGANISMS, RAT LIVER,
LYSOSOMES AND IN THE VACCULES OF
ORGANISMS SUCH AS YEAST, AMINO
ACIDS ARE HIGH CONCENTRATIONS
AND SOME CAN REACH MOLAR AMOUNT.
WE DEVELOPED A HYPOTHESES THEN
THERE IS SOMETHING ABOUT
LYSOSOMES THAT IS KEY FOR AMINO
ACID METABOLISM AND THAT IS WHY
mTORC1 IS IN THIS COMPARTMENT.
NOW IT'S EASY TO ILLUSTRATE THIS
BUT THERE IS A SIMPLE QUESTION
THAT COMES OUT OF THESE KINDS OF
IDEAS THAT TURNS OUT TO BE
EXCEEDINGLY COMPLICATED TO
ANSWER.
AND THAT IS EXACTLY WHERE ARE
THE AMINO ACIDS SENTENCED?
YOU CAN IMAGINE THE SENSING
HAPPENS ON THE OUTSIDE OF THE
LYSOSOME OR THAT IT HAPPENS IN
THE INSIDE.
SO INSIDE-OUT TYPE OF MECHANISM.
NOW THIS IS EASY TO ASK,
EXTRAORDINARILY DIFFICULT TO
ADDRESS PARTICULARLY IN IN TACT
CELLS.
SO WHAT WE DID WAS DEVELOP AN IN
VITRO ASSAY WHERE WE CAN TAKE
HIGHLY PURIFIED LYSOSOMES, AND
HIGHLY PURIFIED mTOR 1 AND MIX
THEM AND ADD AMINO ACIDS AND
FIND THAT THESE NOW INTERACT,
WHICH TO US TELLS US THAT AT
LEAST THE BASIC COMPONENTS OF
THE AMINO ACID SENSING MACHINERY
ARE AT THE LYSOSOMES.
SO USING THE SYSTEM, WE HAVE
DONE A NUMBER OF DIFFERENT
EXPERIMENTS AND I'M GOING TO
GIVE YOU A SUMMARY.
WE ASKED VERY SIMPLE QUESTIONS.
DO YOU NEED A MEMBRANE?
NONE ARE DETERGENT SENSITIVE.
IF YOU POKE HOLES WITH A
DETERGENT, YOU GET NO SENSING.
DO YOU NEED THE AMINO ACIDS TO
BE IN HERE RATHER THAN OUT HERE?
WE HAVE DONE A NUMBER OF
DIFFERENT EXPERIMENTS TO ADDRESS
THIS.
THE ANSWER IS THEY HAVE TO BE
INSIDE.
DO YOU NEED THE V A TP A SE?
SO THIS IS PUMPING PROTONS IN
THE LYSOSOMAL LOOM IN AND THESE
PROTONS ARE USED FOR MANY THINGS
AS WELL AS COUNTER IONS TO BRING
IN OTHER THINGS LIKE AMINO ACIDS
EVEN WHEN WE PUT THE AMINO ACIDS
IN IN, IT IS STILL NECESSARY.
IT SEEMS TO HAVE AN ADDITIONAL
ROLE WE ARE STILL TRYING TO
UNDERSTAND.
SO I WANT TO TRY TO PROVE TO
YOU -- SORRY, WITH THE PROTON
GRADIENT IT TURNS OUT NOT TO BE
IMPORTANT.
ONCE WE GET AMINO ACIDS IN, WE
CAN GET RID OF THE PROTON
GRADIENT BUT THE VATPSE STILL
DOES.
I WANT TO PROVE ONE OF THESE
POINTS, THAT LUMINAL AMINO ACIDS
MATTER INSIDE OF THE CELL.
SO FIRST QUESTION IS, DO AMINO
ACIDS GIVEN ON THE OUTSIDE OF A
CELL GET INSIDE THE LYSOSOME?
TO DO THAT, WE DEVELOP A CELL
LINE WHICH WE CAN QUICKLY PURIFY
LYSOSOMES BECAUSE THEY HAVE A
TAGGED LYSOSOMAL PROTEIN ON
THEM.
WE CAN FEED CELLS IN A SHORT
PULSE AND THESE ARE BEADS THAT
HAVE LYSOSOMES ON THEM AND
MEASURE THE RATE OF ACTIVITY AND
WHEN YOU GIVE AMINO ACIDS, YOU
QUICKLY FIND IN 10 MINUTES EACH
IF YOU BLOCK PROTEIN SYNTHESIS,
YOU FIND THEM INSIDE OF
LYSOSOMES.
IF YOU USED DETERGENTS OR POKE
HOLES IN THE MEMBRANE, OBVIOUSLY
THE AMINO ACIDS COME OUT.
THE MORE INTERESTING CONDITION
IS WHEN YOU OVEREXPRESS THIS
PROTEIN HERE CALLED PAT 1.
SO WHAT IS PAT ONE?
IT'S CLEARLY ELIMINATING THE
AMINO ACIDS INSIDE THE LYSOSOME.
SO PAT ONE IS TURNED OUT TO BE A
VERY USEFUL TOOL.
IT'S A PROTEIN THAT IS
DISCOVERED BY OTHERS THAT
EXPORTS A VARIETY OF DIFFERENT
AMINO ACIDS OUTSIDE OF THE
LYSOSOME PRESUMABLY WHEN
PROTEINS ARE BROKEN DOWN, THE
AMINO ACIDS NEED TO GET OUT.
IT'S COMPLETELY LYSOSOMAL BASED
ON THE LYSOSOMAL MARKER.
AND SO IF OUR HYPOTHESES IS
CORRECT, THAT LYSOSOME -- YOU
NEED TO GET AMINO ACIDS INTO THE
LYSOSOMAL LUMEN, AND I JUST
SHOWED YOU IN THE PREVIOUS SLIDE
IF YOU OVEREXPRESS PAT 1, YOU
REMOVE AMINO ACIDS, THIS SHOULD
BE INHIBITOR OF THE mTOR
PATHWAY.
SO THAT'S EXPERIMENT THAT IS
SHOWN HERE.
HERE IS NO PAT ONE MINUS PLUS
AMINO ACIDS.
NICE ACTIVATION WHEN WE
OVEREXPRESS PAT ONE WE ELIMINATE
THIS ACTIVITY.
NOW, WHENEVER YOU INHIBIT A
SIGNALING PATHWAY, YOU HAVE TO
BE WORRIED YOU'RE NOT CAUSING
TOXIC EFFECTS.
WE CAN EXPRESS MUTANTS OF THE
RAGS, THE ONE I SHOWED YOU
BEFORE IN PARTICULAR, THIS GTP
LOCKED A OR B MUTANT AND BEFORE
I SHOWED THAT YOU RESCUESES
AMINO ACID STARVATION.
SO IT SHOULD THEN RESCUE THE
EFFECTS OF PAT 1 AND IN DEET IT
DOES.
NOT ONLY DO YOU EASE SENSITIVITY
TOW AMINO ACIDS BUT YOU LOSE
SENSITIVITY TOW PAT 1 OVER
EXPRESS.
SO OUR INTERPRETATION FOR THESE
RESULTS, AND I ADMIT ONE CAN
HAVE OTHER INTERPRETATIONS BUT
THE SIMPLE VEST ONE NEEDS TO GET
AMINO AS BOYS THIS LOOM TOINE
ACTIVATE THIS.
