SO WELCOME, EVERYBODY AND THANK
YOU FOR COMING.
AS WE BEGIN PLEASE NOTE THAT
THIS LECTURE IS BEING RECORDED.
I AM JOHN LORSCH AND I WANT TO
WELCOME YOU TO THE
DEWITT LECTURE, HE WAS THE THIRD
DIRECTOR TO NIGMS AND HAD A
STRONG BASIS IN CELLULAR, AND
MOLECULAR CHEMISTRY.
HE SPENT MOST OF HIS CAREER
SERVING AT 2 INSTITUTES AND AS
THE OFFICE OF DIRECTOR OF NIH
AND PARTICULARLY WELL KNOWN AS A
CO-AUTHOR OF THE EARLY ADDITIONS
OF THE TEXTBOOK PRINCIPLES OF
BIOCHEMISTRY.
IN RECOGNITION OF HIS MANY
ACHIEVEMENTS AND CONTRIBUTIONS,
BOTH WITHIN AND OUTSIDE OF NIH,
HE HAS THE RARE DISTINCTION OF
HAVING 2 NIH ENTITIES NAMED IN
HIS HONOR, THIS LECTURE AS WELL
AS THE DEWITT ST ADTMAN
BUILDING.
THE WAY THE LECTURE WORKS IS
THAT THE SPEAKER DR. RY YOUNG
WILL GIVE A 50 MINUTE TALK ON
HIS RESEARCH WHICH WILL BE
FOLLOWED BY A 10 MINUTE QUESTION
AND ANSWER SESSION.
YOU CAN ASK YOUR QUESTIONS BY
USING THE MICROPHONE IN THE ROOM
IF YOU'RE HERE WITH US IN PERSON
OR BY SELECTING THE ACCEPTED
LIVE FEEDBACK FROM THE VIDEOCAST
SCREEN IF YOU ARE YOINING US
VIRTUALLY.
IF YOU ARE SEEKING CONTINUE
MEDICAL EDUCATION CREDIT THE
CODE IS 5 WHERE ARE 098.
LET ME REPEAT.
FIVE WHERE .
5009ATE.
SO TODAY'S SPEAKER RY YOUNG JOIN
US A VERY DISTINGUISHED LIST OF
STADTMAN LECTURERS, HE IS THE
FOUNDER AND DISTINGUISHED
PROFESSOR EMERITUS AT
BIOCHEMISTRY AND BIOPHYSICS AT
TEXAS A&M UNIVERSITY.
HE STUDIED BACTERIAIA PHAGES AND
HOW THEY LYS THE CELLS.
EVEN WHEN OTHERS TURNED AWAY IN
THE FIELD HE SAW THE BENEFIT TO
USING THEM WITH THEIR BIOCHEMIST
RADIOY TO ASK SPECIFIC AND
IMPORTANT BIOLOGICAL QUESTIONS.
DURING TODAY'S LECTURE, RY WILL
DISCUSS HOW THEY CONTROL LYSIS
IN A TIMELY FASHION AND HOW THEY
MIGHT BE HARVESTED FOR
DEVELOPING NEW ANTIBIOTIC ANDS
THERAPY.
HE RECEIVED HIS Ph.D. IN ON
MOLECULAR BIOLOGY FROM THE
UNIVERSITY OF TEXAS AT DALLAS
ALSO A VETERAN OF THE NAVY AND I
THINK HE PROBABLY IS THE
STADTMAN LECTURE AND WALS
LECTURE WHO HAS THE MOST
IMPRESSIVE JOURNEY FOR HOW HE
GOT HERE, HE DIDN'T FLY, HE
DIDN'T TAKE A TRAIN.
HE ACTUALLY WITH HIS WIFE, DROVE
OR PILOTED HIS BOAT ALL THE WAY
FROM TEXAS, THROUGH THE GULF,
THROUGH FLORIDA, UP THE WATER
WAYS INTO THE CHESAPEAKE BAY.
[ APPLAUSE ]
SO THAT IS SOME SERIOUS
DEDICATION TO GET HERE.
SO THANKS FOR COMING RY.
IT'S IMRAIT --GREAT TO HAVE YOU
WITH US.
>> SO FIRST OF ALL, I'M SORRY
ABOUT WEARING MY GLASSES LIKE
THIS, I'M NOT TRYING TO IMITATE
THE OLD FUDY DUDY PROFESSOR BUT
I LOST MY BI FOCALS, MY WIFE
SAYS I RIDICULOUS, I HOPE THEY
DON'T FALL OFF.
THIS SLIDE IS TO MAKE SURE I
ACKNOWLEDGE PEOPLE WHO HAVE DONE
THE WORK, SEVERAL HERE IN THE
BUILDING AND I WILL COME BACK TO
THE VERY END BECAUSE THIS WAS
KIND OF THE WORK PRODUCT, THIS
NIGMS GRANT FUNDED THIS WORK FOR
OVER 40 YEARS AND PART OF
PRODUCT IS ACCIDENT WELL TRAINED
PEOPLE HAVE TBOAN ON TO
SUCCESSIVE SCIENCE IN MANY WAYS.
THE 1S IN RED ARE MEMBERS OF THE
BIOTECH COMPANY AND ALMOST ALL
THE OTHER 1S ARE ON THEIR WAY,
PHAGE BIOLOGY IS A GREAT SYSTEM
FOR TRAINING BECAUSE CAN YOU GET
SPRMS DONE QUICKLY, NOBODY THERE
IS STILL WORKING ON PHAGE LYSIS
OR EACH PHAGE OF THE IT'S BEEN A
GOOD SYSTEM IN THAT REGARD.
OKAY, SO IT'S A GREAT HONOR TO
BE OVER 40 YEARS AGO BEFORE THIS
STARTED THERE'S TILL NOT BEEN AS
FAR AS I CAN TELL, I'M THE FIRST
PHAGE GUY, WHICH IS REALLY WEIRD
SINCE NIGMS IS THE FIRST PLACE
TO GIVE A FAINL TALK, THE FITTER
1 WAS MARK [INDISCERNIBLE] BUT
HE MIGHT HAVE BEEN TALKING ABOUT
YEAST TRANSCRIPTION SO WE'RE
WRITING A HISTORICAL WRONG HERE.
ALL RIGHT, SO I CAN REMEMBER
THIS, IS A RECONSTRUCTION, NOT
THE ACTUAL, BUT BACK IN 1969 OR
70, NO IT WAS EARLIER THAN THAT,
IT WAS LATER THAN THAT IT WAS
72, I THINK, I WAS STANDING IN
JOHN [INDISCERNIBLE]'S LAB AT
HARVARD MEDICAL SCHOOL,IME 5
YEARS OLD, OLD FUDY DUDYS HAVE
TROUBLE WITH DECADES.
ANY RATE, I BORROWED HIS FLASK
AND HIS SPEC TOM TERTO FOLLOW
LAMDA INDUCTIONS, I WON'T TELL
YOU WHY I WAS DOING THESE LAMDA
INDUCTIONS BUT YOU CAN HEAT
SHOCK THE BACTERIA IN A CULT IRB
FLASK AND THEN THE PROPHAGE WILL
SUDDENLY INDUCE AND YOU GET
SYNCHRONOUS INFECTION THIS IS
WAY.
AND THAT'S THE HIGH VALUE.
SO I STARTED 5 CULTURES, I
LOOKED DOWN AND DISCOVERED, 3
MINUTES AGO THAT I HAD NOT
STARTED 1 OF THE CULTURES SO I
SHIFTED IT, SO 1 OF THESE TARTED
LATER, SO IF YOU LOOK AT IT ALL
THE CELLS IN THE ORIGINAL FLASK
HAVE LYSED AND NONE OF THE IN
THE ORIGINAL HAVE LYRK SED AND
IT IS ENACTED BY LAMDA, IT'S
HIGHLY CONTROLLED IN TERMS OF
TIME, SO THIS IS A--THIS IS AN
EVENT THAT CAN KEEP TIME ON THE
ORDER OF LOW MINUTES TO SECONDS.
AND I'VE HAD CHILDREN KEEP TRACK
OF TIME AND DAYS.
THIS TURNS OUT ALL TO BE DUE
SINGLE PROTEIN, THIS LECTURE, BY
THE WAY HAD IS A LECTURE SO YOU
WILL HEAR MOSTLY MY THEORIES ON
WHAT IS HAPPENING OVER SOMETHING
THAT HAS NO CONTROLS AND THEN I
WILL TRY TO COVER THE
ENTIRE--HOW MY UNDERSTANDING IS
OF THE ENTIRE SYSTEM THAT MAKES
GOING FROM THERE TO THERE.
LEGALITYEE SEE IF I CAN NOT
CRASH THE COMPUTER.
