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WHAT I'D LIKE TO DO IS TELL YOU
A LITTLE BIT ABOUT THE LECTURE
NAMED AFTER G. BURROUGHS MIDER,
THE FIRST DIRECT OUR OF NIH
LABORATORIES AND CLINICS, A
PREDECESSOR FOR THE POSITION I'M
IN AS DEPUTY DIRECTOR.
SO THIS LECTURE IS CLOSE TO MY
HEART.
AND THE OTHER INTERESTING THING,
IT'S THE ONLY ONE OF THE
LECTURES PRESENTED BY AN
INTRAMURAL SCIENTIST IN
RECOGNITION AND APPRECIATION OF
OUTSTANDING CONTRIBUTIONS TO
BIOMEDICAL RESEARCH.
SO, IT SEEMS ONLY NATURAL THAT
OUR SPEAKER IS
JENNIFER LIPPINCOTT-SCHWARTZ.
FOLLOWING BACHELOR'S DEGREE, SHE
TAUGHT CHEMISTRY AND BIOLOGY
BOTH IN KENYA AND CALIFORNIA.
AND THEN, BECAME A RESEARCH
ASSOCIATE SPARKING WHAT HAS BEEN
OBVIOUSLY A LIFETIME ENGAGEMENT
WITH SCIENCE SCIENCE.
SHE DID HER GRADUATE STUDIES
WITH DOUG FAMILIAR BRO AT JOHN'S
HOPKINS AND WAS A POSTDOC
SUPPORTED BY THE PRATT AWARD
WITH RICK CLOUZNER ALSO AT
NICHD.
SHE HAS GONE THROUGH THE TENURED
TRACK AND SENIOR INVESTIGATOR
PROCESS AND NOW A NIH
DISTINGUISHED INVESTIGATOR IN
THE CELL BIOLOGY METABOLISM
BRANCH.
SO, IN LOOKING AT THE HISTORY OF
JENNIFER LIPPINCOTT-SCHWARTZ'S
SCIENCE, IT IS HERE IS THAT HER
INTERESTS IN CELL BIOLOGY WAS
EARLY ABOUT YOU WHEN SHE WORKED
WITH RICK, SHE DID SEMINOLE WORK
ON THE RECYCLING PATHWAY FROM
THE GOALING TOW THE
ENDOPLASMICRI TICK LUM.
AND I THINK AT THAT POINT, SHE
BEGAN TO APPRECIATE THE NEED FOR
MUCH MORE QUANTITATIVE WAYS OF
MEASURING EVENTS IN CEIL
BIOLOGY.
UNTIL THAT TIME, MOST CELL
BIOLOGY WAS RELATIVELY
DESCRIPTIVE.
AND I THINK IT IS FAIR TO SAY
SHE LED A REVOLUTION IN
UNDERSTANDING HOW LIPIDS AND
PROTEINS MOVE IN CELLS USING
QUANTITATIVE APPROACHES.
AMONG OTHER THINGS, SHE
PIONEERED THE DEVELOPMENT OF
PHOTO ACTIVATABLE FLUORESCENCE
IMAGING AND LIGHT MICROSCOPY,
AND THAT IS WHAT ALLOWED THE
QUANTITATIVE WORK THAT YOU'LL
HEAR ABOUT A LITTLE MORE TODAY.
WITH GEORGE PATTERSON, SHE
INVENTED PHOTO ACTIVATABLE GFP,
A REAGENTED IN COMMON USE IN
CELL BIOLOGY LABS AND WITH ERIC,
DEVELOPED SUPER RESOLUTION
FLUORESCENCE MICROSCOPY, KNOWN
AS POM, ANOTHER TECHNIQUE NOW IN
GENERAL USE.
BOTH OF THESE ARE AMAZING
BREAKTHROUGH AND ANY ONE OF THEM
WOULD HAVE BEEN A MAJOR ARK
ACHIEVEMENT.
THESE TECHNIQUES HAVE BEEN USED
TO PRODUCE THREE DIMENSIONAL
SUPER RESOLUTION IMAGES OF HOW
SHELL SHAPE IS DETERMINED AND
HOW CELLS MOVE.
THE WORK LED TO HER ELECTION TO
THE NATIONAL ACADEMY OF SCIENCES
AND INSTITUTE OF MEDICINE AND
MANY, MANY AWARDS.
ONE OF THE THINGS I WAS GOING TO
DO IF WE COULDN'T GET THIS
SLIDESHOW GOING WAS JUST READ
HER AWARDS DURING THIS REMAINING
HOUR.
SHE HAS GIVEN HUNDREDS OF
LECTURES, THROUGHOUT THE WORLD.
AND SERVED ON SCIENTIST ADVISORY
BOARDS FOR THE HOWARD HUGHES
UNTIL INSTITUTE, WEITZMAN
INSTITUTE, SORREL PROGRAM AND
THE PSALM INSTITUTE.
AND MOST RECENTLY ELECTED AS
PRESIDENT OF THE AMERICAN
SOCIETY OF CELL BIOLOGY.
SHE IS ROYALTY, AS FAR AS WE ARE
CONCERNED.
HER TALK IS ENTITLED, NAVIGATING
THE CELLULAR LANDSCAPE WITH
OPTICAL PROBES AND IMAGING
STRATEGIES AND TECHNICAL
INNOVATIONS.
I HOPE OUR TECHNICAL INNOVATIONS
WORK.
JENNIFER?