AND WE ARE VERY INTERESTED IN
WHAT THE SENSOR MIGHT BE.
SO THE MODEL THEN THAT WE HAVE
FOR THE SENSING MECHANISM IS
SOMETHING LIKE THIS.
WHEN THERE IS NO AMINO ACIDS,
mTOR ONE IS AN UNKNOWN
COMPARTMENT.
WHEN YOU ADD AMINO ACIDS, THEY
GET INTO THE LYSOSOME AND THERE
ARE TRANSPORTERS KNOWN TO DO
THIS.
YOU CAN IMAGINE OTHER
MECHANISMS, IT DOESN'T GO
THROUGH AMINO ACIDS BUT PEPTIDES
THAT GET DEGRADED OR PROTEINS
THAT ARE INTEGRATED IN THE
LYSOSOME.
ALL OF THESE ARE POSSIBLE AND
PROBABLY ALL GOING ON TO
DIFFERENTENTS AND DIFFERENT
CELLS.
NOW THE AMINO ACIDS, ONCE THEY
ARE HERE, WE KNOW THEY TALK TO
THE VACULAR ATP.
WE KNOW THAT THE STRUCTURE
CHANGES.
THERE IS CONFIRMATIONAL CHANGES
IN THE ATP ACE WHEN YOU ADD
AMINO ACIDS.
THE RAG LATER IS BOUND TO THE
VATPASE.
IT'S WELL BOUND.
AND WHEN YOU ADD AMINO ACIDS,
THERE IS CHANGE HERE WHICH THEN
REFLECTED IN A CONFIRMATIONAL
CHANGE IN RAG.
AND THESE ARE ALL THINGS THAT WE
HAVE LOOKED AT.
THIS SEEMS TO ACTIVATE THE
GEFFEN SO THIS NOW BECOMES
LOADED WITH GTP.
 GEFFEN.
AND IN A PART OF THE MODEL THAT
IS SPECULATIVE, WE THINK THE
RAGS COME OFF AT THAT POINT AND
THEY FIND mTORC1 AND BRING IT
BACK WE KNOW THIS EXISTS AS WE
CAN ISOLATE IT FROM CELLS.
AND THEN THIS GETS TURN TURNED
ON AND THIS DRIVES CELL GROWTH.
PART OF THE 39 POPS OFF IS THAT
SOME OF MY COLLEAGUES SAID,
DAVID, mTOR MOVES WAY TOO
QUICKLY TO THE LYSOSOME TO
SIMPLY BOUNCE AROUND BY
DIFFUSION TO GET THERE.
THERE MUST BE SOME ACTIVE
MECHANISM TO BRING IT THERE.
MOREOVER, WE HAVE SOME DATA FROM
PHOTO BLEACHING EXPERIMENTS THAT
THE RAG SEEMS TO BE DOING SOME
KIND OF CYCLING OFF THE SURFACE
OF THE LYSOSOME IN AN AMINO ACID
DEPENDENT FASHION.
NOW THIS IS OF COURSE A VERY
COMPLICATED MECHANISM AND THIS
IS WHERE WE HAVE GOTTEN.
SO IT'S NOT WHERE WE WANTED TO
GO NECESSARILY BUT THIS IS THE
DATE TELLING US.
BUT ONE OF THE THINGS THAT IT
MADE US THINK ABOUT IS A
QUESTION THAT IS IN THE BACK OF
OUR MIND FOR A VERY LONG TIME
AND THAT I FEEL THAT IF WE HAD
AN ANSWER TO IT, WE WOULD HAVE A
LOT OF CLARITY TOW WHAT IS GOING
ON.
AND THAT IS WHY ARE THE RAGS
HETERODIMERS?
NOTHING I JUST TOLD YOU MAKES
THEM HALF TO BE HETERODIMERS.
SO IF YOU THINK ABOUT RAS, A
PROTOTYPICAL SMALL PROTEIN, IT
COULD HAVE TWO STATES.
IF YOU THINK ABOUT THESE GUYS,
THEY COULD HAVE 4 STATES.
SO ONE OF THE WAYS WE ARE
THINKING ABOUT THIS SYSTEM IS
THAT WE NEED TO IDENTIFY WHICH
EACH OF THESE STATES DO.
WE ARE CONFIDENT ABOUT THESE TWO
AT THE END.
ONE IS AN mTOR BINDING STATE
AND THIS IS AN mTOR RELEASE
STATE.
BUT WE ARE STARTING TO THINK
THAT MAYBE THE CYCLING THAT I
MENTIONED HAS SOMETHING TO DO
WITH THESE TWO OTHER STATES.
SO, AGAIN, THIS IS A MODEL THAT
IS USED TO ILLUSTRATE HOW WE ARE
STARTING TO THINK ABOUT THIS
SYSTEM.
I WANT TO SWITCH GEARS BEFORE I
TELL YOU THAT THE CANCER STORY
ABOUT THESE MICE THAT I
MENTIONED IN WHICH WE KNOCKED
IN -- WE HAVE TWO ALLELES OF THE
GTP LOCKED FORM OF RAG A.
AND WHEN WE STARTED MAKING THESE
MICE, WE WERE HAVING LOTS OF
WHAT I WOULD SAY, RETROSPECT,
PIPE DREAMS.
WE IMAGINED WE WERE GOING TO
MAKE A MOUSE THAT WAS RESISTANT
TO AMINO ACID STARVATION  AND DO
AGING AND SIZE STUDIES BUT WE
NEVER GOT A LIVE MOUSE.
SO THAT HURT THOSE PLANS AND HE
COULD NEVER FIND A MOUSE.
HE FINDS PIECES OF MICE BUT
NEVER FOUND A LAB MOUSE.
SO HE STARTED GOING BACK IN
DEVELOPMENT SAYING WHEN DID
THESE MICE DIE?
HE NEVER FOUND THEM DEAD.
THEY WERE ALWAYS THERE.
THEY WERE ALWAYS AT MENDELIAN
RATIOS.
SO IF HE DIFFERED THEM BY
CESAREAN, NO PROBLEMS.
HERE ARE THE MUTANT MICE, THEY
ARE IN DISTINGUISHABLE FROM THE
HETEROZYGOUS OR WILDTYPE.
THEY ARE A LITTLE BIT SMALLER AS
YOU CAN SEE HERE, VERY, VERY
LITTLE BUT STATISTICALLY
SIGNIFICANTLY SMALLER.
WE HAVE DONE LOTS OF HISTOLOGY
ON THESE MICE AND CANNOT FIND
ANY DEFECT IN THESE ANIMALS YET
WE NEVER HAD A LIVE MOUSE.
WE HAD TO CONCLUDE THESE ANIMALS
WERE BEING BORN, DYING AND THE
MOTHER WAS LIKELY EATING THEM
BEFORE WE COULD FIND THEM.
AND SO TO DO THAT, TO TEST
WHETHER THAT IDEA WAS TRUE, HE
ISOLATED THEM BY CESAREAN AND
RESUSCITATED THEM AS YOU
TYPICALLY DO, AND HE PUT THEM IN
A CAGE, HUMIDIFIED CAGE AND
ASKED WHAT HAPPENS TO THESE
ANIMALS?
THESE ARE THE TYPE OF
EXPERIMENTS THAT ARE ROUTINELY
DONE WITH MICE, YOU PUT THEM IN
THE ABSENCE OF THE MOTHER AND
LOOKING AT THE LONGEVITY OF THE
MOUSE IN A STARVATION SITUATION.