THIS IS WHAT THAT LOOKS LIKE AT
THE SINGLE CELL LEVEL AND THIS
IS ALL IN SECONDS HERE SO WE
LOOK AT IT IN MILLISECOND FUEL
AND NOTICE WHAT'S GOING ON,
NOTICE IT'S NOT JUST A GENERAL
BLOW OUT OF THE WHOLE THING,
THERE'S A SPECIFIC LESION THAT
OCCURS ISSUES AT 1 POINT IN THE
CELL, WE CALL THIS LOCAL BLOW
OUT SO WHATEVER THAT TIMING
EVENT THAT WE LOOK AT THE
MACROSCOPIC LEVEL OCCURS ALSO AT
THE SINGLE CELL LEVEL, THIS
HAPPENS AT A SINGLE PLACE AT A
SINGLE TIME AND FOR SEVERAL
SECURITIZATION.S AFTER THAT THE
REST OF THE STRUCTURE OF THE
CELL IS NORMAL AND THAT FLOW
OUT, PRESUMABLY ALTHOUGH WE
CAN'T SEE IT, ALL THE HUNDRED OR
SO PHAGE PARPARTICLES ARE
EXPELLED FROM THAT END OF THE
CELL IN THIS CASE, ABOUT 80% OF
THE TIME IT DOES OCCUR AT THE
POLL.
THIS DOES DONE BY
JCERTAINLY--CERTAINLY SSE
CAHILL, WHO HAS HIS OWN LAB AT
SANDIA.
SOIME PRETTY SURE THAT
[INDISCERNIBLE] WAS THERE WHEN
THEY DID THIS, THIS IS NOT
LAMDA, THIS IS T4 SO IT'S
UNUSUAL FOR ME TO SHOW T4 SLIDES
I'M A LAMDA TRIBE PERSON BUT THE
DELBRUCK GROUP DID THIS
EXPERIMENT, AND NOTICE THAT VERY
SHARP TEMPERRAL CONTROL OF LYSIS
EXISTS ISSUES IT'S IN A
DIFFERENT TIME BECAUSE IT'S A
DIFFERENT PHAGE BUT IT'S VERY
ABRUPT AND SO IT'S THE GRAPHICAL
REPRESENTATION OF WHAT WE
ALREADY SAW.
SO IF YOU-1 OTHER THING WE HAVE
TO EXPLAIN IS THE UNIVERSAL
CHARACTER OF PHAGE INDUCED LYSIS
AND THAT IS IF YOUADAD CYANIDE
EARLY IN INFECTION, IT JUST
STOPS ALL ENEMBRYONIC STEMY
METABOLISM AND NOTHING HAPPENS
IF YOU LOOK INDEED THIS CELL,
PHAGES AREN'T REPLICATING, NO
ASSEMBLY IS GOING ON, NOTHING'S
HAPPENING AND THAT SHOWS UP AS
NO CHANGE IN O. D. FOR A LONG
TOWARD OF TIME BUT IF YOU WAIT,
SAY 15 OR 20 MINUTES AND NOW,
INSIDE THIS CELL PHAGE PARTICLES
ARE BEING ASSEMBLED, ROBUST GENE
EXPRESSION, THE LYSISSED
PROTEINS ARE BEING EXPRESSED,
THE INSTANT YOU ADD ANYTHING
ELSE THAT CAN DEPOLARIZE THE
MEMBRANE, YOU TRIGGER LYSIS
IMMEDIATELY AND THIS IS CALLED
PREMATURE LYSIS, THIS WAS THAT
TERMINOLOGY INVENTED BY MAX
DELBRUCK, SO IT'S A PHENOMENON,
WHEN WE TRY TO FIGURE OUT HOW
THEY DO IT AND WE HAVE TO
EXPLAIN TO EXPLAIN THE TIMING
AND IN ADDITION YOU CAN DISRUPT
THAT BY COLLAPSING THE MEMBRANE
POTENTIAL, I'M SORRY, I'M SO
HORSE, I'VE ANSWERED SO MANY
QUESTIONS TODAY.
OKAY, SO, THE MODEL HAS TO
EXPLAIN BOTH THINGS.
ALL RIGHT, SO THIS IS THE LAMDA
LYSIS SET.
SO IF YOU DRAW TO SCALE THIS IS
AT THE RIGHT END OF LAMDA.
IS THIS WORKING IN YEAH.
SO THERE'S THE LATE PROMOTER AND
THE FIRST GENES ARE THE LYSIS
YEENS THAT ARE COUNTER ENTUMOR
SPECTRUMMATIVE AND THEN THERE'S
THE HEAD YEENS AND TAIL GENES
AND THIS WOULD BE ALL THE WAY IN
THE NEXT BUILDING FOR THE NEXT
20 YEARS BUT FROM 1 TRANSCRIPT,
IT TURNS OUT AFTER 8 MINUTES OF
INFECTION, AND IT MAKE ITS EASY
IN TERMS OF KINETICS BECAUSE
THERE'S NOT A COMPLEX
TRANSCRIPTIONAL O THIS IS EITHER
ON OR OFF AND AFTER THAT IT'S A
MATTER OF COPY NUMBER AND THERE
ARE 4 GENES, SR, RZ, AND RZ1.
WE WILL SPEND MOST OF OUR TIME
TALKING ABOUT THE GENES ARRANGED
BY THE WAY IN THE ORDER OF
ASSAULTING THE ENVELOPE.
SO THE FIRST GENE, GENE S, GENE
S ENCODES 2 PROTEEPS.
IT'S 107 CODONS LONG, MAKES 1
PROTEIN FULL LENGTH AND THAT'S
THE ACTUAL ANTIHOLEN AND MAKES A
SHORTER BY EARLIERINNISHIATION
AND THAT WILL BE THE HOLE AND
THAT WILL BE THE START OF THE
SHOW, WE WON'T TALK ABOUT HOW
THIS WORKS BUT YOU CAN TELL FROM
ITS NAME THAT IT WORKS IN
DIRECTLY INHIBITING THE HOLE.
WE WILL TALK ABOUT THIS AT THE
END OF THE SEMINAR, IT THE NEXT
GENE IS THE ENDOLIESIN AND THE
LYSOSIEM, IT'S THE ENZYME THAT
DEGRADES THE CELL WALL AND OF
COURSE IT ATTACKS THE PEPTIDE O
O GLYCAN AND THEN THE 2 GENES IN
THE SAME DNA, RZ, AND RZ1, 153
AND 160 CODONS LONG, COMPLETELY
DIFFERENT PROTEINS, BOTH
REQUIRED WHICH RAISES ALL KINDS
OF EVOLUTIONARY QUANDARYS.
THEY'RE ENCODING THE 2 SUBUNITS
OF THE SPANIN PROTEIN AND
THEY'RE REQUIRED FOR DISRUPTING
THE OUTER EMPLOYMENT PROGRAMS
BRAIN AND IT'S ESSENTIAL FOR
LYSIS.
SO IMPORTANT TO NOTE THAT YOU
CAN EITHER USE THE PHAGE OR YOU
CAN CLONE THOSE 4 YEENS INTO AN
INDUCIBLE BACK DROP TO GET THE
SAME PHENOTYPES.
YOU CHANGE THE TYPES JUST BASED
ON COPY NUMBER BUT EVERYTHING
ELSE IT DOESN'T MATTER IF
YOU'RE--THERE'S NO OTHER YEENS
IN THE PHAGE THAT HAVE ANY
EFFECT ON FUNCTION OR TIMING.
SO SOMETIMES I WILL SHOW YOU
DATA FROM PLASMA AND IT WON'T
EVEN TELL YOU WHEN I'M DOING
THAT.
OKAY, THE FIRST THING, TO DO IS
TO GET RID OF THE LAMDA
LYSOSIEM, THIS IS THE BASIC
STRUCTURE OF THE BACTERIAL CELL
WALL, AND ALL YOU NEED TO KNOW
IS AMONG THE 4 MOST IMPORTANT
SCIENTIFIC TRIBES, THE T7 T4
LAMDA AND T5 TRIBES, EACH 1 OF
THEM HAD A DIFFERENT GENE CALLED
LYSOSIEM, AND SO IT TURNS OUT
THAT ALL 4 OF THESE LYSOSIEMS
ARE COMPLETELY DIFFERENT
ENZYMES, SO LAMDA MAKES A
TRANSGLYCOSYLASE AND T4 MAKES A
GLUE MARIOUS COSDACE O IT'S A
BOND, IT MAKES THE BOND BETWEEN
THE PEPTIDE IS THE CELL WALL AND
THE TBLIEKOCIDE, AND THEN T5
MAKES AN ENDOPEPTIDE O
EDUCATIONAL ACE, AND THEY'RE
THEY'RE NOT MUCH INTEREST,
THEY'RE INTERCHANGEABLE, THEY'RE
ALWAYS PRODUCED IN 100 FOLD
ACCESS.
AND THEY'RE INTERCHANGEABLE AND
THEY'RE BORING, SO THE--THE
INTERESTING GIVING--YOUS HERE
ARE THE WHOLE 1S.
SO I MADE THE WORD UP, HOLEN, IF
EVERYBODY GENERATES A WORD
THAT'S TOO MUCH ADDITIONS TO THE
ENGLISH LANGUAGE.
BUT SO LAMDA IS TO EMPHASIZE THE
FACT THAT THEY ALL HAVE AT LEAST
1 TRANSMEMBRANE DOMAIN WHICH IS
NOT SURPRISING IF IT'S DOING
SOMETHING FOR THE INNER
MEMBRANE, AND THEN, SO LAMDA,
HAS 3 TRANSMEMBRANE DOMAINS,
IT'S AMINO TERMINUS IS OUT AND
THE C-TERMINUS IS IN, AND HAD IS
THE TOPOLOGY, THE OTHER--THE
OTHER 4 TOPOLOGYS, THESE ARE THE
ONLY 4 TOPOLOGYYS THAT HAVE BEEN
EXPERIMENTALLY CONFIRMED SO
THERE'S TYPE 1, 2, 3, 4, AND IF
WE HAD BEEN SMART THIS WOULD BE
TYPE 1 CAN THAT WOULD BE TYPE 3
BECAUSE THEN THE TYPE NUMBER
WOULD BE THE NUMBER OF
TRANSMEMBRANE DOMAINS.