>> THANK YOU VERY MUCH FOR THAT
VERY NICE INTRODUCTION, MICHAEL.
IT'S REALLY A GREAT HONOR TO BE
HERE AND TO GIVE THIS TALK.
AS MICHAEL MENTIONED, REALLY MY
WHOLE CAREER HAS BEEN FOCUSED ON
THIS CELL AND HOW THE ORGANELLES
AND PROTEINS THAT REALLY
COMPRISE THIS STRUCTURE ARE
ORGANIZED TO REALLY CREATE THIS
AMAZING SYSTEM.
AND FOR THOSE OF YOU WHO ARE
WONDERING WHAT THIS TENTED IS
FOR, THIS IS A PROP FOR A CELL,
IN WHICH I WILL TALK ABOUT LATER
AT THE END OF THE TALK.
BUT, MY LAB IS REALLY BEEN
FOCUSED ON MANY OF THE DIFFERENT
ORGANELLES COMPRISING THE CELL,
INCLUDING, AS MICHAEL MENTIONED,
THE SECRETORY PATH A ENDOPLASMIC
METIC LUM, GOLGI, MOST RECENTLY
MITOCHONDRIA AND HOW THEY CHANGE
THEIR DYNAMIC FORM UNDER
DIFFERENT CELLULAR CONDITIONS.
WE HAVE ALSO BEEN INTERESTED IN
THE NUCLEUS, ASPECTS OF NUCLEAR
ENVELOPE BREAKDOWN AND
DISASSEMBLY AND PHENOMENON
OCCURRING ON THE PLASMA
MEMBRANE, INCLUDING BUDDING OF
VARIANCE.
BUT TO DATE, I WANT TO TALK
ABOUT THE CYTOSKELETON, AND WHAT
WE HAVE RECENTLY LEARNED USING
THESE MODERN SUPER RESOLUTION
IMAGING APPROACHES REGARDING HOW
THE CYTOSKELETON REALLY
ORGANIZES THE CELL AND ACTS AS A
CONTRACTILE ENGINE REALLY UNDER
THE HOOD OF CELLS TO ALLOW CELLS
TO HAVE PARTICULAR SHAPES THAT
PLAY CRITICAL ROLES FOR ALLOWING
CELLS TO PROTRUDE INTO DIFFERENT
ENVIRONMENTS, TO MIGRATE OVER
DIFFERENT SURFACES.
NOW, A KEY ASPECT OF THIS
CONTRACTILE ENGINE THAT I'M
GOING TO BE TALKING ABOUT IS THE
ACTIN AND MICE INCOME OPPONENTS
WITHIN THE CELL -- MY SIN
COMPONENTS WITHIN THE CELL.
WHAT THE SYSTEM CAN DO IS PRETTY
SPECTACULAR.
IF WE JUST TAKE CYTOPLASMA FROM
EGGS FROM A FROG, AND COVER THE
DROP WITH OIL AND LOOK AT IT IN
DIC, WHAT YOU CAN SEE IS A
INCREDIBLE SELF ORGANIZING
CAPABILITY OF THIS CYTOPLASM
THAT IS DRIVEN BY THE
CYTOSKELETAL ELEMENTS, ACTIN AND
MICE IN WITHIN THIS SYSTEM.
THIS IS A DIC IMAGE IMAGING OVER
SEVERAL HOURS AND YOU CAN SEE --
THIS IS JUST A SHORT TIME WINDOW
BUT THIS PERIODIC CONTRACTION,
CONTRACTILE WAVES OF THE
CYTOPLASM YOU CAN SEE HERE WILL
CAREO FOR HOURS, AS SOON AS THE
CYTOPLASMIC DROP LET HAS BEEN
WARMED UP TO ROOM TEMP OR 37
DEGREES.
NOW THIS BEHAVIOR OF THE
CYTOPLASM HAS CONSEQUENCES FOR
VESICLES AND MEMBRANES THAT ARE
WITHIN IT, AND THAT IS
ILLUSTRATED HERE WHERE WE
ESSENTIALLY LABELED, WITH DIOC6,
A FLORESCENT TAG FOR MEMBRANES,
THAT WERE ALSO CARRIED INTO THIS
LITTLE DROP WILL THE HERE.
AND YOU CAN SEE -- DROPLET.
AND THE CONTRACTION CAPABILITY
OF THIS CYTOPLASM IS ABLE TO
CENTER IN A SELF-ORGANIZED
FASHION, THE MEMBRANE ORGANELLES
OF THE CYTOPLASM.
SO THIS IS JUST ILLUSTRATES THE
REALLY LIVING NATURE OF
CYTOPLASM IN THE ABSENCE OF ANY
SORT OF OUTSIDE MEMBRANE AND
OUTSIDE OF THE CELL.
SO THE QUESTION IS, WHAT WE
THINK IN TERMS OF HOW THIS
CONTRACTILE ENGINE IS ACTUALLY
WORKING, CAN BE SUMMARIZED FROM
THE FRAMEWORK OF THIS VERY
SIMPLE MODEL PROPOSED BY CHRIS
FIELDS AND TIM MITCH EN SON, AND
SUPPORTED BY MANY OTHER
RESEARCHERS, WHERE ESSENTIALLY
THIS PERIODIC GEL CONTRACTION IS
DRIVEN BY THREE COMPONENTS, AN
ACTIN FILAMENT WHICH IS
NUCLEATED BY A PARTICULAR
NUCLEATOR, SO THE FILAMENT CAN
GROW, AND A MOTOR PROTEIN MY SIN
TWO, CAPABLE OF WALKING ALONG
THESE ACTIN FILAMENTS AND
BECAUSE IT CAN BIND TO MORE THAN
ONE FILAMENT, WHAT WILL QUICKLY
HAPPEN AS THESE FILAMENTS START
GROWING, IS THAT THE MYCIN2 WILL
COLLECT THE FILL COMMENTS
REORGANIZING THEM INTO A TIGHT,
BUNDLED-LIKE STRUCTURE.