AND VERY, VERY TYPICALLY, A
MOUSE WILL TYPICALLY LIVE
SOMEWHERE BETWEEN 20-24 HOURS IN
THIS SITUATION.
A WILDTYPE MOUSE.
THESE MICE LIVE ABOUT HALF THAT
PERIOD OF TIME.
VERY, VERY CONSISTENTLY.
NOW, THIS IS NOT SOME CONSULT
DEFECT THAT e-LICITS ITSELF
POSTNATALLY BECAUSE WE CAN
RESCUE THIS BY GIVING THEM
RAPAMYCIN.
SO IF YOU GIVE RAPAMYCIN, THIS
LINE SHIFTS OVER HERE.
SO THIS IS ABARENT mTOR 1
ACTIVITY.
MANY WILL RECOGNIZE THAT THIS
TYPE OF PHENOTYPE, THIS SORT OF
PERINATAL LETHALITY, DIFFICULTY
WITH DEALING WITH STARVATION, AS
WELL AS THAT SMALL CHANGE IN
SIZE THAT I MENTIONED THAT IS
SIGNIFICANT, THAT THIS IS
CHARACTERISTIC OF MICE THWART
MUTANT FOR THE AUTOPHAGY
PATHWAY.
THEY HAVE THESE TACT TWO
PHENOTYPES.
MICE BORN WITHOUT VERY MUCH FAT
OR ANY FAT AT ALL, REQUIRE AWE
TOUGH GEE TO SURVIVE THE
PERINATAL PERIOD TO BREAKDOWN
MUSCLE TO GENERATE ENERGY AND
RESOURCES TO LIVE FOR SURVIVOR
OF ANIMAL IN THE WILD, BEING
ABLE TO LIVE AFTER YOU'RE BORN
WITHOUT FEEDING MIGHT BE
SOMETHING THAT IS REALLY
SELECTIVE FOR QUITE STRONGLY
BECAUSE THE MOTHER MIGHT NOT BE
ABLE TO FEED THE PUPS RIGHT
AWAY.
SO, I'LL TELL THAW THESE MICE
HAVE A SIGNIFICANT, I WOULD SAY
COMPLETE DEFECT IN AUTOPHAGY AT
THIS POINT AND WE HAVE DONE ALL
THE TYPE OF EXPERIMENTS, EM.
LC3.
ALL THOSE EXPERIMENTS.
I'M NOT GOING TO SHOW YOU THOSE.
I'M GOING TOY SHOULD YOU THE
OUTPUT, AMINO ACIDS IN THE BLOOD
T BREAKS DOWN PROTEIN AND
RELEASES AMINO ACIDS.
YOU CAN SEE THAT TIMES 0 RIGHT
AFTER BIRTH, CESAREAN BIRTH, NO
DEFECTS IN THE AMINO ACIDS.
DOESN'T MATTER ON THE GENOTYPE.
BY 10 HOURS, THERE IS A
SIGNIFICANT DEFECT IN THE AMOUNT
OF AMINO ACIDS IN THE BLOOD OF
THE MUTANT ANIMALS.
NOW BY 10 HOURS, THIS IS
STARTING TO GET QUITE CLOSE WHEN
THESE ANIMALS ARE GOING TO START
TO DIE.
A COUPLE OF HOURS AFTER THIS.
SO WE DECIDED TO GO FURTHER BACK
AND ASK, WHAT HAPPENS TO THE
ACTIVITY OF THE mTOR PATHWAY
RIGHT AFTER BIRTH?
AND THIS WAS QUITE SURPRISING TO
US.
WHAT YOU'LL SEE HERE, THIS IS IN
THE LIVER.
HERE ARE SEVERAL GENOTYPES.
YOU CAN SEE THAT THERE A LITTLE
BIT OF NOISE BUT AT THYMES NOISE
THERE IS ACTIVATED PATHWAYS.
BY ONE HOUR OF FASTING, THE
HETEROZYGOUS AND WILDTYPE
ANIMALS DOWN REGULATED THEIR
MTOR PATHWAY AND THE MUTANT HAS
NOT.
THIS IS IN THE LIVER, THIS IS IN
THE HEART, PRETTY MUCH EVERY
TISSUE SHOWS THIS.
SO WHAT WE HAVE TO CONCLUDE FROM
THIS EXPERIMENT IS THAT WITHIN
ONE HOFFURE LIFE, SOMETHING HAS
DROPPED IN THESE ANIMALS THAT
THE mTOR PATHWAY HAS SENTENCED
AND -- SENTENCED AND TURNED OFF
THE PATHWAY.
WHATEVER THAT THING IS, THESE
MICE HAVE NOT SENTENCED AND HAVE
NOT TURNED OFF THE PATHWAY.
SO THE QUESTION BECAME, WHAT IS
THIS THING?
IF YOU LOOK AT AMINO ACID LEVELS
AT ONE HOUR OF LIFE, THERE IS
NOT A HUGE EFFECT.
RELATIVELY LITTLE EFFECT ON
AMINO ACIDS.
WE WERE SURPRISED THOUGH WHEN WE
LOOKED AT GLUCOSE LEVELS.
THAT IS SHOWN HERE.
HERE ARE THE CONTROL ANIMALS.
YOU CAN SEE THAT YOU STRUGGLE
RELATIVELY LOW AMOUNTS OF
GLUCOSE, CHARACTERISTIC FOR
NEWBORN MICE.
BY ONE HOUR, THIS PLUMMETS.
THIS IS UNDERESTIMATION OF HOW
FAR IT GOES BECAUSE WE ARE USING
A FLEW COM TERWHICH HAS THIS
SENSITIVITYEE - GLUE COM TER.
IF YOU CHEMICALLY MEASURE
GLUCOSE, WE HAD SOME ANIMALS
THAT WE CAN'T MEASURE GLUCOSE AT
THIS POINT AT 24 HOURS THIS IS
WHEN THESE MICE DIE.
WHY DOES THIS GO UP?
AUTOPHAGY IS PRODUCING AMINO
ACIDS AND THE LIVER IS TAKING
THEM IN AND MAKING GLUCOSE AND
RELEASING IT.
THE GLIKE JEN STORES IN THE
LIVER ARE BEING BROKEN DOWN TO
MAINTAIN THIS BLOOD GLUCOSE.
IF WE GIVE THEM GLUCOSE, WE CAN
PROLONG THE SURVIVAL.
SO WE THAN A DEFECT IN
GLUCOHOMEOSTASIS IS LEADING TO
THEIR DEMISE.
SO THESE ARE CONTROLS.
WHAT HAPPENS TO THE MUTANTS?
THE BEGINNING RESPONSE IS
IDENTICAL.
THEY PLUMMET.
BUT THEY NEVER RECOVER.
AND THE REASON IS THEY NEVER
SUPPORT THE AMINO ACIDS WHICH IS
NOT SUFFICIENT TO THE DRIVE
60s R. SINT SIS.
WE CAN RESCUEY THEM BY GIVING
THEM GLUCOSE.
NOW, IF YOU HAVE BEEN PAYING
ATTENTION, YOU'LL THINK THERE IS
SOMETHING WRONG WITH WHAT I SAID
GLUCOSE PRETTY MUCH PLUMMETS TO
ZERO BY ONE HOUR OF LIFE AND YET
THIS MUTATION IN THIS AMINO ACID
SENSING PATHWAY IS PREVENTING
THIS RESPONSE TO THESE
EXCEEDINGLY LOW LEVELS OF
GLUCOSE.