BUT WE DIDN'T DO IT SMARTLY SO
TYPE 1 WILL HAVE 3 AND THE LAST
TIME I LOOKED WHICH WAS LAST
WEEK, THERE ARE 215,000 RECORDS
IN THE IDENTICAL PROTEIN GROUP
AND MY EXPERTISE IS NOT
ENVIRONMENTAL PHYSICS BUT I
LOOKEDDA THE FIRST 600 OR SO AND
THEY'RE ALL DIFFERENT.
SO THERE ARE THOUSANDS, AT LEAST
THOUSANDS OF UNRELATED GENE
FAMILIES AND THEY ALL HAVE AT
LEAST 1 TRANSMEMBRANE DOMAIN BUT
NOTICE, 2 THAT 1 OF THEM WHICH
IS THE T-4 WHICH IS ALSO SHARED
BY T-5 HAS A PERIPLAZ MIDSIC
DOMAINS OF ABOUT AMINO ACIDS AND
HAD IS A SIGNATURES 95
TRANSDUCTION RECEPTOR AND THIS
IS THE ONLY CASE WE KNOW IN
BIOLOGY WHERE EXTERNAL
INFORMATION IS PUT INTO LYSIS
TIMING, UNFORTUNATELY, WE'RE NOT
GOING TO BE ABLE TO TALK ABOUT
THIS UNLESS I GET A PREGNANT
QUESTION NEAR THE END BUT IT'S A
WONDERFUL STORY THAT'S RECENTLY
MADE PROGRESS AT THE STRUCTURAL
LEVEL, BUT IT'S NOT RELATED TO
THE BASIC STORY OF HOW THE
TIMING OCCURS.
SO THIS IS ONLY 1 OF THE 2
PROTEINS MADE BY THE S-GENE,
WHICH IS THE TRUE
[INDISCERNIBLE].
SO THE INITIAL--BACK WHEN I WAS
STARTING TO GET MY LAB TOGETHER
I GOT A COUPLE PAPERS IN GRANT
REVIEW WAS THAT THE SIMPLEST
IDEA WAS KNOWN THAT CYANIDE
COULD CAUSE PHAGE INFECTIONS,
YOU GIVE CYANIDE, EVERYTHING
COLLAPSES, EVERYTHING LYSIS,
THAT WAS ALREADY KNOWN, SO
HOLEIN CONTRIBUTE OUT THE
MEMBRANE, IF YOU EXPRESS
MEMBRANE PROTEINS YOU POISON THE
PROTEIN, AND WHATEVER WAS
HAPPENING WAS HAPPENING THE SAME
AND NORMAL INDUCTION HAS A
PRACTICES MATURE LYSIS.
SO WE ANSWERED THAT QUESTION
QUITE A LONG TIME AGO.
THIS IS WORK DONE BY
[INDISCERNIBLE] FROM THE COLLEGE
OF LONDON AND COLLABORATOR WITH
MIKE [INDISCERNIBLE] AT TEXAS
A&M.
LET'SEE IF I CAN DO THIS,
IMAGINE THESE ARE CELLS THAT ARE
TETHERED TO THE MEMBRANE BY
THEIR FLAGELLA TO TELL IT TO THE
FLAGELLA AND BEGINNING OF THE
DIRECT READ OUT OF THE MEMBRANE
POTENTIAL BECAUSE THE FLAGELLA,
BY THE WAY, THESE ARE LOCK
INDEED A CLOCKWISE ORIENTATION
SO WE KNOW THEIR PMF IS 170
MILLI VOLTS RIGHT NOW BECAUSE
THEY'RE SPINNING AT A REGULAR
NORMAL RATE AND NOW THEY--WITHIN
A FEW SECONDS OF EACH OTHER,
THEY BOTH STOP AND THEN IF
ANOTHER 30 OR 40-SECONDS THEY
ARE GOING TO LYSE, AND 25
MINUTES BEFORE WE TARTED THIS
REALTIME VIDEO, WE EDIT IT OR
INDUCE THE GENES SO WHAT WE SAW
WAS ANOTHER REPRESENTATION OF
THE SAME TIMING EVENT BEFORE
WHERE AFTER A LONG PERIOD OF
TIME IN THE ORDER OF MINUTES, 25
MINUTES PERKAY, IN THIS CASE
THERE WAS A PLAZ MIDS MID, AND
SUDDENLY INTED OF TITRATING IT
OUT SLOWLY, IT ALL HAPPENED AT
ONCE.
SO WE CALLED THIS PAPER, HOLIN'S
KILL WITHOUT WARNING SO THIS WAS
A REPUTATION OF THIS PUKE OUT
MODEL EVERYBODY WAS SUGGESTING.
OKAY.
THIS FTION AN ACCIDENTAL
EXPERIMENT DONE BIAN JELLICCA,
SO BY ELIMINATING THAT YOU COULD
PUT CYSTINES WHEREVER YOU WANT
AND THE IDEA WAS TO USE THESE
CYSTINES TO VARIOUS TYPES OF
BIOCHEMICAL REACTIONS, INCLUDING
DIMERIZATION,OXIDATION, SO ON SO
SHE WEPT THROUGH AND PUT SINGLE
CIS RESIDUES ISSUES THESE ARE
ALL MISSED CHANGES.
SO WE NOTICED THAT THE COLOR
COATED MUSEUM TAIGS IN THE
SECOND AND THIRD TRANSMEMBRANE
DOMAIN THAT FOR EVERY SINGLE
INITANCE CHANGED CAUSED A TIMING
CHANGE IN LYSIS, EVERY SINGLE 1,
SOMETIMES THEY WERE EARLIER,
SOMETIMES THEY WERE LATER AND WE
CALL THIS THE RAINBOW, BECAUSE
THIS GOT US THINKING ABOUT HOW
THIS TIMING CLOCK COULD WORK
EMPLOY SO THAT MEANS THAT--AND
THEN SUPER IMPOSED ON THIS, IF
YOU--THIS WAS DONE BY INDUCING A
LAMDA PHAGE IN TRANSTO THIS PLAZ
MIDS MID.
SO IF YOU COUNT THE PARTICLES
THAT WERE BEING GENERATED AND
REMEMBER, THE PARTICLES ARE
BEING ASSEMBLED ALL THE TIME,
THIS IS GOING ON, AND NORMALLY
AT ABOUT 50 MINUES YOU WOULD
HAVE A HUNDRED OR SO, BUT IF YOU
GO TO 3 HOURS, YOU WOULD 600 SO
IT DOES RAISE THE QUESTION, WHY
DOES LAMDA QUIT AT A HUNDRED, IS
THAT GOOD ENOUGH OR WHAT'S THE
RATIONAL FOR IT AND THE OTHER
THING IS HOW CAN WE EXPLAIN
EVERY SINGLE CHANGE IN 3
DIFFERENT TRANSMEMBRANE DOMAINS
ALTERING THE TIMING AND BY THE
WAY WE CHECKED AND NONE OF THEM
HAVE ANY ACCUMULATION OF THE
PROTEIN.
SO THAT LED TO A PROJECT THAT
REBECCA WHITE RAN AND REBECCA
WAS--SHE'S NOW THE CEO OF A
COMPANY CALLED AVALO, I THINK
IT'S THE THIRD 1 SHE'S RUN BUT
HE MADE THIS INTO A STANDARD
UNDERGRAT GRADUATE LAB, SO EVERY
UNDERGRADUATE THAT WOULD COME TO
THE LAB WOULD GET A MISSENSED
MUSEUM TAIG EMPLOY SO WE HAD 2
ORIGINALS BACK IN THE 70S AND
THESE WERE ALANINE 52 AND
ALA1952 TO GLY.
AND THEY WERE CONSERVATIVE IN 1
SENSE BUT 1'S IMING TO BE A
LARGE OUTFIELD GROUP AND THE
OTHER 1 IS A HYDROGEN ATOM AND
NEITHER 1 OF THEM, SO BOTH OF
THESE MUTATIONS WERE ISOLATED AS
NONPLAQUE FORMERS, SO THAT'S
PROVE THAT IT HAS 2 FUNCTIONS
BECAUSE YOU COULD KNOCK OUT BOTH
OF THEM.
SO IF YOU LOOK AT THIS, SO WHEN
SHE FIRST STARTED OUT WAS TO
HAVE EVERY SINGLE POSSIBLE OTHER
AMINO ACID CHANGE DONE AT ALA52,
AND WE DISCOVER THAD A 4 OR 5 OF
THEM LIESED AT A RIDICULOUSLY
EARLY TIME, SO PHAGES ARE
PRODUCED BY ANY OF THESE, AND
THAT'S WHY YOU DON'T GET ANY
PLAQUES BUT YOU ALSO HAVE A
NUMBER THAT WAS SLOWER AND IF
YOU PUT, YOU KNOW CHARGE
RESIDUES IN THIS THE MIDDLE OF
THE MEMBRANE, YOU WILL KILL THE
MEMBRANE SO THAT WASN'T
SURPRISING THAT A LOT OF
PROTEINS WERE ENACTIVATED SO
THEN SHE EXTEND THAD TO ALL
OTHER, TO THE 3 RESIDUES NEXT TO
THAT, 49, 50, 51 AND 52.