THAT UNDERLICE THE CONTRACTILE
BEHAVIOR OF THOSE CONTRACTILE
WAVE THAT IS YOU'RE SEEING, WE
BELIEVE REPRESENTS THIS PERIODIC
CONTRACTILE FORMATION OF THIS
ACTIN FILAMENT MESH WORK DRIVEN
BY MICE IN TWO ACTIVITY.
AS THESE FILAMENTS CONTRACT,
ULTIMATELY, THEY START NUKE
LATERS AND FILAMENT COMPONENTS
START TO DISASSOCIATING FROM
THAT AND YOU GET A WHOLE NEW
CYCLE FORM IN A NEW WAVE.
SO IS THIS OPERATING WITHIN THE
CONTEXT OF A LIVING CELL?
IF SO, HOW?
THIS IS A PTK1 CELL WHERE IT'S A
CRAWLING CELL.
IT CRAWLS ON A SUBSTRATE, IN
PARTICULAR, FIBER IN EFFECT IN,
AND WHAT CAN YOU SEE IS THAT THE
ACTIN LABELED IN RED, HAS A OR
LOOKS LIKE IT'S UNDERGOING THIS
TYPE OF CONTRACTILE ASPECT TO
IT.
AND IT APPEARS TO BE COORDINATED
WITH ASSEMBLY OF THESE ADHESION
PROTEINS LABELED IN GREEN.
AND YOU CAN SEE THESE ARE
SUBSTRATES OR ADHESION COMPLEXES
THAT ARE FOUND ON THE BOTTOM OF
THE CELL THAT ACT AS FEET TO
ALLOW THIS CELL TO ADHERE TO THE
SUBSTRATE AND TO CRAWL.
NOW, IN MY TALK TODAY, I WANT TO
FOCUS ON TWO ASPECTS OF THIS
DYNAMIC CELL SYSTEM, WHICH WE
THINK IS REALLY MANY ASPECTS OF
IT ARE BEING DRIVEN BY THIS
GELATION CONTRACTION ACTIVITY
DUE TO MYCON AND PUMMERRIZATION
CYCLES.
THE FIRST PART OF THE TALK IS
GOING TO BE FOCUSED ON THIS
LEADING EDGE, WHICH IS THE
DYNAMIC PORTION OF THIS CELL
THAT IS ESSENTIALLY ACTIVE IN
THE DIRECTION THAT THE CELL IS
CRAWLING.
THE SECOND PART OF THE TALK IS
GOING TO RELATE TO THE OVERALL
SHAPE OF THIS CELL.
AND WHETHER THIS GELATION
CONTRACTION PROCESS IS IN ANY
WAY IMPORTANT FOR HOW THE CELL
THREE-DIMENSIONALLY ORGANIZES
ITSELF.
SO LET'S START WITH JUST
FOCUSING ON THAT LEADING EDGE
AND THE ACTIVITY OF THE ACTIN
AND THE MYCOIN TWO IN THIS
LEADING EDGE.
THIS IS A MOVIE SHOWING ACTIN
MRFP AND WHAT YOU CAN SEE IS THE
LEADING EDGE UNDERGOES PERIODIC
PROTRUSION AND RETRACTION
CYCLES.
AND THEY SEEM TO BE COUPLED TO
TWO DIFFERENT OVERALL
APPEARANCES OF ACTIN.
A DIFFUSE MESH WORK ACTIN IN
THIS PROTRUDING AND RETRACT ZONE
OF THE LEADING EDGE, AND AN
ACTIN POOL THAT IS IN A MUCH
MORE BUNDLED FORM HERE.
NOW, IF WE TAKE THIS CELL AND
FIX IT, AND LOOK AT THE ACTIN
FILAMENTS, AND THIS HAS BEEN
DONE IN MANY, MANY LABS AND IT
HAS BEEN STUDIED OVER DECADES,
WHAT YOU SEE IS THE FOLLOWING.
YOU SEE A CRISSCROSS PATTERN OF
ACTIN IN THAT DYNAMIC REGION, WE
CALL THE LAMELLIPODIA UNDERGOING
THE PROTRUSION AND RETRACTION
CYCLES AND THEN WE SEE A
PARALLEL BUNDLED POOL OF ACTIN
IN THIS ZONE WHERE THE FOCAL
ADHESIONS THAT ATTACH THE CELL
TO THE SUBSTRATE ARE LOCALIZED.
NOW, ELEGANT WORK FROM MANY LABS
HAVE REALLY PROVIDED INSIGHT
INTO THESE TWO ACTIN SYSTEMS.
IN THE CASE OF THE LAMELL
APODIA, WE KNOW THAT THIS MESH
WORK IS CONTINUOUSLY BEING
FORMED AND PLIMMORIDES AND
DISASSEMBLED AND ITS PURPOSE IS
TO ESSENTIALLY CREATE A STIFF
SURFACE THAT CAN LEAD TO
PROTRUSION OF THE CELL.