SO THIS SENSES GLUCOSE BUT WE
EXPECT IT TO BE OFF.
AND WE MADE A MUTATION.
SO THIS STRUCK US AS ODD.
THESE ARE RESIST TONIGHT THIS
FACTING AFFECT BY ONE HOUR.
WE THOUGHT THAT GLUCOSE IN THIS
PATHWAY WAS SENTENCED AS AN
ENERGY STORE.
BUT WE HAVE A LOT OF EVIDENCE
NOW THAT THIS IS NOT ONLY THE
CASE, THAT GLUCOSE MIGHT BE
SENTENCED AS A MOLECULE FOR
ITSELF, CARBON SOURCE AND THAT
THIS IS HAPPENING THROUGH THIS
VATPASE RAG LATER RAG PATHWAY
AND THIS IS A SLIDE WE HASN'T
DONE SO MUCH BUT WE ACTUALLY
KNOW THIS IS THE CASE.
SO THE STUDY REVEALED A KEY ROLE
FOR THE RAGS IN NUTRIENT
HOMEOSTASIS AND REVEALED THE
PATHWAY WE WERE BEING MY OPTIC
AROUND AMINO ACID SENSING AND
SEEMS TO BE GLUCOSE AND AMINO
ACID SENSOR.
THESE ANIMALS AT THIS EARLY
STAGE OF LIFE WHEN IT IS KNOWN
THERE IS NO INSULIN, SO IT
DOESN'T COME ON UNTIL THEY START
TO SUCKEL, THESE ANIMALS ARE
ACTINGS AS YEAST CELLS BECAUSE
THEY ARE CARING FOR NUTRIENTS,
GLUCOSE AND AMINO ACIDS AND THEY
ARE DOING IT THROUGH WHAT SEEMS
TO BE THROUGH A DIRECT NUTRIENT
SENSING TYPE OF PATHWAY AS A
SINGLE CELL ORGANISM SUCH AS
YEAST WOULD DO IT.
AFTER WEANING AND AFTER THE
INTRODUCTION OF GROWTH FABBER
SIGNALING, THIS WHOLE BRANCH
MIGHT BECOME MORE IMPORTANT.
OKAY, I'M GOING TO TELL YOU A
NEW STORY THAT HAS BEEN
SPEARHEADED BY TWO REALLY
FANTASTIC MIT STUDENTS.
THEY COLLABORATED FULLO THIS AS
AN EQUAL COLLABORATION.
AND WE ALWAYS HAVE BEEN
INTERESTED IN THIS QUESTION.
IS THE RAG PATHWAY DEREGULATING
CANCER?
ONE DOESN'T HAVE TO BE ANY KIND
OF SAGE TO POSE THIS QUESTION
BECAUSE I HAVE ALREADY SHOWN YOU
THIS SLIDE.
EVERY OTHER INPUT TO THIS
PATHWAY HAS A TUMOR SUPPRESSOR
IN IT.
IF THERE IS ONE ON THE ENERGY
SENSING PATHWAY, WHY SHOULDN'T
THERE BE ONE IN THE AMINO ACID
SENSING PATHWAY?
OF COURSE WE LOOKED FOR
MUTATIONS AND ALL THE COMPONENTS
I TOLD YOU ABOUT, PARTICULARLY
IN THE RAGS AND THERE ARE A
COUPLE OF VERY RARE MUTATIONS IN
THE RAG PROTEINS.
THIS STARTED BEFORE WE
DISCOVERED THIS RAG LATER
COMPLEX BUT IT MOVED SLOWLY FOR
A NUMBER OF DIFFERENT REASONS
AND IT STARTED WITH THE
IDENTIFICATION OF THIS PROTEIN.
SO WE HAVE DONE VERY EXTENSIVE
PROTEOMICS ON PROTEINS THAT
INTERACT WITH THE RAGS.
AND AT THE TIME WE IDENTIFIED
THIS PROTEIN, THERE WAS NOTHING
KNOWN ABOUT IT.
IT WAS IDENTIFIED GENETICALLY IN
A SCREEN, ORGANISM THAT HAD
PROBLEMS.
THE SEQUENCE TOLD US NOTHING.
THE PHENOTYPE TOLD US NOTHING SO
WE DIDN'T KNOW WHAT TO DO WITH
IT.
WE DECIDED TO WORK ON IT A
LITTLE BIT.
WE GENERATED ANTIBODY AND
STARTED KNOCKING IT DOWN AND
WHAT WE FOUND IS THAT QUITE
STRONGLY INHIBITED THE ACTIVITY
OF THIS PATHWAY.
SO THIS IS IN HUMAN CELLS AND WE
DID IN DROSOPHILA CELLS AND
KNOCKING DOWN ONE OF THE RAGS IT
INHIBITED THIS PATHWAY.
A CHARACTERISTIC SIGN WE ALWAYS
GET WHEN WE INHIBIT THE TORE
PATHWAY IN DRASOPHLA CELLS.
WE HAD TO CONCLUDE THAT THIS
PROTEIN WAS A POSITIVE
COMPONENT.
BUT THAT'S ALL WE KNEW.
AND SO WE DID WHAT WE REALLY
KNOW BEST HOW TO DO AND THAT IS
WE PUT THIS BACK IN CELLS AND
SAID DOES IT BIND TO ANYTHING?
I SHOULD SAY, WE COULD NEVER GET
THESE TO INTERACT INCH VITRO
TOGETHER.
THE COIP WAS WEAK AND WE COULD
NEVER GET INTERACTION.
SO MAYBE THERE ARE THINGS
MISSING.
THERE WERE WHEN WE PULLED OUT
MIOS, THERE WERE OTHER PROTEINS
BOUND TO IT.
THIS SAY COMPLEX FOR REASONS WE
CALL THE GATOR COMPLEX.
THEN WE FAGGED THE PROTEINS AND
ASKED WHO THEY BIND TO --
TAGGED -- THERE ARE TWO
COMPLEXES HERE.
IF YOU TAG ANY OF THE RED
PROTEINS FAVORING DOWN MOSTLY
THE RED PROTEINS IF YOU DOT
OPPOSITE EXPERIMENT, TAG THESE
PROTEINS, THEY MOSTLY BRING DOWN
THE RED PROTEINS AND LOW AMOUNTS
OF THESE LIGHT BLUE ONES.
SO THE CONCLUSION WE DREW FROM
THIS IS THERE ARE TWO
SUBCOMPLEXES THIS IS WHY WE
COULD NEVER GET THESE TO
INTERACT WITH THE RAGS.
SO THIS COMPLEX WAS QUITE
INTERESTING BECAUSE A SIMILAR
COMPLEX ALTHOUGH NOT EXACT, HAS
BEEN IDENTIFIED IN YEAST AS
HAVING A ROLE IN AUTOPHAGY IN
CERTAIN STRAINS AS WELL IN
NITROGEN SENSING.
SO THIS LED US TO BELIEVE THAT
THIS WAS AN INTERESTING CONFLICT
ALTHOUGH IN YEAST IT SEEMS TO BE
A COMPLEX WHILE HERE THERE ARE
TWO COMPLEXES.
WE HAD FOUND THIS ONE PROTEIN AS
A POSITIVE COMPONENT OF THIS
PATHWAY.
HOW MANY ALL OF THESE OTHERS?