AND BOTTOM LINE IS THAT IT'S THE
SAME FOR 4950 AND 52, BUT IN MED
50, IT WAS SILENT BUT WE GOT
VERY LARGE NUMBER OF EARLY LYSIS
MUTANTS.
SO TAKE HOME NOTES IS THAT LAMDA
COULD EXPLORE TIME SPACE, IF IT
HAS A REASON TO EXPLORE LYSIS
TIME SPACE, IT CAN DO THAT BY
SINGLE CHANGES AND A SINGLE
CONSERVATIVE CHANGE CAN
DRAMATICALLY AFFAIRS TEAM
LEADERRER TIME IN EITHER EARLIER
OR LATER.
OKAY, SO HERE COMES THE FIRST
MODEL.
SO THE MODEL FOR HOLIN'S WORK IS
THAT THE INNER MEMBRANES, I'M
NOT HOEING YOU ANYTHING ABOUT
THE PHAGE.
PHAGE PARTICLES AS LONG AS
THERE'S LYSIS PROTEINS BEING
PROTUSSED THAT'S ALSO THE MORPH
O GENERIC PARTICLE FOR PHAGE
PRODUCED BUT WE'RE NOT SHOWING
THAT IN THE CARTOON, SO BLUE IS
THE WHOLE, LYSOSIEM IN THE GREEN
AND THEY REACH WHAT WE CALL A
CRITICAL CONCENTRATION.
THIS IS THE MODEL ANYWAY.
AND THEN AT THAT CRITICAL
CONCENTRATION, THE PROTEINS
AGGREGATE INTO WHAT WE CALL A
RAFT AND THEN SOMEHOW THAT RAFT
IS CONVERTED INTO HOLES AND
THESE HOLES ARE UNFORTUNATELY
CALLED S-HOLES.
[LAUGHTER]
IT NEVER FAILS TO--ANYWAY, THESE
ARE MIRROR IMAGE CROHN SCALE,
THE LARGEST HOLES FORMED IN
BIOLOGY BY NORMAL MEANS.
AND SO, THESE GIANT HOLES ALLOW
THE LYSOSIEM TO ESCAPE AND
ATTACK THE CELL WALL AND OF
COURSE WE ALREADY SAW THAT LYSIS
IS FUNDAMENTALLY A PUNKITATIVE
END AND THAT'S REFINED BY WHERE
THE HOLE IS FORMED.
OKAY, SO WERE, THE UNIVERSAL
PHENOTYPE IS THE TRIGGERING ALSO
HAS TO BE EXPLAINED SO WHAT WE
ARGUE IS THAT IN THE--IF YOU HAD
SIGH NIGHED AT THIS POINT
INSTEAD OF GOING THROUGH THE
WAITING FOR THE CRITICAL
CONCENTRATION BEFORE YOU FORM
THE RAF, THE ADDITION OF CYANIDE
INITANTLY ARK LOWS WHATEVER THE
LEVEL OF HOLIN IS, TO FORM, AND
IT TURNS OUT IF 1 SIDE
PREDECLARATION OF THAT IS THAT
THE HOLE SHOULD BE SMALLER
BECAUSE THERE'S LESS LYSISSED
PROTEIN AND YOU'LL SEE THAT IS
THE CASE, SO THE FIRST THING YOU
WANT TO SHOW IS TRUE, THAT IT'S
ACTUALLY MOBILE AND SO, IN MORE
CLARRABRAITORS FROM
[INDISCERNIBLE] LAB, THESE ARE
EXPERIMENTS, SO IF YOU TURN ON
THE HOLIN GFP FUSION WHICH WORKS
PRETTY WELL AND IF YOU USE A52
IT JUST STAYS MOBILE SO IT
IMMEDIATELY FOLLOWS OR BECOMES
FLUORESCENT AND IF YOU DO THE
BLEACHING IN THE S105 GFP BEFORE
TRIGGERING, YOU GET REPOPULATION
OF FLUORESCENCE BUT AFTER
TRIGGERING IT'S NO LONGER, IT
DOESN'T RECOVER AS YOU CAN SEE.
THIS THING'S NOT WORKING NOW.
OH YEAH.
SO S105 AND MOBILE AND BEFORE
TRIGGERING AND IMMOBILE AFTER
TRIGGERING.
THIS WAS PROBABLY LE THE HARDEST
EXPERIMENT WE EVER DID, THIS IS
S-BIOCHEMISTRY, I WON'T ANY INTO
THE DESPERATION MODE TO PURIFY
HOLEINS, TURNS OUT YOU CAN GET
HALF A GRAM PER LITER BUT WE
WERE ABLE TO PURIFY THE LAMDA
HOLIN, AND I HAVE TO TELL YOU WE
HAD TO EXPLORE TREAN DIFFERENT
POSITIONS TO PUT THAT TAG IN
BEFORE WE FOUND 1 THAT WOULD
WORK.
SO THIS WAS WORKED ON MY JOHN
AND DAVE SMITH.
O YOU BASICALLY PURIFY IT, DUMP
IT INTO A KOTROAP WHERE IT'S
TOTALLY UNFOLDED AND THEN YOU
DILUTE THAT DOWN INTO SUSPENSION
OF LYSOSOMES THAT HAVE
FLUORESCENT CALCIUM IN THEM AND
THEN WHAT YOU SLEEP APNEA AND
OBESITYY IS RELEASE THE
WILD-TYPE RELEASES THE
FLUORESCENCE, THE MUTANT DOESN'T
AT EVEN TWICE THE CONITRATION
AND EVEN MORE IMPORTANTLY
PURIFIED MATERIAL FROM A
TS-ALLELE WHICH IS ALOE 19 TO 3
A19, WORKS AT THE HIGH
TEMPERATURE, NOT THE LOW
TEMPERATURE WHICH FORMERLY
EXCLOUDS THE POSSIBILITY OF SOME
CONTAMINANT OF SOME PREPARATION
THAT WAS CAUSING FORMULATION SO
IT DOES WHAT IT'S SUPPOSED TO DO
AND CAN PERMIALIZE THE MEMBRANES
IN AN ARK LEGAL SPECIFIC MANNER
I THINK THIS IS SO JUST TO
REMIND YOU WHAT HAPPENSA THE
MILLI SECURITIZATION. LEVEL,
THERE'S GOING TO BE A EXPULSION
OF THE INNER--CYTOPLASMIC
MATERIAL FOR THE LOCAL BLOW OUT.
OKAY, SO OUR NEXT GOAL WAS TO
VISUALIZE THE S-HOLE AND IT
SHOULD BE BIG ENOUGH, WELL THIS
IS HOW WE LEARN IF IT'S MICRON
SCALE.
THIS WAS A COLLABORATION WITH
ANDRE'S LAB AT [INDISCERNIBLE],
AND [INDISCERNIBLE] DID THE WORK
AND SO BASICALLY YOU'RE LOOKING
AT INTERRUPTIONS IN THE MEMBRANE
OF THE E.COLI AND THE AVERAGE
AND THE AVERAGE SIZE OF THESE,
ACTUALLY LET ME SHOW YOU THE
NEXT SLIDE, WHICH IS--YEAH, THIS
IS ACTUALLY A TOMOGRAM.
IT TOOK US A YEAR TO DO THIS
EXPERIMENT AND THEN A REVIEWER
RYED ANOTHER 1 AND IT TOOK
ANOTHER 1 TO DO THE EXPERIMENT
BUT THIS WAS ABOUT A HUNDRED
MICRON HOLD, ON AVERAGE THERE'S
1 TO 3 PER CELL AND ON AVERAGE
THEY ARE 350-MICRONS.
SO EVERYBODY WANTED TO SEE THE
HOLE AND WE DID IT.
SO THEN THE NEXT THING WAS,
WHAT'S THE STRUCTURE OF THE
HOLE, AND SO WE KNEW THAT THE
S-PROTEIN STARTED OW AS A MOBILE
PROBABLY DISCIPLINARY MERRIC
FORM THAT FOLDED AROUND THE
MEMBRANE AND ENDS UP IN AN
IMMOBILE LARGE MICRON SCALE HOLE
BUT WHAT'S THE ACTUAL STRUCTURE
AND WHAT'S FACING WHAT EMPLOY SO
WHAT WE WANTED TO DO WAS TO FIND
OUT IN THE S-MOLECULE, REMEMBER
I TOLD YOU ABOUT ANGELICA'S WORK
FOR PUTTING CYSTINES EVERYWHERE,
CAN YOU SEE ALL THE CYSTINES WE
USED AND THEN YOU TREAT THIS
WITH A REAGENT AND IT BLOCKS AND
IF THE REAGENT MODIFIED PROTEIN
IT'S IN THE SOLUBLE PHASE AND
YOU CAN DETECH THAT BY WESTERN
BLOTS BECAUSE YOU PAY FOR THAT
AFTERWARDS, THE BOTTOM LINE WAS
THAT THERE WERE HALF OF TMD 1
AND 3 THAT WERE EXPOSED IN THE
HOLE, WERE EXPOSED TO THE
AQUESTIONNAIRESUS SOLUTION
WHEREAS IN THE MEMBRANE WHEREAS
TMD2 IN THE MIDDLE WAS EXPOSED
SO THAT,A LOWED US AND KENNETH
TOW WHO NOW RUNS [INDISCERNIBLE]
WHO DID THIS WORK AND THIS LED
TO THIS MODEL WHERE HYDROPHILIC
PHASES OF TMD'S 1 AND 3 IN THE
DIMER AND I'M NOT SHOWING YOU
THE EVIDENCE, IT'S A DIMER IT'S
VERY SIMPLE CROSS LINKING
EXPERIMENTS, SO, BASED ON WHAT
WE JUST SAW, THE SYSTEM WITH THE
NOTION THAT THE HYDROPHILIC
PHASES AND MULTIPLE DIMER ARE
FACING EACH OTHER SEQUESTER ON
THE MEMBRANE, SO WE'RE LOOKING
DOWN, SO THIS,A COUNTS FOR THE
ABILITY OF THE PROTEIN TO
FREELYY MIGRATE BECAUSE IT'S
NOT--ITS HYDROPHILIC PHASES ARE
NOT FACING ANY OTHER SIDE.