AS IT EXPANDS, BECAUSE THE FOCAL
ADHESIONS AND THE REST OF THE
CYTOPLASM ARE BACK HERE, THEY
ACT AS A RESISTOR SO THE ONLY
PLACE THIS MESH WORK GROWS THAT
THE STRUCTURE CAN EXPAND IS IN
THIS FORWARD DIRECTION.
THAT IS REALLY THE BASIS FOR HOW
THESE CELLS ARE MOVING FORWARD.
THEY ARE PUSHING THROUGH THE
ACTIVITY OF THIS, GROWING ACTIN
MESH WORK, THE PLASMA MEMBRANE
FORWARD.
NOW THIS IS A CARTOON
ILLUSTRATING THE MOLECULAR
PLAYERS THAT ARE INVOLVED IN
REGULATING THIS ACTIN
POLYMERIZATION PROCESS.
INITIALLY YOU HAVE STIMULI ON
THAT PLASMA MEMBRANE THAT CAN
PRESENT PET 2 AND THEN RECRUITS
VARIOUS MOLECULES THAT LEAD TO
THE POLYMERIZATION OF THESE
ACTIN FILAMENTS.
THESE ARE BRANCHED FILAMENTS
THAT CAN BECOME EXTREMELY DENSE
AND STIFF AND AS THEY GROW, THEY
PUSH THE WHOLE CELL SURFACE
FORWARD.
NOW ONE OF THE KEY PREDICTIONS
IN THIS MODEL THAT IS SUPPORTED
BY MANY DIFFERENT DYNAMIC
FLORESCENT IMAGING ASSAYS.
THAT ALL OF THE FILAMENTS WITHIN
THESE -- ALL THE ACTIN MOLECULES
WITHIN THESE INDIVIDUAL
FILAMENTS ARE UNDERGOING WHAT IS
CALLED, RETROGRADE FLOW.
ACTIN IS DEPOLYMERIZING HERE AND
POLYMERIZING AT THESE POINTS
HERE.
A VAR TALENTED POSTDOC IN MY LAB
WHOSE WORK REALLY IS GOING TO BE
OR THE REAL TOPIC OF TODAY,
VIRTUALLY ALL THE EXPERIMENTS
THAT I'M TALKING ABOUT HAVE
REALLY BEEN PIONEERED BY DYLAN.
ANYWAY, WHAT DYLAN DID WAS, HE
USED A PHOTO CONVERTIBLE
FLORESCENT PROTEIN TAGGED TO
ACTIN TO LABEL ALL OF ACTIN
FILAMENTS WITHIN THE CELL AND
THEN HE SELECTIVELY USING 405
LASER LIGHT PHOTO CONVERTED TO
RED, A SUBSET OF THE ACTIN
MOLECULES.
WHEN ONE DOES THAT, YOU CAN SEE
THE VERY QUICK REPLACEMENT OF
THE PHOTO CONVERTED MOLECULES IN
RED WITH GREEN MOLECULES, WHICH
REPRESENT ACTIN THAT HAS BEEN
REPOLYMERIDES ON TO THIS GROWING
AMERICAN WORK.
WHAT THIS DATA IS SUGGESTING IS
INCREDIBLY DYNAMIC TURNOVER OF
THESE MOLECULES IN THAT LEADING
EDGE.
AND THIS ALSO CAN BE SEEN USING
A VARIATION OF SPECKLED
MICROSCOPY, WHICH WAS DEPENDENT
BY CLAIR WATERMAN.
HERE WE ARE USING A PHOTO
CONVERTIBLE EOS MOLECULE TOO
GOOD ACTIN AND WE CAN
ESSENTIALLY SWITCH ON THESE
MOLECULES USING 405 LIGHT AND AS
WE SWITCH THEM ON, INDIVIDUAL
MOLECULES LIGHT UP AND BECAUSE
WE ARE LIGHTING UP REALLY SPARSE
POPULATIONS AT ANY PARTICULAR
TIME, WE CAN FIT THE CENTROID OF
THE POINTS FUNCTION OF THE
INDIVIDUAL MOLECULE THAT WE HAVE
SWITCHED ON AND TRACK IT.
AND THIS TECHNIQUE IS CALLED
SINGLE PARTICLE TRACKING P PALM.
ONE OF THE SINGLE MOLECULES
SUPER RESOLUTION IMAGING
APPROACHES.
IT ALLOWS US TO TRACK THE
MOVEMENT OF INDIVIDUAL ACTIN
MOLECULES ALONG THESE BRANCHED
STRUCTURES THAT ARE CREATING
THAT LAMELLA PEDAL REGION OF THE
CELL THAT ALLOWS THE CELL TO
DRIVE FORWARD.
EACH LINE REPRESENTS A TRACK OF
A ACTIN MOLECULE WITHIN A FIBER.
THE BEGINNING OF THE TRACK IS
LABELED IN THE COLOR AS A SPOT,
AND THE TRAIL OF THE TRACK
FOLLOWS THAT.
YOU CAN SEE THAT ALL OF THESE
TRACKS ARE MOVING INWARD.
IN FACT, YOU CAN CREATE A FLOW
MAP TO LOOK AT THE VELOCITY OF
THESE ACTIN MOLECULES IN
DIFFERENT REGIONS OF THE CELL
USING THIS APPROACH.
VERY SIMILAR TO SPECKLED
MICROSCOPY BUT AT THE -- BUT
INSTEAD OF LOOKING AT A LARGE
POPULATION OF ACTIN MOLECULES
THAT HAVE BEEN SPECKLED, WE ARE
LOOKING AT INDIVIDUAL ACT INCH
MOLECULES WITHIN THESE ACTIN
FIBERS.