SO WE STARTED DOING LOTS OF LOSS
OF FUNCTION EXPERIMENTS HERE IN
HUMAN CELLS AND KNOCKED DOWN
THIS ONE AGAIN LOOKING LIKE A
POSITIVE COMPONENT AND HERE IS A
KNOCK DOWN OF ANOTHER ONE AND IT
LOOKS LIKE IT'S POSITIVE.
WE DID ALL THE OTHER ONES IN
HUMAN CELLS.
WE ALSO DID IN FLY CELLS AND SO
AT THE END OF THE DAY, ALL OF
THESE GUYS ACT AS POSITIVE
COMPONENTS OF THIS PATHWAY.
THEN WE TURNED TO THIS COMPLEX.
AND HERE I THINK THINGS GOT MORE
INTERESTING FOR US.
WE STARTED SEEING THIS A RESCUE.
HERE IT IS WEAK AND IT'S NOT A
GREAT KNOCK DOWN.
SO HERE IS WC5 AND YOU CAN SEE
THE KNOCK DOWN IS BETTER AND WE
GET BETTER RESCUE AND THE SAME
THING WITH THE OTHER GENE AND WE
GET EXACTLY THE SAME THING IN
FLY CELLS EXCEPT WHEN WE DO THE
FLY CELLS WE GET HYPERACTIVATION
IN THE -STATE WHICH I'LL SHOW
YOU AFTERWARDS -- A MINUS STATE.
WE THINK THIS IS A FLY-SPECIFIC
PHENOMENON.
THE CONCLUSION IS THIS IS A
NEGATIVE REGULATOR OF THE
PATHWAY AND THIS IS POSITIVE
REGULATOR.
SO THESE ARE INTERACTING SUB
COMPLEX THAT IS HAVE THESE TWO
PROPERTIES.
THE QUESTION BECOMES WHAT IS THE
RELATIONSHIP BETWEEN THESE TWO?
AND SO THE WAY WEED LIKE DO DO
THIS IS WHERE WE HAVE MUTANTS OF
EACH ONE BUT BEE DON'T HAVE
THAT.
WE HAD TO DO THE NEXT BEST
THING, DOUBLE RNAI IN DROSOPHILA
CELLS WHEN YOU GET GOOD
KNOCKDOWNS AND CONFIDENT THAT
VERY LITTLE AMOUNT OF THE
MESSAGES ARE LEFT.
THIS GIVES MEET OPPORTUNITY TO
SHOW THE YOU PHENOTYPE IN
DRASOPHLA CELLS.
HERE IS WHEN WE KNOCKED DOWN A
GATOR ONE COMPONENT.
THIS IS TRUE OF ALL OF THEM.
THERE IS HYPERACTIVATION IN THE
-STATE.
AND WE THINK THIS REFLECTS A
FEEDBACK LOOP THAT IS FLY
SPECIFIC.
YOU WILL SEE A TRUE NULL HUMAN
CELL AFTERWARDS.
IN CONTRAST, WHEN WE KNOCKED
DOWN THESE GUYS, IT'S A CLEAR
POSITIVE COMPONENT.
NOW WE CAN COMBINE THIS RNAI
REAGENTED WITH THESE AND ASK WHO
WINS?
AND IF YOU DO THAT, YOU GET A
CLEAR ANSWER.
VERY CLEAR THAT THE GATOR 1
KNOCKDOWNS WIN.
NOW AGAIN, THE SIMPLEST
INTERPRETATION OF THIS IS THAT
GATOR 1 IS DOWNSTREAM OF GATOR 2
BUT IT'S NOT THE ONLY ONE.
I FULLY ACCEPTED THE OTHER
INTERPRETATIONS BUT THAT'S THE
CONCLUSION THAT WE HAVE GONE
WITH IS THIS IS THE RELATIONSHIP
BETWEEN THESE TWO COMPLEXES.
AND USING THE RAG MUTANTS, IF
YOU DO THOSE KINDS OF
EXPERIMENTS COMBINING LOSS OF
GATOR WITH EXPRESSION OF THE
RAGS OR OVEREXPRESSION OF GATOR
WHICH INHIBITS THIS PATHWAY, YOU
GENFIND THE RAGS ARE DOWNSTREAM
OF GATOR 1.
SO I TOLD YOU THAT LOCALIZATION
SAY REALLY IMPORTANT THING IN
THIS PATHWAY SO DO THESE GATOR
PROTEINS FIT THAT MODEL?
AND THEY DO.
WELL AND THIS MIGHT BE HARD TO
SEE BUT HERE IS IN THE ABSENCE
OF AMINO ARSENIDES A CONTROL
KNOCKDOWN YOU SEE THIS DIFFUSE
mTOR PATTERN AND ADD A AMINO
ACID -- THIS IS A MARKER OF
LYSOSOMES.
KNOCK DOWN THESE COMPONENTS, YOU
SEE A DIFFUSE PATTERN EVEN WHEN
AMINO ACIDS ARE PRESENT IN
CONTRAST IN THE CONTROL
SITUATION.
IN CONTRAST, IF WE KNOCK DOWN
THIS POSITIVE COMPLEX, A
COMPONENT OF THIS, YOU CAN START
TO SEE THESE PUNK THAT APPEAR
EACH WHEN THERE IS NO AMINO
ACIDS -- FURTHERRA.
EYE WILL SHOW YOU BETTER DATA IN
A TRUE NULL SITUATION.
SO, WHAT MIGHT BE THE MOLECULAR
FUNCTION OF THIS COMPLEX?
SO PARTICULARLY OF THE GATOR 1
COMPLEX.
THIS IS THE FIRST TIME IN THIS
PATHWAY THAT WE HAVE IDENTIFIED
WHO I WOULD SAY IS A BONA FIDE
NEGATIVE REGULATOR IN THISES IS.
NOW IN ALL SMALL -- IN THIS
SYSTEM.
IN ALL PROTEINS IN PARTICULAR,
WE KNOW SOME OF THE KEY
REGULATORS OF THE NUCLEOTIDE
STATE AND SO FOR EXAMPLE,
GEFs, HELP THE PROTEIN GO FROM
THE GDP STATE TO THE GTP STATE
AND WE KNOW THAT THE RAG LATER
IS THE GEF FOR RAG A AND B NOT
FOR C AND D.
THE OTHER SIDE TO THIS IS THE
GAP.
AND THESE STIMULATE WHAT CAN BE
VERY LOW INTRINSIC GTP ACTIVITY.
MOREOVER, GAPS ARE TRA DISHY
MUTATED IN CANCER PATHWAYS.
SO NF1 ON THE RAS PATHWAY AND
TSC2 IN THE mTOR REG PATHWAY.
SO WE ASKED COULD GATOR ONE BE
THE GAP FOR THE REG?
SO WE WANTEDDED TO ASK, DO THEY
HAVE HIGH INTRINSIC GTPASE
ACTIVITY?
BECAUSE THESE ARE HETERODIMERS,
THIS COMPLICATES THE ANALYSIS OF
NUCLEOTIDE STATE TREMENDOUSLY.
SO WHAT WE HAVE DONE IS MUTATE
ONE OF THEM INTO A BINDING G
PROTEIN N-THIS CASE RAG C.
SO IT DOESN'T BIND THE
NUCLEOTIDE WE PUT IN THAT IS
LABELED.
HERE IS CONTROLLED PROTEIN T
ALSO HAS NO ACTIVITY.