SO IN THE LUMEN, IN THE FINAL
TRUCTURE AFTER COLLAPSE, THIS IS
THE MODEL WE SAY BECAUSE WE'RE
SHOWING HALF THE FACE OF 1 AND 3
ARE ACCESSIBLE TO THE SOLUBLE
REAGENT.
AND, BY THE WAY, WE MEASURED THE
AMOUNT OF S-PROTEIN BY
QUANTITATIVE WESTERN BLOT AND
ALSO BY SOMETHING MEASURING THE
PERIMETER OF THE HALL ANDS IF
YOU FILLED ON THE FACT THAT EACH
1 OF THESE ALPHA HELIXES IS
ABOUT 1 NANOMETER IN DIAMETER
THERE'S A CONSISTENT NUMBER OF
S105 MOLECULES IN THE TOTAL
PERIMETER OF THE HOLES.
HERE IS THE GRAPED MODEL HERE,
SO WE HAVE A MUTANT THAT DOESN'T
DIMERIZE SO WE SUSPECT
THAT--WELL WE DON'T KNOW WHAT
CONTROLS THE DIMERIZATION, BUT
THE DIMER IS THE MOLECULE THAT
UNDERGOES THE TIMING BECAUSE
IT'S NOT UNTIL THE DIMER REACHES
CRITICAL CONCENTRATION THAT IT
SUDDENLY AGGREGATES TO FORMER
RAF AND WE'RE ARGUING THAT THE
TOTAL POPULATION OF S-MOLLULES
IN RAF IS ABOUT 5000 AND IT'S
THE EXISTENCE OF THE RAF THAT
CAUSES THE NEXT PHASE AND HERE'S
WHERE--I WILL BEG YOUR
INDULGENCE SO WHAT WE THINK IS
HAPPENING IS, YOU HAVE A SHORT
CIRCUIT BECAUSE YOU HAVE A LARGE
RELATIVELY LARGE SURFACE OF
LIPID DEPLETED ASHES JACENT
PROTEIN MOLECULES WHICH ARE
FEATURED BY TRANSMEMBRANE
DOMAINS EACH 1 WITH AN ACTIVE
POLAR, SEVERAL ACTIVE POLAR
UNITS SO WE THINK THERE'S A
SHORT CIRCUIT LOCAL DEPOLAR
EYATION IN THE RAF, AND NOW YOU
ARE BACK TO OUR ORIGINAL IDEA IF
YOU DEPOLARIZE THE MEMBRANE THAT
CAUSES THE SECONDARY, TERTIARY
AND QUANDARY CHANGE THAT STARTS
THE FORMATION OF THE HOLE.
SO IT'S THE FORMATION OF RAF
THAT LEADS TO THE SMALL HOLES
AND RAPIDLY EXPAND AND FEET
FORWARD MECHANISM TO THE LARGE
SCALE 300-MICRON HOLES.
SO THAT'S THE MODEL.
AND THEN EITHER PREMATURE LYSIS
OR A LIAISONSIS MUTEITANT THAT
HAD AN EARLIER TRIGGER CHICAGO
WAS EASY TO CREATE.
SIMPLY CAUSES THE CRITICAL
CONCENTRATION TO BE LOWER SO YOU
END UP WITH A SMALLER HOLE WITH
FEWER S-PROTEINS ON THE
PERIMETER APPROXIMATE WE'VE
PUBLISHED THAT.
THAT'S EXACT LYE WHAT HAPPENS TO
THE IMMUNE DISORDER IN PREMATURE
TRIGGERING, YOU REDUCE THE SIZE
OF THE HOLES.
OKAY SO WHAT DO WE MEAN BY
CRITICAL CONCENTRATION.
I DID NOT MAKE THISSA WORD UP.
SO THIS IS WORK DONE BY MARK
KREB'S LAB QUITE A LONG TIME
AGO, HE WAS FROM [INDISCERNIBLE]
LAB AND HE WORKED ON THE P A RVO
VACCINE XTHEY DORPHED VERY EARLY
ON IF YOU PURIFIED IT AND THEN
DILUTED IT INTO A DETERGENT OF
SOME PERCENTAGE THEN IT WOULD
FORM--IS IT FINALLY NOT WORKING
AT ALL NOW.
WHOOPS.
SO THE BOTTOM LINE IS AT
WHATEVER CONCENTRATION YOU MADE
THE PROTEIN, LOW CONCENTRATIONS
IT WOULD ALL BE IN MONITOR MER,
BUT AS YOU WENT TO HIGHER AND
HIGHER CONCENTRATIONS THESE ARE
SUCROSE GRADIENTS, YOU GET MORE
AND MORE IN THE LATTICE FORM SO
IF YOU PLOT THE AMOUNT IN THE
LATTICE VERSUS THE
CONCENTRATION, CAN YOU OCCUR
LIKE THIS, IS WHERE IT
INTERSECTS THE LINE IS THE
CRITICAL CONCENTRATION AND SO IT
TURNS OUT THAT THEY HAD A ROUGH
CRYOEM STRUCTURE OF HOW IT WAS
INTERACTING WITH THE UNITS
INTERACTING WITH EACH OTHER AND
THEY DID SOME SATURATED IMMUNO
GENESIS, AND SO NORMALIZED TO
THE WILD-TYPE OF I HIGHLIGHTED
WOTHAT ARE SIMPLE RELATIVELY
CONSERVATIVE CHANGES THAT EITHER
CHANGE THE CRITICAL
CONCENTRATION OF A FOLD LOWER OR
6 FOLD HIGHER WHICH WHEN IT DOES
THAT IT COMPLETELY CHANGES THE
PERCENTAGE THAT CHANGES THE
CRITICAL CONCENTRATION.
SO I HOPE IT VALIDATED IN YOUR
MIND IT'S THE SINGLE REASON WHY
SINGLE AMINO CHANGES IN THE
HOLIN, YOU ARE CHANGING THE
CONCENTRATION OF WHICH IT
SUDDENLY FORMS THE RAFT.
SO FINAL ACT OF THIS IS TO SHOW
THERE'S ACTUALLY BEHAVES LIKE WE
SAY IT DOES SO THESE ARE FGFP
SPRPTS WHERE IT'S ATTACKED WITH
THE LINKER TO THE C-TERMINAL END
AND CYTOPLASMIC DOMAIN OF THE
HOLIN AND THESE ARE JUST NOW
COMING OUT IN M-BIOSO IT'S
RELATIVELY NEW EMPLOY CAN I GET
THIS CLIENT?
SO THE IDEA IS THAT YOU WILL SEE
SUDDENLY WE START WITH DISPERSED
SGFP AND THEN AG TBREIGATE AND
THEY BOTH FOLD BO RAFTS WHICH
THEN DISAPPEAR APPROXIMATEEE
THINK THE DISAPPEARANCE OF THE
RAFT IS WHEN THE ELONGATION
OCCURS AND THEN YOU GET THE
EXPLOSIVE DISRUPTION OF CELL IN
BOTH OF THESE POSITIONS.
SOONER OR LATER.
THIS IS ONLY SECONDS, IT SEEMS
LONGER WHEN I'M LOOKING AT IT.
THERE WE GO.
IT LOOKS PAINFUL ACTUALLY.
SO THERE'S ANOTHER 1 EMPLOY THIS
WAS A LITTLE BIT MORE CONVINCING
BECAUSE IT'S AT THE POLE, BUT
80% OF THE TIME IT'S AT THE
POLE, IN THIS CASE THEY FORMED
AND THEY WERE NOT AT THE POLAR.
AFTER ALL THAT WORK.
SORRY.
WE'RE RUNNING OUT OF TIME SO LET
ME GO ON TO THE NEXT 1.