SO TA VERY NICELY SUPPORTS THIS
REALLY CLASSIC CONCEPT FOR HOW
THIS LAMELL APODAL SYSTEM IS
GROWING AND TURNING OVER ITS
COMPONENTS IN AN ACTIVE WAY.
WHAT ABOUT THIS BUNDLED POOL OF
ACTIN IN THE LAMELLA WHERE THE
FOCAL ADHESIONS ARE LOCALIZED?
TO WHAT EXTENT IS THIS POOL OF
ACTIN RELATED TO THE BRANCHED
MESH WORK OF ACTIN THAT WE SEE
CLOSE TO THE EDGE OF THE CELL?
SO THIS IS SOMETHING THAT IS
EXTREMELY IMPORTANT TO
UNDERSTAND IF ONE IS GOING TO
TRY TO DEVELOP A MODEL FOR HOW
THE CELL IS REALLY CONTROLLING
ITS EDGE BEHAVIOR IN ORDER FOR
THE CELL TO EXPLORE DIFFERENT
ENVIRONMENTS AS IT IS MOVING.
SO THE FIRST HINT FOR WHAT MIGHT
BE THE RELATIONSHIP BETWEEN
THESE TWO POOLS OF ACTIN IN THE
LEADING EDGE OF THESE CRAWLING
CELLS CAME WITH THE OBSERVATION
THAT FROM THESE FLOW MAPS, THAT
AS WE TRACK THE FLOW VELOCITY OF
THESE INDIVIDUALS ACTIN
FILAMENTS, WE SEE THAT THE
VELOCITY CHANGES DEPENDING ON
WHETHER THE EDGE OF THE CELL IS
PROTRUDING OR RETRACT.
WHEN THE EDGE OF THE CELL
RETRACTS, THE ACTIN MOLECULES IN
THE FILAMENTS ARE ACTUALLY
MOVING MUCH FASTER BACKWARDS.
AND SO THAT SUGGESTS THAT THERE
IS SOME FORT FORCE THAT IS
OPERATING ON THIS SYSTEM TO
DRIVE THE EDGE OF THAT CELL
BACKWARDS.
AND IF ONE GOES BACK TO JUST
CONVENTIONAL CONFOCAL IMAGING,
ONE CAN START GETTING A SENSE OF
WHAT MIGHT BE UNDERLYING THIS.
SO HERE IS THE EDGE OF THE CELL,
WE ARE ZOOMED UP ON THE PORTION
OF THE CELL AND YOU CAN SEE THE
PLASMA MEMBRANE IS CONTINUALLY
PROTRUDING AND RETRACT COUPLED
WITH THE LAYING DOWN OF THESE
ACTIN BUNDLES IN THE REAR HERE.
AND IF ONE ACTUALLY DOES A LINE
SCAN THROUGH ANY PORTION OF THE
MRS. MEMBRANE OF THIS CELL, AS
SHOWN HERE, YOU CAN SEE THAT THE
AMPLITUDE OF THIS PROTRUSION
RETRACTION CYCLE IS CONSERVED
IT'S REALLY REMARKABLE.
SO YOU CAN SEE IT IS ESSENTIALLY
LIKE A SYSTEM OF WAVES THAT ARE
CONTINUOUSLY WORKING.
YOU CAN SEE IN THIS PERIODIC
MOTION THAT AS THE EDGE RETRACTS
BACK, THE VELOCITY IS MUCH
FASTER.
SO IT IS SAW TOOTHED INSTEAD OF
TRIANGULAR INSTEAD OF PURELY
PERIODIC.
SO THAT SUGGESTS THAT SOMETHING
IS ACTUALLY PULLING THIS SURFACE
BACK.
ONE WAY TO ADDRESS THAT IS TO
LOOK AT IT MORE CAREFULLY AT THE
SPACIAL TEMPORAL RELATIONSHIP
BETWEEN THE ACTIN IN THAT
PERIPHERAL REGION VERSUS THE
ACTIN THAT IS IN THE BUNDLES.
HERE IS AN ADDITIONAL
PHOTOACTIVATION EXPERIMENT WHERE
WE ARE ASKING THE QUESTION OF,
WHAT IS THE SPACIAL TEMPORAL
RELATIONSHIP BETWEEN THE ACTIN
HERE AND ACTED IN FOUND HERE
WHEN THE EDGE OF THE CELL IS
UNDERGOING RETRACTION MODE?
SO, HERE IS RIGHT BEFORE PHOTO
CONVENTIONER, AND ALL OF THE
ACTIN WITHIN THAT EDGE REGION IS
LABELED WITHIN GREEN AND WITHIN
JUST A SECOND AFTER PHOTO
CONVENTIONER, WHERE WE SWITCH ON
ALL OF THESE RED MOLECULES, YOU
CAN SEE WE HAVE A SELECTIVE POOL
THAT IS LABELED.
NOW IF WE TRACKED THAT SELECTIVE
POOL, WHAT WE CAN SEE IS WITHIN
TWO MINUTES THESE MOLECULES
COLLAPSE DOWN TO FORM THIS
BUNDLE HERE.