YOU CAN SEE WHEN YOU ADD GATOR
1, YOU GET NICE ACTIVITY IN THIS
CASE.
THIS IS AGAINST RAG A AND RAG B.
YOU HAVE IDENTICAL AS A RESULT
NO ACTIVITY AGAINST THE RAG C
FORM SO IF WE MAKE A VERSION OF
RAG A AND CREE ON GTP, WE GET NO
HYDROLYSIS.
SO WE THINK THAT WRONG OF THE
FUNCTIONS AND PERHAPS NOT THE
ONLY FUNCTION FOR GATOR 1 IS TO
ACT AS A GAP FOR THE RAG A AND
B.
AND THIS IS THE FACT WHERE THE
NAME COMES FROM.
GATOR IS GAP ACTIVITY TOWARDS
RAG.
NOT PARTICULARLY ORIGINAL BUT
GETS THE POINT ACROSS.
OKAY.
SO, GIVEN HOW I STARTED THIS AND
GIVEN THE FACT THAT THERE ARE A
NUMBER OF DIFFERENT TUMOR
SUPPRESSORS IN ALL OF THESE
PATHWAYS, WE WANTED TO ASK THE
QUESTION, IS GATOR ONE MUTATING
CANCER?
IS IT APPROPRIATE FOR ME TO
COLOR THIS IN THIS LIGHT BLUE
COLOR SUCH AS THESE OTHER
PROTEINS ARE?
AND SO TO DO THIS, WE STARTED TO
INTERROGATE THE TCGA DATA IN THE
CANCER GENOME ATLAS AND WE DID
THIS WITH MAT MEYERS IN HIS
GROUP AND HIS LAB AND WE HAD THE
BEST DATA OR THEY HAD THE ACCESS
TO THE BEST DATA FOR
GLIOBLASTOMA AS WELL AS OVARIAN
CANCER.
THAT IS THE DATA THAT I'LL SHARE
WITH YOU.
AND INDEED, WHEN WE DO A
RIGOROUS ANALYSIS HERE, WE'RE
ONLY LOOKING FOR MUTATIONS THAT
WE ARE CERTAIN WILL ELIMINATE
THE ACTIVITY OF THE PROTEIN.
WE FIND THAT THERE IS A FAIR
NUMBER, NOT HUGE, ABOUT 3% IN
GLIOBLASTOMA AND 2% IN OVARIAN
CANCER.
THIS IS ONLY ANALYZING THESE
TWO.
THE MPRL3 IS IN A LOCATION FOR
WHICH THEY DID NOT HAVE
CONFIDENCE IN THE CGL DATE TO
KNOW WHETHER THIS GENE WAS BEING
LOST.
SO I THINK THESE NUMBERS WILL GO
UP A LITTLE BIT.
NOW INTERESTINGLY, THERE HAS
ALREADY BEEN CLINICAL TRIALS
WITH RAPPA MICE IN IN
GLIOBLASTOMA AND THERE WERE
RESPONDERS IN SOME OF THESE
TRIALS BUT WE DON'T HAVE
RESPONDER IDEAS IN THAT WE CAN
USE THE ENCYCLOPEDIA, INCREDIBLY
USEFUL RESOURCE AND THERE WE
DON'T HAVE MUTATION DATA BECAUSE
THESE GENES ARE NOT STUDIED BUT
WE DO HAVE VERY GOOD COPY NUMBER
DATA.
WE INTERROGATED THAT DATASET AND
LOOKED FOR CELL LINES THAT HAD
DOUBLE LOSS.
SO THEY WERE HOMOZYGOUSLY
DELETED FOR THESE GENES.
WE DON'T THINK WILL BE A COMMON
MECHANISM OF LOTS.
WE THINK THERE WILL BE A
DELETION PLUS AWE MUTATION.
THAT'S ALL WE COULD LOOK AT AND
WE PULLED OUT OF THAT RESOURCE
CELL LINES THAT WERE DELETED FOR
ANY OF THESE THREE GENES AND
CONFIRMED THIS BY GENOMIC PC R
AND THEY ARE GONE.
SO THE QUESTION BECOMES, WHAT IS
THE EFFECT ON SIGNAL
TRANSDUCTION WHEN YOU DELETE
THESE GENES IN A REAL HUMAN
CANCER CELL LINE?
SO HERE ARE CONTROL CELL LINES.
NICE SIGNALING.
SO IF THE CELL LINE IS
MISSING&YOU CAN SEE THERE IS NO
SIGNALING.
AND THERE IS REALLY TWO
PHENOTYPES WE NOTE.
ONE IS HYPERACTIVATION OF THE
PATHWAY COMPARED TO THE CONTROL
CELLS.
THERE IS REALLY NO SIGNALING AT
ALL.
THESE LOOK VERY, VERY CLOSE TO
THOSE RAG AGDP MESS WHERE WE
KNOCKED IN A GTPASE DEFICIENT
VERSION OF RAG A.
SO THIS IS NPRL2 AND HERE IS 3.
THE SAME PHENOTYPE.
HERE IS DEPC5, THE SAME
PHENOTYPE.
THESE DATA ARE VERY SUPPORTIVE
OF THE LOSS OF FUNCTION DATA WE
DID IN DRASOPHLA CELLS AND HUMAN
CELLS SUGGESTING THAT THESE ARE
MAJOR NEGATIVE REGULATORS OF THE
AMINO ACID SENSING PATHWAY AND
THAT WHEN REMOVING THEM OR
NATURALLY HAVE THEM REMOVED, WE
DON'T HAVE SENSING OF THE
ABSENCE OF AMINO ACIDS.
THIS EXPERIMENT WAS DONE OVER AN
HOUR.
WE HAVE DONE REALLY CRAZY
EXPERIMENTS EACH 24 HOURS OF
AMINO ACID DEPRIVATION.
CELLS BE PROBABLY STARTING TO
GET SICK AND EVEN THEN, THEY ARE
NOT SENSING THE ABSENCE OF AMINO
ACIDS.
THIS SEEMS TO BE A KEY MECHANISM
IN THE DOWNREGULATION OF THIS
PATHWAY.
NOW IT'S FAIR TO SAY, HAVE YOU
ALL THESE CELL LINES BUT THEY
HAVE ALL THE OTHER MUTATIONS IN
THEM.
NOT JUST THESE.
HOW DO YOU KNOW THESE MUTATIONS
ACCOUNT FOR THIS PHENOTYPE?
WELL, WE DON'T BECAUSE WE
HAVEN'T CHECKED ALL THE OTHER
MUTATIONS SO THEY ADD BACK THE
GENES.
NOW IT TURNS OUT THAT FOR MANY
TUMOR SUPPRESSORS, IT'S HARD TO
ADD BACK THE GENE BECAUSE THE
CELL LIKES THE FACT THEY LOST
THAT GENE.
THIS IS THE CASE HERE.
WE HAVE FAILED TO ADD BACK THE
GENES THE VAST MAJORITY OF CELL
LINES.
WE SUCCEEDED TO ADD BACK THIS.
THIS IS BARELY DEDUCTIBLE BY AN
ANTIBODY BUT THAT A LITTLE BIT
OF EXPRESSION STARTS TO RESTORE
SIGNALING.
THE ONE CELL LINE WE CAN ADD IT
BACK TO IS HERE.
IS IT THE DEFECT OF THIS GENE
CAUSING THIS PHENOTYPE?