SO THERE'S JESSE DEVELOPED
ANOTHER ASSAY FOR THE INTEGRITY
OF THE MEMBRANE, OUR MODEL
SPECIFICALLY PREDICS THAT THE
MEMBRANE WILL DEPOLARIZE BEFORE
THE FIRST DEFORMATION OF THE
CELL MORPHOLOGY OCCURS SO HE'S
MAKING USE OF THE FLUORESCENCE
MOLECULE BIOFLAVIN T, WHICH IS
NORMALLY NOT USED BECAUSE IT
BINDS TO SURFACES AND
FLUORESCENCES, IF YOU TREAT
WHOLE CELLS WITH THAT YOU DON'T
SEE FLUORESCENCE UNTIL THE
MISDEMEANOR BRAIN IS COMPROMISED
AND IT TURNS OUT, HOPEFULLY THIS
PARTICULAR MOVIE HAS CRASHED
NUMBERS OF TIMES.
HOPEFULLY IT DOESN'T.
OKAY, SO BIOFLAVIN T IS PRESENT.
VERY NEAR THE END, WITHIN
SECONDS OF TRIGGERING AND WHAT
YOU SEE IS IT'S FAIRLY UNIFORM
IN THE CELL LONG BEFORE THE UDEN
EXPLOSION AT 1 POLE AND WE HAVE
MANY, MANY MOVIES LIKE THIS.
SO WHAT TIME AM I AT NOW?
>> [INDISCERNIBLE]
>> SO I HAVE TO PREVENTIVELY
TAKE A DIVERGENCE BECAUSE AFTER
WE STARRED THIS CRUSADE TO
UNDERSTAND THE LAM TAKEN--THEY
HOLIN, THE MOST IMPORTANT
PROTEIN AND MOST IMPORTANT
ORLGANISM IN THE WORLD.
IT TURNS OUT THAT IF WE HAD
LISTENED TO LAMDA, LAMDA WAS
SAYING ABOUT YOU WAIT, BUT WAIT.
IT TURNS OUT THERE'S 2
FUNCTIONAL TYPES OF HOLINs, WE
CALL THE LAMDA CONONICLE, BUT
THERE ALSO'S PIN HOLINS WHICH
MAKE TINY HOLES, AND LAMDA TOLD
US THIS BECAUSE IT HAS A NEAREST
COULD YOU SAYIN, THIS IS WHAT
YOU GET TO BE 78, I HAVE AN
UNTIED SHOE LACE.
SO THE LAMDA PHAGE IT LOOKS IKE
DENTICAL TO LAMDA BUT WHEN YOU
LOOK AT IT IT'S A HOLEIN AND
ONLY HAS 2 TRANSMEMBRANE DOMAINS
AND THE ENDOLICENSE IS A
LYSOSIEM AND IT HAS WHAT APERS
TO BE A TRANSMEMBRANE DOMAIN OR
SIGNAL SEQUENCE AT THE IMMUNO
TERMINUS, SO THAT WAS A SHOCK.
IF I TRIP AND BREAK MY NECK THEN
I'LL--I WILL NOT GO THROUGH
THIS.
THIS IS 2 SEMINARS, REALLY
AMAZING WORK TO ME BECAUSE WE
DOCUMENTED 2 DIFFERENT TYPES OF
MEMBRANE DYNAMICS, MEMBRANES
YUSMING IN ASK OUT OF MEMBRANES,
TRANSMEMBRANE DOMAINS JUMPING IN
AND OUT OF MEMBRANES SO THE
BOTTOM LINE IS THAT THERE IS
THIS TRANSMEMBRANE COMES OUT AND
IT RELEASES AND ACTIVATES THE
ACTIVITY OF THE ENZYME AND THERE
ARE MULTIPLE PAPERS, I ACTUALLY
HAD A REALLY CUTE, MAYBE NOT SO
CUTE COMMENT FROM 1 OF THE
REVIEWERS WHO MAY BE IN THIS
ROOM, SAID THAT YOU REALLY
ADMIRE OUR WORK BUT HE FELT WE
MIGHT HAVE MORE REVIEWERS THAN
READERS.
SO THIS IS A LOT OF VERY HIGH
PROFILE PAPERS, ABOUT A VERY
SMALL THING BUT IT WAS REALLY
HIGH COOL FACTOR IN MY OPINION.
SO I WILL SUMMARIZE HOW THIS
WORKS.
I'VE FLIPPED THROUGH THE WHOLE
THING SO BOTTOM LINE IS, IT'S
THE SAME STORY, JUDGE UOF THE A
SLIGHTLY DIFFERENT, MUSICAL
SCORE, SO, THE HOLIN
ACCUMULATES, EEIVETTUALLY FOR
THE PURPOSES RAFTS AT CRITICAL
CONCENTRATION, AT THAT POINT THE
ONLY HOLE FORMING DOMAIN IS THE
C-TERMINAL TRANSMEMBRANE DOMAIN
AND YOU END UP WITH INSTEAD OF 1
OR 2 LARGE HOLES, YOU EPPED UP
WITH ABOUT A THOUSAND PIN HOLES,
AND WE HAVE CRYOEM, AND CRYOEM,
NEGATIVE STAIN EM AND ALSO
ELECTRODYNAMICS MODELING AND ALL
SORTS OF BIOCHEMISTRY TO DEMON
TRAIT THIS IS RIGHT.
SO YOU END UP WITH A HEPTAMEROF
THE S105 PROTEIN AND ABOUT A
THOUSAND OF THEM EMPLOY NOW THE
PROBLEM IS, THAT'S TOO SMALL TO
LET THE LYSOSIEM OUT BUT THAT'S
NOT A PROBLEM HERE BECAUSE WHAT
HAPPENS IS, WHEN THESE FORM
THESE HOLES, THEY DEPOLARIZE THE
MEMBRANE CAN THAT ALLOWS THIS
MEMBRANE TO COME OUT OF THE
MEMBRANE WHICH ACTIVATES THE
ENZYME SO IT'S STILL A RAFT
CRITICAL CONCENTRATION DEPENDENT
STORY, JUST A DIFFERENT CHORIO
GRAPH.
SO THAT'S SHOWN BY THIS
PARALLEL.
HERE IS CANNONICLE SYSTEM WE'RE
TALKING ABOUT, AND HERE'S THE
NEW PINHOLE SYSTEM, THE ONLY
THING THAT CHANGE SYSTEM THE
SIZE OF THE HOLE AND THE RAFTS,
EVERYTHING ELSE IS THE SAME AND
THE GENETICS OF S21 ARE EQUALLY
CONVINCING AS THEY ARE FOR
LAMDA.
OKAY AND WE SHOULD HAVE NOTICED
THIS EARLY ON BECAUSE WHAT YOU
EXPECT SINCE IN THIS CASE, THE
ENDOLIES IS NOT COMING OUT
THROUGH 1 BIG HOLE, IT'S ALL THE
WAY AROUND THE HOLE ALREADY, SO
YOU'RE LOOKINGA THE DESTRUCTION
OF THE CELL WALL AND THAT'S WHAT
YOU SEE.
SO I WILL TAKE A RISK AND TRY TO
RUN THIS FORWARD MUCH THE
CONTROLLER OF BANKED 1S WILL ALL
GO ARK ROUND BEFORE THEY BLOW
UP.
AND NOTICE ALSO THAT ONLY 7 OF
10 CELLS THAT WE'RE HEARING HERE
DID THIS AND THIS TELLS US
SOMETHING WE ALREADY NOTICED
THAT THE PIN HOLE ASSESSMENTS
ARE NOT QUITE AS WELL TIMED AS
THE CANNONICLE WHOLE SYSTEMS
ARE, SO IF YOU DID THE LAMDA
S-PROTEIN WITH THIS, THEY WOULD
ALL BE LIESING WITHIN A
MACHINEUTE OR 2 O IT'S NOT QUITE
AS WELL CONTROLLED.
SO IN THE LAST 5 MINUTES HERE WE
WILL TALK ABOUT SPANINS, 2 OF
THE SPANIN MIEOF THERS ARE HERE,
IS IS HE THERE IN MANOY, ARE YOU
THERE IN OH WELL, SHE'S IN
[INDISCERNIBLE] LAB ANYWAY SO
MAYBE HE FELT LIKE HE KNEW IT
ALL.
BUT THESE ARE THE 4 PEOPLE WHO
DID THIS WORK, LIZ SUMMER DID
THE BIO-INFORMATICS AND
EVERYBODY ELSE DID GENETIC ANDS
BIOCHEMISTRY.
THIS IS THE WEIRDEST YEEN IN THE
WORLD WITH THE WEIRDEST PROTEINS
IN THE WORLD SO THE BOTTOM LINE
IS THE MAJOR PROTEIN, RZ PROTEIN
HAS A TRANSMEMORY RESPONSE BREAN
DOMAIN AND ABOUT A HUNDRED AMINO
OOH SIDS OF ALMOST TOTALLY ALPHA
HELICAL PROTEIN AND THEN
EMBEDDED IN THE DIFFERENT
READING FRAME IS THE RZ1 PROTEIN
WHICH ONLY HAS 40 RESIDUES IN
THE MATURE FORM POPULATED BY
PROLANES AND HAS AN OUTER
MEMBRANE LIPOPROTEIN SIGNAL
WHICH MEANS FOR THOSE WHO ARE
NOT FAMILIAR WITH LIPOPROTEINS,
BY THE WAY, BEFORE I GET TO--IT
TURNS OUT THAT PHAGE T1 HAS THE
SAME SYSTEM, PINHOLIN, AND
DIFFERENT TYPE OF SPANIN, THIS
CASE IT WAS [INDISCERNIBLE] BABY
WHILE HE WAS IN THE LABORATORY,
IT DOES HAVE A LIFE PROMOTING
SIGNAL BUT IT'S ALL BETA SHEET
PREDICTION AND HAS A C-TERMINAL
TMD.