THAT IS JUST A CYME OR GRAPH
THAT SHOWS THAT PROCESS OVER
TIME THIS IS INDICATING THAT
UNDER THIS CONTRACTION MODE,
WHEN THAT CELL EDGE IS
CONTRACTING, THE ACTIN FILAMENTS
IN THIS THAT HAVE BEEN BUILT
THROUGH THIS DYNAMIC BRANCHING
OFF THAT PLASMA MEMBRANE AND
OTHER MODULATORS, THIS SYSTEM IS
BEING BROUGHT DOWN AS A WHOLE,
AS A STRUCTURE, TO POTENTIALLY
CREATE THE BUNDLES FOUND IN THE
REAR.
THAT IMMEDIATELY SUGGESTS
THAT -- SOME KIND OF MOTOR
PROTEIN OR ENERGIZER IS
MEDIATING THIS SO WE LOOKED AT
THE MOTOR PROTEIN AT WHETHER IT
COULD POTENTIALLY JUST AS WE
KNOW IN THE CYTOPLASMIC
EXTRACTS, CAN DRIVE THESE
RHYTHMIC CONTRACTILE PHASES,
WHETHER IN FACT MICE IN TWO IS
DOING THE SAME THING AT THE EDGE
OF THE CELL.
YOU IF WE LOOK AT MYOSIN 2 IN
THIS CELL AND LOOKING AT THE
LEADING EDGE OF THE CELL, YOU
CAN SEE THE MYOSIN 2, ALTHOUGH
ENRICHED ON THE ACTIN FIBERS,
THESE ARE -- ACTIN ARKS WHERE
FOCAL ADHESIONS ARE LOCALIZED.
IT IS LOADING ON TO THE ACTED IN
UP IN THIS REGION AND LOOKS LIKE
IT IS DRAWING DOWN THE ACTIN
MESH WORK INTO THIS ZONE.
SO THIS IS A MODEL DESCRIBING
WHAT WE THINK IS HAPPENING AT
THAT LEADING EDGE THAT UNDERLIES
THIS TYPE OF DYNAMIC MOTION THIS
IS GROWING OFF SURFACES OF THIS
BRANCHED MESH WORK.
MICEO SIN TWO HAS THE AFFINITY
FOR THIS BATCHED SYSTEM AND
COMBINED TO ONE OR MORE
FILAMENTS.
ONCE THIS MESH WORK GUESS DENSE
ENOUGH, IT CAN START WALKING A
LOSS THAT THEY NOW BECOME
PARALLEL.
THE MYOSIN TWO WAS A TWO HEADED
MOTOR THAT CAN WALK ALONG TWO
FIBERS AND DRAW THEM INTO A MORE
CONTRACTILE BUNDLE AND THAT IS
WHAT WE THINK IS HAPPENING
DURING THIS PHASE.
AND MA DOES IS ESSENTIALLY
CONVERT THIS SYSTEM OF ACTIN
MESH WORK FROM ONE THAT IS A
PROTRUSION MACHINE TO ONE THAT
IS CONTRACTILE THAT IS LOCALIZED
IN THIS REGION HERE WHERE ALL
THE FOCAL ADHESIONS ARE
LOCALIZED.
WELL, WHAT IS THE FUNCTION OF
THIS SYSTEM?
SO IF WE GO BACK TO THIS
CRAWLING CELL, THERE SEEMS TO BE
A CORRELATION OF THE FEET AT THE
BOTTOM OF THE CELL THAT ALLOW
THE CELL TO CRAWL ON A SUBSTRATE
AND THIS DYNAMIC PROTRUSION
RETRACTION ACTIVITY.
SO WHAT DYLAN DID IN ORDER TO
LOOK MORE CAREFULLY AT WHETHER
THE LAYING DOWN OF THESE FOCAL
ADHESIONS COULD BE COUPLED TO
THIS DYNAMIC PROTRUSION
RETRACTION CYCLE, WAS TO IMAGE
VERY CAREFULLY IN A TEMPORAL
PALTERERN, THE LOCALIZATION OF
THE FOCAL ADHESIONS AND WE ARE
LOOKING AT THIS AND YOU SEE THIS
FOR MANY OTHER MOLECULES AS WELL
RELATIVE TO ACTIN.
IF WE TAKE A CYMEO GRAPH THROUGH
THIS EDGE AND PLAY IT OUT IN
TIME.
WHAT YOU CAN SEE IS THE FOCAL
ADHESIONS ARE LAYING DOWN AT A
TIME WHEN THEY LAY DOWN, IT'S
COINCIDENCE WITH THE PROTRUSION
CYCLE OF THE LEADING EDGE.
WHAT IS INTERESTING IS WHEN THE
EDGE RETRACTS BACK, IT ONLY
RETRACTS BACK TO WHERE THAT
FOCAL ADHESION IS LOCALIZED.
AND THEY ARE ACTING AS A BARRIER
TO PREVENT FURTHER RETRACTION.
AND SO, WHAT THESE FOCAL
ADHESIONS ARE DOING IS SERVING
AS A BASE FOR A NEW PROTRUSION
CYCLE.
NOW IF THESE ADHESIONS HAVE
ATTACHED TO THE SUBSTRATE AT A
PLACE THAT IS UPSTREAM FROM
EARLIER FOCAL ADHESION THAT IS
HAVE BEEN LAID DOWN IN AN
IDENTICAL FASHION, THE NET
RESULT IS THE CELL STARTS MOVING
FORWARD BECAUSE THE BASE IS
CONTINUALLY SHIFTING FORWARD FOR
THIS DYNAMIC RETRACTION
PROTRUSION AND RETRACTION CYCLE.