HERE IS THAT CELL LINE.
WE PUT A CONTROLLED PROTEIN AND
NO SENSING AND WHEN WE ADD BACK
THIS PROTEIN, SENSING COMES
BACK.
SO I'M CONFIDENT SAYING FOR THIS
ONE CELL LINE THIS GENE ACCOUNTS
FOR THIS DEFECT AND I WOULD BET
A LOT THAT THIS IS THE CASE AS
WELL.
NOW THIS IS NOT ONLY AT THE
SIGNALING LEVEL BUT ALSO AT THE
LOCALIZATION LEVEL.
YOU CAN SEE HERE IN THE CONTROL,
THE PARENTAL CELL LINE AND IN
THE ABSENCE OF AMINO ACIDS,
MTOR IS PURCHASING INDICATED.
NO DIFFERENCE.
WHEN WE ADD BACK THIS GENE,
MTOR BECOMES DIFFUSE WHEN YOU
REMOVE AMINO ACIDS.
OKAY.
SO IS THERE ANY POTENTIAL
PREDICTIVE VALUE IN TERMS OF
THERAPY FOR TUMORS THAT MIGHT
HAVE THESE MUTATIONS?
OF COURSE THERE ARE A NUMBER OF
DIFFERENT mTOR INHIBITORS
THERE.
THE RAPAMYCIN CLASS.
THERE IS LOTS OF WORK ON
CATALYTIC INHIBITORS AND THOSE
ARE STARTING TO GO INTO TRIALS.
OFES THO ARE mTOR SPECIFIC AND
SOME ARE PI3 KINASE AND mTOR
AS WELL.
WE ASKED, RAPAMYCIN, ALREADY
BEING USED IN CANCER, DO THESE
CELL LINES THAT I SHOWED YOU
HERE, ARE THEY PARTICULARLY
SENSITIVE TO RAPAMYCIN?
SO, MANY CELL LINES DON'T CARE
AT ALL ABOUT RAPAMYCIN.
WHO ARE TWO EXAMPLES OF THEM.
SO YOU CAN SEE, REALLY THE IC50
IS IN THE MICROMOLAR RANGE.
SOME CELL LINES DO CARE A LOT.
SO THIS IS ARGUED TO BE A
BIOMARKER FOR RAPAMYCIN
SENSITIVITY.
NOW HERE IN THE GATOR NULL CELLS
THEY ARE EXTREMELY SENSITIVE.
SO WE ARE HOPEFUL THAT AT LEAST
THIS IN VITRO EXPERIMENT MAY
HAVE SOME CORRELATE IN VIVO AND
SO WE HAVE DEVELOPED NOW A DEEP
SEQUENCING APPROACH TO SEQUENCE
THESE GENES PARTICULARLY
STARTING TO COLLABORATE A NUMBER
OF DIFFERENT GROUPS HAVE
PATIENTS IN RESPOND TO RAPAMYCIN
BUT WE DON'T KNOW WHY THEY
RESPOND.
SO AT LEAST SOME FRACTION WE'LL
SUSPECT WE'LL BE MUTATIONS IN
THESE GENES.
SO I TOLD YOU ABOUT THIS
NUTRIENT SENSING ARM, THIS
GLUCOSE AND AMINO ACID SENSING.
WE DON'T KNOW THE SENSOR YET.
OUR NUMBER 1 CANDIDATE THAT THE
VATPASE IS THE SENSOR.
THIS IS EXTREMELY DIFFICULT
COMPLEX TO WORK WITH SO WE HAVE
NOT HAD LUCK IN PURE TUIFYING
IT.
AND I ALSO TOLD YOU ABOUT THIS
NEW COMPONENT.
WE THINK THESE ARE MAJOR
REGULATORS OF THIS PATHWAY AND I
WOULD ARGUE THEY MAY BE SOME OF
THE MORE RELEVANT ONES IN THE
CANCER SITUATION.
THE CLOSEST TO THE mTOR
PATHWAY IN REGULATION AND
DEREGULATION IN CANCER.
NOW, THERE ARE 8 PROTEINS HERE
AND SO CLEARLY IT CAN'T BE THAT
THERE IS NOTHING UP STREAM OF
THIS.
WE HAVE NO IDEA WHAT THAT MIGHT
BE.
MOREOVER, THESE PROTEINS HAVE
NOTHING IN THEM THAT TELL WHAT
THEY MIGHT DO.
WE DON'T HAVE BEEN HOW THEY WORK
AT ALL.
SO WE HAVE A TREMENDOUS AMOUNT
OF WORK TO DO.
NOW IN THIS PATHWAY, REMEMBER
THE TSC COMPLEX?
THEIR THREE PROTEINS THERE AND
WE KNOW OF 10 DIFFERENT
REGULATORS.
SO I BET THESE EIGHT PROTEINS
THERE WILL BE A TREMENDOUS
NUMBER OF REGULATORY MECHANISMS
THAT COME INTO PLAY WITH THEM.
SO JUST IN CONCLUSION, I TOLD
YOU ABOUT THIS INSIDE-OUT MODEL
OF AMINO ACID SIGNALING AND
ABOUT HOW THIS POINT MUTATION IN
THE RAGS HAS A DRAMATIC EFFECT
ON AUTOPHAGY AND I DIDN'T SHOW
YOU THE DATA.
ANDNY NATAL NUTRIENT
HOMEOSTASIS.
THE AUTOPHAGY PATHWAY IS
SUPPOSED TO BE REGULATED BY MANY
DIFFERENT PATHWAYS BUT YET ONE
SEEMS TO ELIMINATE AUTOPHAGY
INDUCTION.
AND I TOLD YOU ABOUT A COMPLEX
MUTATING CANCER AND WE THINK
THIS IS THE GAP.
IT HAS GAP ACTIVITY TOWARDS THE
RAGS.
IT MAY NOT BE THE ONLY GAP OF
COURSE.
THE WORK ON THE RAGS WAS
INITIATED BY YASEMIN AND IS THE
ONE WHO FOUND THE RAGS AS PART
OF THE mTOR PATHWAY.
LIRON DISCOVERED THE RAG LATER
COMPLEX.
ROBERTO HAS DONE A LOT OF WORK
ON THE INSIDE-OUT MODEL AND LYNN
AND ALEJO DISCOVERED THE MOUSE
AND THE COMPLEX.
I WANT TO THANK OUR FUNDING
SOURCES AND COLLABORATORS AS
WELL AS ALL OF YOU FOR PAYING
ATTENTION.
THANK YOU VERY MUCH.
[ APPLAUSE ]
Y.
>> THANK YOU FOR A VERY
INTERESTING PATHWAY.
WE HAVE TIME FOR QUESTIONS SO
PLEASE USE THE MICROPHONES IN
THE AISLES.
WHILE PEEP ARE -- PEOPLE ARE
GETTING THERE, WITH NOTION THAT
THERE IS SENSITIVITY TO THE
AMINO ACID CONCENTRATION, INSIDE
LYSOSOME, NOT ALL ARE CREATED
EQUAL.
DIFFERENT SHAPES.
THE KIDNEY HAS DIFFERENT PUMPS
FOR DIFFERENT CLASSES.
DO YOU KNOW WHETHER THERE IS A
DIFFERENCE IN WHICH AMINO ACIDS
ARE THERE AND HOW MUCH RESPONSE
YOU GET?