SO THE BOTTOM LINE IS IN 1 CASE
YOU HAVE A PROTEIN COMPLEX THAT
SPANS THE ENTIRE PERIPLASM, IN
THIS CASE IT'S 2 PROTEINS ISSUES
THE 1 PROTEIN AND THE RZ1
PROTEIN, IN THE CASE OF T1 YOU
HAVE A SINGLE MOLECULE WITH A
COMPLETELY DIFFERENT TOPOLOGY
AND IT'S ALL IN THE SHEET, SO
NOTICE IN BOTH CASES THEY'RE
THREADED THROUGH THE GLYCAN MESH
WORK AND THEREFORE KOOBT DIFFUSE
LATERALLY.
AND THAT WE THINK IS HOW THEY'RE
CONTROLLED.
SO THE BOTTOM LINE IS, FIRST OF
ALL, WE IT SHOW THAT THREAR
REQUIRED AND SO I WILL START TE
CLOCK.
CAN I MAKE IT GO FASTER, THERE
WE GO.
YOU CAN GET THE WHOLE FORM, THE
OUTER MEMBRANE LEFT INTACT SO IT
TURNS INTO A FEAR AND WE ARE A
PICTURE OF THAT.
THIS THE ASSUME THING, FOR THE
SAME STORY, SO THIS IS A
CRYOPICTURE OF 1 OF THOSE CELLS
THAT HAS NO PANIN, THE OUTER
MEMBRANE IS INTACT AND HERE THE
NICE THING IS YOU CAN SEE,
HERE'S THE INNER MEMBRANE AND
THE ACTUAL S-HOLE, SO WE CAUGHT
IT IN THE ACT, 1 OF THE THINS
YOU LEARN FROM THIS IS THAT
ABSENT PHYSICAL STRESSES, THE
OUTER MEMBRANE IS CAPABLE IF
NOT--IF LEFT ALONE IS CAPABLE
FOR STANDING THE ENTIRE LINE OF
THE CELL WITHOUT--EACH WITHOUT
THE TBLI KAN--KANA.
OKAY O THIS IS WHAT WE THINK
WHAT HAPPENS WITH THE SINGLE OR
DOUBLE, ONCE THE ENDOLIESIN
DEGRADES THE FLI COLSIS CON,
WITH REARAINCHLMENT
OLIGMERRIZATION OCCURS AND THAT
LEADS TO COLLAPSE OF THE
MEMBRANE AND THESE 2 MODELS, WE
DON'T HAVE ANY EVIDENCE FOR
THESE, FOR THE ACTUAL FORMATION
OF THESE MODELS BUT WE DO HAVE
EVIDENCE FOR THE CLAPS IN
CONDENSATION OF THESE DOMAINS,
AND IF THIS LOOKS LIKE VIRAL
MEMBRANE PROEN TOOS TO YOU IF
YOU'RE INTO HA KIND OF THING,
THEY LOOK LIKE THE SAME TO US.
SO 1 STRONG POSITION OF
PREDICTION, IF YOU--IF YOU
EXTRAPOLATE THIS INTO 3
DIMENSIONS, WHAT THIS SAYS IS
THAT DURING LYSIS, THE LAST STEP
MODIFIED BY--MEDIATED BY SPANIN,
YOU OUGHT TO SEGGREGATE THE
PERIPLAZ MIDSIC CONTENTS IN
SECULAR FORM AND AT LEAST
INITIALLY THE PERIPLASMIC
CONTENTS DO NOT PARTICIPATE,
THEY'RE NOT RELEASED SO THAT'S 1
WAY WE CAN TEST THIS MODEL.
FIRST THIS IS TO SHOW FUSION
OCCURS, SO THIS IS--THIS IS
MAMOY, AND WHO ELSE DID THIS
WITH YOU, HE'S NOT HERE RIGHT
NOW.
SO THE BOTTOM LINE IS YOU CAN
IMET WITH THOSE--WE FOUND A
TRICK FOR PUTTING THE SPANIN
PERIPLASMIC DOMAINS ON THE
MEMBRANES, IT ALLOWS THEM BY
PUTTING DIFFERENT COLORS IN
DIFFERENT VESICLES WE CAN SEE
DIRECTLY WHETHER OR NOT THOSE
DOMAINS ARE CAPABLE OF FUSION IN
THE MEMBRANE AND IF THEY ARE,
AND IF YOU HAVE EITHER DEFECTIVE
MUTANTS OR SPANINS, YOU CAN
STABLE ABSORPTION BUT NO FUSION.
OKAY, SO LYSIS JEERN ROSTER, 2
TYPES OF HOLINs, 2 TYPES OF
ENDOLIESIN, THE CANNONICLE
ENDOLIESIN THERE ARE 2 TYPES OF
THOSE AND 2 TYPES OF SPANINS.
I DON'T KNOW WHY THERE'S ALL
THESE DIFFERENT POSSIBILITIES
AND WE'VE EITHER FOUND IN NATURE
OR FOUND OR CONSTRUCTED IN A
LABORATORY ALMOST EVERY
COMBINATION OF THESE 3
COMPONENTS BUT THAT'S NOT ALL.
AND WE PROMISE TO JUF MENTION
ANTIHOLIN, ABOUT HALF THE
SYSTEMS WE STUDY HAVE
ANTIHOLINs I DON'T HAVE TIME
TO TALK ABOUT WHAT THEY DO BUT
IN EACH CASE, 1 ACTIVATES 1
HOLIN FOR THE PURPOSE OF THE
LYSIS CLOCK AND THE MOST
INTERESTING 1 IS T4 WHICH HAS A
AS YOU REMEMBER IT HAS A
CYTOPLASMIC DOMAIN AND A
PERIPLASMIC DOMAIN.
IT TURPS OUT THERE ARE 2
DIFFERENT HOLEINS A SOLUBLE 1
AND AND OACH MORE REMARKABLY,
THE CONTEXT BETWEEN THOSE AND
THE HOLEIN AND THE T-4 CASE THAT
COMPLEX IS DENSATIVE TO DNA IN
THE PERIPLASMA, SO THE MODEL IS
THAT IF YOU INRECYCLING, IF YOU
HAVE A SECONDARY PHAGE INFECTING
DNA, THE DNA GOES IN, IF YOU
PERMIT THAT DMA FROM GETTING TO
THE CYTOPLASM, IT THEN CAN BE
ACT AS A HARBINGER THE
PARTICULAR INFECKED T4 CELL IT'S
IN AN REQUIREMENT WHERE IT'S NOT
A GOOD IDEA TO LYSE.
BECAUSE IT'S BEING SUPER
INFEBBED SO A DNA BINDS TO IT,
STABILIZES AND PREVENTS THE
HOLIN FROM AG TBREIGATING INTO
HOLES AND TRIGGERING LYSIS, SO
IT'S AN INTERESTING STORY IN ITS
OWN WRITE AND GOOD
CRYSTALLOGRAPHY BY WHY
IMPEDIMENTS' LAB AND IN
PARTICULAR FOR THAT UP.
SO THIS I WANT TO SHOW YOU WHAT
WE JUST PRELIMINARY EXPERIMENTS
WE TRY TO SHOW WHETHER OR NOT
IT'S TRUE, THAT AFTER HISPANIN
FUNCTION THE PERIPLASMIC
CONTENTS ARE SEGREGATED.
SO IN THIS EXPERIMENT WE
SECRETED THE SUPER FOLDER GFP TO
THE PERIPLASM, USING A SIGNAL
SEQUENCE AND THEN LET IT TAKES
IT COURSE.
THIS IS A REGULAR LAMDA LYSIS
WITH ALL THE GENES NOW WE'RE
WATCHING GFP IN THE PERIPLASM,
AND IT SHOULD STAY IN THE
PERIPLASM, BECAUSE IT'S NOT
ATTACHED TO ANY LYSIS PROTEIN.
SO--SO WHAT'S HAPPENING IS WE
THINK VERY NEAR THE END OF LYSIS
AS THE PEPTIDE O O GLYCAN IS
DESTROYED, THE PERIPLASM IS
SQUISHED OUT INTO THE HOLES OF
THE CELL, I DON'T KNOW THE
PHYSICS OF IT, BUT AS YOU CAN
SEE, AT LEAST IN THIS--BOTH
CASES, LET ME BACK THIS UP, 1 OF
THEM KIND OF FELL APART AFTER A
WHILE, BUT ALL OF THE INTENSITY
THAT WAS THERE, IN THE
PERIPHERY, IS NOW THERE THIS
THOSE 2 VESICLES AND JUST TO
MAKE SURE THAT THIS ISN'T AN
ARTIFACT, WE WENT ON TO SHOW
THAT IF YOU GET RID OF THE
SPANINS, NOW THERE'S NEVER GOING
TO BE A FUSION OF THE INNER OR
OUTER MEMBRANE.