THIS IS WHAT IS GOING ON IN A
CRAWLING CELL.
WHAT IS HAPPENING IN A CELL THAT
IS NOT CRAWLING?
IN A CELL THAT IS NOT CRAWLING
FAST OR AT ALL, WE SEE THE SAME
RETRACTION OF THIS LEADING EDGE
AND WHEN THAT NEW FOCAL ADHESION
IS LAID DOWN.
IT SLIPS BACK.
THIS WILL IS PRESUMABLY BECAUSE
THE FOCAL ADHESION HAS NOT MADE
A TIGHT ENOUGH ASSOCIATION WITH
THE SUBSTRATE TO HOLD IT AGAINST
THIS REAR WARD FLUSH OF ACTIN
AND MEMBRANE BACKWARDS.
AS A RESULT IT SLIPS BACK AND
THE NET RESULT IS THE NEXT
PROTRUSION CYCLE IS NOT THE BASE
OF THE NEXT CYCLE HAS NOT
SHIFTED FORWARD.
SO THIS CELL SITS THERE
UNDERGOING THIS DETRACTION AND
NOT GOING ANYWHERE SO THE
PURPOSE OF THIS DYNAMIC
CONTRACTILE ACTIVITY OF THE
CYTOPLASM IN THIS LEADING EDGE
IS ALL ABOUT IS TO ALLOW THE
CELL TO CREATE AN ORGANELLE THAT
CAN PROTRUDE FORWARD, THAT CAN
LAY DOWN FOCAL ADHESIONS LIKE A
TANK MOVING THROUGH A JUNGLE AND
ESSENTIALLY ALLOW THE CELL TO
MOVE THROUGH A PARTICULAR AREA.
SO THE NEXT PART RELATES TO IS
THIS CONTRACTILE ENGINE PLAYING
ANY OTHER ROLE IN THE WAY THAT
THE CELL IS SHAPED OR BEHAVES
DYNAMICALLY?
SO FOR THAT, DYLAN DECIDED TO
LOOK IN 3D IN A MORE SORT OF
THREE-DIMENSIONAL PERSPECTIVE IN
TERMS OF THIS GEL CONTRACTION
CYCLE.
UP TO NOW, ALL OF THE MOVES THEY
HAVE SHOWN YOU HAVE BEEN LOOKING
TOP DOWN AT THE CELL AND THE
DYNAMIC MOTION.
WHAT IS HAPPENING IN THE SIDE
VIEW?
SO IN ORDER TO DO THAT, WE
NEEDED TO BEGIN DOING 3D IMAGING
OF THESE CELLS.
NOW, IF YOU USE JUST
CONVENTIONAL CONFOCAL
MICROSCOPY, AND COLLECT Z
SECTIONS TO RE-CREATE IN Z, THE
SHAPE OF THE CELL AS SHOWN HERE,
WHAT YOU YOU QUICKLY SEE IS THAT
THE BEAUTIFUL ACTIN STRUCTURES
THAT YOU CAN SEE IN XY
PROJECTION, WHICH INCLUDES THESE
ACTIN ARCS AS WELL AS THESE
DORSAL STRESS FIBERS, IF WE
ROTATE THIS ON ITS SIDE, WE NOW
SEE ZERO RESOLUTION IN Z.
ALL OF THIS STUFF IS JUST LOOKS
LIKE, THERE IS NO ORGANIZATION.
BUT WE WANT TO UNDERSTAND HOW
THESE STRUCTURES, THESE DORSAL
STRESS FIBERS IN THESE ACTIN
AARE ORGANIZED IN Z IN THIS
PORTION OF THE CELL BECAUSE THIS
IS A REALLY IMPORTANT PART OF
THE CELL.
THIS IS WHERE THE FOCAL
ADHESIONS ARE BEING LAID DOWN.
THIS IS THE DIRECTION OR THE
LEADING EDGE OF THE CELL THAT IS
PUSHING THROUGH SUBSTRATES AS A
CELL CRAWLS IN DIFFERENT PLACES
WITHIN YOUR BODY OR ON A
SURFACE.
SO IN ORDER TO BE ABLE TO GET
INSIGHT INTO THE Z DIMENSION,
THE VERTICAL ORGANIZATION OF
THESE ACTIN FILAMENTS, WHAT
DYLAN DID WAS APPLY STRUCTURE
ELIMINATION MICROSCOPY.
THIS IS A SUPER RESOLUTION
IMAGING APPROACH THAT USES
MODULATED STRUCTURE LIGHT
PATTERNS ON A SAMPLE TO GENERATE
INTERFERONS PATTERN THAT ALLOW
RECONSTRUCTION OF AN IMAGE WITH
DOUBLE THE X, Y AND Z
RESOLUTION.
WHAT THAT ALLOWED US TO SEE IS
THE ARRANGEMENT OF THESE ACTIN
TYPERS AT INCREDIBLES RESOLUTION
AND THAT IS ILLUSTRATED HERE.
THIS IS THE SAME CELL I SHOWED
YOU BUT NOW LOOKING WITH
STRUCTURE ELIMINATION AND EACH
OF THE COLORS THAT YOU SEE HERE
REPRESENTS THE POSITION OF THE
ACTIN FILAMENT THAT YOU'RE
LOOKING AT IN Z.
AND IF WE JUST ROTATE THIS
IMAGE, THE LINE HERE, THIS IS A
TOP-DOWN VIEW OF THE CELL, AND
THIS WHAT WE SEE.