>> YES, SO THAT'S THE NUMBER 1
QUESTION WE ARE TRYING TO SUNDAY
WHICH AMINO ACIDS MATTER AND
WHY.
AND IT TURNS OUT THAT THERE ARE
MANY THAT MATTER.
SO THE CLASSIC SON LEWIS IN.
SO MANY, MANY PEOPLE HAVE SHOWN
THAT IT IS A KEY COMPONENT.
BUT IT'S NOT SUFFICIENT.
SO YOU CAN REMOVE MANY AMINO
ACIDS BUT YOU CAN'T ADD THEM
BACK AND GET REACTIVATION.
WE DON'T QUITE UNDERSTAND THAT.
SO WE CAN DO NECESSITY BUT WE
HAVEN'T BEEN ABLE TO DO
SUFFICIENCY.
NOW WE ARE NOW DOING METABOLITE
PROFILING IN LYSOSOMES SO WE CAN
PURIFY THEM OUT AND ASK WHAT IS
INSIDE THEM.
AND IT'S TECHNICALLY AS YOU
MIGHT IMAGINE, QUITE DIFFICULT
TO DO.
WE FIND LOTS OF AMINO ACIDS.
BUT IN VACCULES THERE IS LOTS OF
THE BASICS, ARGININE, LYSINE,
HIST TEEN.
THERE IS LESS OF THE ACIDIC
AMINO ACIDS BECAUSE OF THE
ENVIRONMENT.
WE ARE INTERESTED IN THAT AND IT
COULD BE THAT THE GATOR PATHWAY
SENSES ONE AMINO ACID AND THE
RAG LATER ARM SENSES ANOTHER AND
THERE IS INTEGRATION BUT WE
DON'T KNOW THAT.
>> YOU SHOWED THE CHANGES IN
AMINO ACIDS -- [ INDISCERNIBLE ]
I WAS WONDERING IF IT HAS ON
TOC2 AND HAVING EFFECT ON THE
Akt PATHWAY.
>> SO ASKING WHETHER AMINO ACIDS
AFFECT mTOR 2 LOCALIZATION?
>> YES.
>> WE LOVE TO DO THAT
EXPERIMENT.
THE BIG PROBLEM IS THAT WE NEVER
EVER BEEN ABLE TO LOCALIZE
MTOR 2.
SO WE DON'T KNOW WHERE IT IS.
AND THERE IS A NUMBER OF PAPERS
REPORTING -- LIKE BCL2 AT ONE
POINT.
mTOR 2 APPARENTLY IS
EVERYWHERE.
SO, I DON'T THINK ANY OF THAT IS
CORRECT, ACTUALLY.
IF YOU LOOK AT NULL CELLS, NONE
OF THE SIGNALS DISAPPEAR.
WE DON'T KNOW WHERE IT IS.
WE CAN'T DO THAT EXPERIMENT.
BUT WE WOULD VERY MUCH LIKE TO.
WE HAVE LOTS OF PEOPLE HAVE
TRIED THAT AND HAVE NOT
SUCCEEDED.
>> SECOND QUESTION.
YOU ALSO SHOWED THAT GLUCOSE
MIGHT HAVE TWO FUNCTIONS IN
TERMS OF EFFECTING THE mTOR 1
IN TERMS OF METABOLISM AND
AFFECTING ATPAs.
SO IN THE SECTION FUNCTION IN
THE ATPASE, DOES
NONMETABOLIZABLE CARBOHYDRATE OR
SO CAN FUNCTION IN THAT PATHWAY?
>> THAT'S AN INTERESTING IDEA.
SO LIKE GLUCOSE AND STUFF LIKE
THAT.
THE ONLY PROBLEM WITH THAT IS
THIS SYSTEM IS BUILT TO
INTEGRATE ALL THOSE SIGNALS SO
IF YOU GIVE SOMETHING
NONMETABOLIZABLE WHICH IS BE
INHIBITABLE, YOU WILL INHIBIT
THROUGH ENERGY SENSING.
SO WITH THESE MOLECULES THEY
WOULD THEY COULD HAVE MORE THAN
ONE FUNCTION.
IT'S HARD TO TEASE APART THOSE.
YOU MIGHT BE ABLE TO
GENETICALLY, BUT WE KNOW OF SO
MANY WAYS THAT THIS SENSES
ENERGY THAT THAT MIGHT BE QUITE
CHALLENGING.
BUT IT'S AN INTERESTING IDEA.
IN YEAST, THERE IS DATA
SUGGESTING THAT THE VATPASE
FALLS APART WHEN YOU DO GLUCOSE
STARVATION, IT ACTUALLY
SEPARATES.
WE HAVE TESTED THAT IN MAMMALIAN
CELLS AND DON'T SEE THAT
HAPPENING.
SO, IT DOESN'T SEEM THAT THAT IS
THE MECHANISM.
>> THANK YOU.
>> CAN YOU SAY SOMETHING ABOUT
THE DIVISION OF  LABOR BETWEEN
mTOR 1 AND 2, YOU SORT OF
IMPLIED THAT mTOR C1 WAS
SENTENCING GLUCOSE LOCALLY
WHEREAS 2 WAS SENTENCING IT
GLOBALLY THROUGH INSULIN.
IS THIS CONSISTENT FEATURE IN
TERMS OF DEVELOPMENT, PRE-POST
SUCKELLING AND ALSO
EVOLUTIONARILY SINGLE CELLS
VERSUS MULTICELLS?
>> SO LOTS OF INTERESTING IDEAS
THERE.
SO mTOR 2 IS IN YEAST IT'S NOT
SENSING INSULIN IN YEAST.
PROBABLY SOMETHING FROM
NUTRIENTS.
WE HAVE NICE DATA SUGGESTING A
SENSE FOR MEMBRANE STRESS IN
YEAST.
IN MAMMALIAN SYSTEMS, I THINK
mTOR 2,A THE LEAST IN THE STUFF
WE HAVE DONE IS QUITE A BIT LESS
INTERESTING THAN mTOR 1.
IT DOES LOOK LIKE A GROWTH
FACTOR REGULATED KINASE.
SO A NUMBER OF DIFFERENT GROWTH
FACTORS, INSULIN BEING THE BEST
STUDY AND ANY GROWTH FACTOR THAT
SEEMS TO TURN ON PI3 KINASE
SEEMS TO REGULATE ITS ACTIVITY.
WE HAVE NO EVIDENCE FOR
SENTENCING THINGS LIKE GLUCOSE
IN ALL IN ANY KIND OF DIRECT
WAY.
SO THAT IS WHY WE HAVE SOMEWHAT
FOCUSED MORE ON mTOR 1.
IT'S WEIRDER.
WHICH IS MORE FUN.
mTOR 2 THE BIG QUESTION IS HOW
IT IS REGULATED.
WHAT IS UP STREAM OF IT?
IT SEEMS MOSTLY FITS INTO GROWTH
FACTORS WHILE THE OTHER ONE
SENSES ALL OF THIS WEIRD STUFF,
IN INTERESTING WAYS.
>> WE ARE GOING TO HAVE A
RECEPTION IN THE LIBRARY SOY
THOSE WHO WOULD LIKE TO CONTINUE
THE CONVERSATIONS WITH THE
SPEAKER, PLEASE COME ALONG.
THERE MIGHT BE SOME EDIBLES
THERE AS WELL.
LET US THANK ONCE MORE TIME OUR
SPEAKER DR. SABATINI.