JUST HAVE A HOLE IN THE
ENDOLIESIN FUNCTION, SAME
EXPERIMENTAL DETAIL, SO NOW OF
COURSE YOU END UP WITH THE
SPHEREICAL CELL AND AGAIN
WITHOUT THE PEPTIDE O O FLI
COLSIS KAN--KANA, FOR SOME
REASON THE PERIPLASM IS ALL
SQUISHED INTO 1 DOMAIN ISSUE THE
1 PART WE'RE NOT QUITE
UNDERSTANDING IS WHY IT'S
LEAKING GFP BACK INTO THE
CYTOPLASM AND THERE MIGHT BE
SMALL S-HOLES MOST OF THE HOLES
OF THE HOLEIN IS IN LARGE HOLES
AND MAYBE A FEW OF SMALL HOLES
SO IT ALLOWS GFP TO LEAK BACK IN
BUT WE HAVEN'T DONE ENOUGH
EXPERIMENTS TO DEAL WITH THAT
YET.
OKAY, SO THE LAST SLIDE IS WHAT
I KAW LYSIS EFTHIMIOS MOLOGY,
WHY DO HOLINs EXIST AT ALL
EXCEPT IT GIVE ME 40 YEARS OF
FUNDING BUT THAT'S GOOD ENOUGH
FOR ME.
BUT PUT A SIGNAL AND SECRETE IT
WILL DO WHATEVER YOU NEED TO DO
AND SO, THAT'S POSSIBLE AND IT
TURNS OUT IN 1 OF THE MOST
COLOSSAL HYPOTHESIS CANCELING
THAT I'VE EVER--I LOOKED AT THE
SEQUENCE OF THE MUSE GENOME, 1
OF THE CLASSIVE PHAGES, 1 OF THE
LAST 1S TO HAVE THE LOISIS
STUDIED AND THERE WAS A CLEAR
HOLEIN AND ANTIHOLEIN, RIGHT
NEXT TO THE LYSIS YEENS AND SO I
ACTUALLY LABELED IN THE REVOW,
THIS LIKELY HOLEIN AND ANTIHOLIM
AND IT TURNS OUT THEY'RE NOT
RELATED TO LYSIS AND IT HAS A
ENDOLYSIS AND HAS A SPANNED
SYSTEM BUT IT DOESN'T HAVE A
HOLIN, SO HAD THIS CASE, THE USE
SYSTEM USES AN ANTIENDOLIESIN
THAT PREVENTS THE ACTIVITY OF
THE ENDOLIESIN AFTER SECRETION.
SO THIS IS WORK THAT'S BEING
DONE BY JO LEAN RAMSEY WHO JUST
SET HER LAB UP AT TEXAS A&M AND
CARRYING ON A LOT OF MY
MULTIYEEN LICENSE WORK.
AND EVEN MORE SIMPLE IS WHY NOT
USE THE SINGLE GENE LYSIS
AUTOLYSIS SYSTEM?
SO WE'VE BEEN TALKING ABOUT THIS
SYSTEM, WE CALL IT MULTIGENE
LYSIS, BUT ALL SMALL PHAGES LIKE
ALL SINGLE STRAND PHAGES AND
SINGLE STRAND RNAA PHAGES, THEY
ACCOMPLISH THIS WITH YEEPS,
THESE YEENS ARE INHIBITING STEPS
IN THE CELL WALL SIJT SIS AND
STIEPS NOT.
SO, BUT WHATEVER THEY DO,
THERE'S NO ENZYME, CARRIED BY
THE BACTERIA PHAGE SO INSTEAD
THESE ARE AUTOLYSIS EVENTS WE
SHOW IF YOU IMK OUT THE LAMDA
AND REPLACE IT WITH THE E-YEEN
OF 5 FORWHICH WE KNOW WORKS BY
INHIBITING THE GLYCAN, LAMDA
MAKES PERFECTLY GOOD PLAQUES SO
AT LEAST AT THE LEVEL OF PLAQUE
FORM EGG THE LAMDA EGENE COMP
LEAPTS OF DEFECT, SO--BUT YET,
SO FAR AS FAR AS WE CAN TELL,
ALL DOUBLE STRAND DNA FAINLS USE
THE SYSTEM AND SUSPECT THEY MUST
BE POWERFUL EVOLUTIONARY FORCES
TO MAKE THAT HAPPEN.
AGAIN, WE'RE BACK TO THE PICTURE
OF THE PEOPLE WHO DID IT ALL AND
I APPRECIATE YOUR PATIENCE.
[ APPLAUSE ]
ANYONE HERE IS FREE TO USE THE
MIC'S FOR QUESTIONS AS WE WAIT
ONLINE.
NTHANK .
>> THANK YOU VERY MUCH.
FASCINATING.
MY MIND WENT ON WHEN THEY ARE
BURSTING IS THERE ANY, ANY
ENERGY DISPENSED AND/OR CAN YOU
PUT THESE UNDER HIPOXIC OR HYPER
OXIC CONDITION TO SEE WHETHER
THEY CHANGE THE TIMING OF THE
BURSTING?
>> THE ONLY THING THAT MATTER
SYSTEM THE MEMBRANE POTENTIAL
BUT THERE'S A LOT OF WAYS CAN
YOU MESS UP THE MEMBRANE.
FOR EXAMPLE, EVEN IN
[INDISCERNIBLE] LABORATORY BEAN
IN THE 1800S, WE KNEW FOR A LONG
TIME IF YOU INFECT E.COLI WITH
ANYTHING, ANY LAMDA T-4 ANY
PHAGE, YOU CAN'T TOP THE SHAKER,
YOU CAN'T SPIN THEM DOWN AND
THAT'S BECAUSE IF YOU STOP THE
SHAKER FLASK IN DENSE CULTURE
LIKE OD.2, THE ATTENTION DROPS
IMMEDIATELY BECAUSE IT'S BEING
SUCKED UP BY ALL THE CELLS, SO
THE BOTTOM LINE IS, ANYTHING
THAT ASHINGS THE MEMBRANE
POTENTIAL AND WE'VE SHOWN USING
THAT, SPINNING BUG EXPERIMENT
THAT WE SHOWED, THE YOU CAN USE
THAT TO MEASURE THE MEMBRANE
POTENTIAL AND AT WHICH TIME IT
TRIGGERS AND IT ONLY REQUIRES A
30% REDUCTION IN MEMBRANE
POTENTIAL FROM ABOUT 150 MILLI
VOWELS DOWN TO LIKE 100 AND
THAT'S ENOUGH TO TRIGGER.
SO YOU CAN GROW, CAN YOU DO
THESE EXPERIMENTS IN ANOXIC
CONDITIONS AS LONG AS YOU'RE
FERMENITATING THE METHODS.
NCOULD THEY BEHAVE SOMEWHAT LIKE
MITOCHONDRIAOXIDATIVE PROCESS.
>> I AGREE, BCL IS 1 OF THE
PROTEINS IN THE MITOGENIC
PATHWAY, IT TURNS OUT THE BCL
PROTEIN COMPLEMENTINGS LAMDA S,
SO MY NAME IS ON A PAPER THAT
SAYS THOSE PROTEINSOR HOLINs,
BUT IT'S NOT TRUE BECAUSE THEY
DON'T HAVE THE--THE KEY ELEMENT
OF THE HOLINs IS THEY HAVE TO
BE TRIGGABLE BY THE POISON
BECAUSE WE THINK THAT'S INTEGRAL
TO THE MECHANISM AND THE WHOLE
TIMING THING, NOTHING WILL WORK
WITHOUT THAT, BUT I COULDN'T
TURN DOWN GETTING A PAPER.
>> VERY NICE, THANK YOU.
>> I HAVE A QUESTION OVER HERE.
>> YEAH.
>> HI, HOW DO THE HOLINS GO TO
THE CURVEATURE OR TO THE POLES.
>> THEY DON'T ALWAYS DO THAT I
WAS SORT OF PISSED OFF THAT 80%
OF THEM HAPPEN AT THE POLES BUT
THEY DON'T ALWAYS DO THAT, I
THINK ITIA A DECISION EARLY ON
BECAUSE WHEN THEY SHOW UP
ELWHERE, THEY DON'T SHOW UP AT
THE POLES, EITHER 1 SO I SUSPECT
THERE'S SOME SORT OF SORTING
EVENT THAT OCCURS, FOR IT'S NOT
LYSIS SPECIFIC, THERE'S A LOT OF
STILL A LOT OF CONTROVERSY ABOUT
HOW ANY PROTEINS FINDS THE
POLES.
THE ANSWER IS I DON'T KNOW.
NWELL, THERE ARE NO QUESTIONS
ONLINE SURPRISINGLY.
LOTS OF PEOPLE WATCHING SO
DR. LORSCH--I'M SORRY, SOMETHING
CAME IN, THAT WAS NOTHING, THAT
WAS MY TEST TO SEE IF IT WAS
WORKING.
>> ACTUALLY SINCE THIS IS A
LECTURE THERE IS ACTUALLY A
TEST.
[LAUGHTER]
LET'S THANK RY FOR A
FANTASTIC TOUR DEFORCE TALK.
AND THERE WILL BE A SHORT
RECEPTION OUTSIDE FOR THOSE WHO
ARE HERE IN REAL LIFE AND THOSE
OF YOU WHO ARE VIRTUAL, YOUR ON
YOUR OWN, GO GET A COOKIE DOWN
STAIRS OR WHATEVER YOU WANT TO
DO BUT THANKS AGAIN RY.
[ APPLAUSE ]