THE REMARKABLE THING IS THAT ALL
OF THE ACTIN THAT IS COMPRISING
THOSE ARK-LIKE STRUCTURES IS
LOCALIZED AT THE TOP SURFACE OF
THE CELL, THE DORSAL SURFACE OF
THE CELL.
THE DORSAL OR STRESS FINERS
RUNNING PERPENDICULAR ARE
LOCALIZED THROUGHOUT.
YOU CAN SEE FROM THE BASE AS
WELL AS THROUGH THE TOP AND THAT
IS ILLUSTRATED IN THIS LAYERING
IMAGE HERE.
SO THIS IS JUST A MAXIMUM
PROJECTION IMAGE OF ESSENTIALLY
THIS PORTION OF THIS CELL AND
THIS IS THE MAXIMUM PROJECTION
IMAGE.
IF WE JUST MOVE UP IN Z THROUGH
THAT IMAGE, YOU CAN SEE THE
DIFFERENT TYPES OF ACTIN THAT
ARE REVEALING THEMSELVES IN
DIFFERENT POSITIONS ACROSS THAT
VERTICAL SPACE.
THE STRESS FIBERS ARE CROSSING
MANY DIFFERENT Z PLANES AND IT
IS ONLY THE PLANE AT THE TOP OF
THE CELL WHERE ALL OF THESE ARCS
ARE LOCALIZED.
SO IF WE JUST LOOK THAT THE FROM
ANOTHER PERSPECTIVE, HERE ARE
THE STRESS FIBERS I MENTIONED
THAT MOVE THROUGHOUT THIS CASE
IS THEY LOOK LIKE THEY ARE
ACTING OR ESSENTIALLY JUST
CONNECTING STRUTS BETWEEN ACTIN
AT THE TOP OF THE CELL AND ACTIN
AT THE BOTTOM OF THE CELL-BASED
ON THIS STRUCTURE ELIMINATION
MICROSCOPIC IMAGING.
INTERESTINGLY, ALL OF THOSE AAS
WELL AS THE STRESS FIBERS ARE
LOCALIZED IN THE FLAT PORTION OF
THE CELL THE DIRECTION THE CELL
IS MOVING.
THIS CELL IS MOVING IN THIS
DIRECTION AND ALL OF THESEARS
AND FIBERS ARE LOCALIZED HERE.
NOTICE THIS PORTION OF THE CELL
IS VERY FLAT AND IT'S FLAT WE
THINK, BECAUSE THAT ALLOWS THE
CELL TO PROTRUDE INTO NEW PACES
AND MAKES IT VERY EASY FOR CELL
TO CREATE A LAMELLA PODIUM THAT
CAN PROTRUDE OUT IN ESSENTIALLY
IN A SINGLE DIRECTION TO EXPAND
THE DIRECTION OF THE CELL,
ESSENTIALLY SQUEEZE THAT CELL
FORWARD.
A KEY QUESTION IS, HOW DOES THE
CELL GET TO THIS SHAPE?
HOW DOES THE CELL BECOME FLAT IN
THIS REGION HERE?
NOW THE FACT THAT THESE ARCS AND
STRESS FIBERS ARE LOCALIZED
HERE, LED DYLAN TO START
THINKING MAYBE THERE ARE
SOMETHING ABOUT THESE FIBERS
THAT ARE RESPONSIBLE FOR DRIVING
OR SHAPING THIS CELL, MAKING
THIS PORTION OF THE CELL SUPER
FLAT.
SO IT CAN BE OPTIMAL FOR CELL
MOTILITY.
IN ORDER TO START LOOKING INTO
THIS, WHAT WE STARTED LOOKING AT
AGAIN, ALL OF THESE, EVERYTHING
I'M SHOWING YOU FROM NOW ON IS
SUPER RESOLUTION STRUCTURAL
ELIMINATION MICROSCOPY.
THE FIRST THING WE WANTED TO
KNOW IS WE SEE ACTIN IN THESE
DIFFERENT TYPES OF STRUCTURES,
STRESS FIBERS VERSUS ARCS.
WHERE IS THE MYOSIN 2?
IT'S KEY BECAUSE MYOSIN IS THE
CONTRACTILE SORT OF ALLOWS THE
SYSTEM TO BECOME A CONTRACTILE
ENGINE.
WHEN DYLAN LABELED CELLS WITH
MYOSIN 2 SHOWN IN GREEN HERE,
AND LOOKED AT HOW IT DISTRIBUTED
RELATIVE TO ACTIN, HE FOUND THAT
THE MYOSIN 2 IS ALL ALONG THE
DORSAL ARCS.
SO THE MYOSIN TWO IS ON TOP OF
THE CELL ON THOSE ARCS.
VERY LITTLE -- THERE IS
ESSENTIALLY NO MYOSIN 2 ON THOSE
STRESS FIBERS THAT ARE THE
STRUTS CONNECTING THE BOTTOM OF
THE CELL WHERE THE FOCAL
ADHESIONS ARE TO THE TOP OF THE
CELL WHERE THE ARCS ARE.
NOW IF WE GO IN AND ZOOM IN ON
THE SPACIAL DISTRIBUTION OF THE
MYOSIN TWO, ALONG THESE ACTIN
ARCS, WHAT WE SEE FOR THE FIRST
TIME IS TWO HEADS.
WE CAN ACTUALLY SEE A MYOSIN 2
MOTOR COMPLEX OR FILAMENT WHERE
WE SEE THE TWO HEADS OF THESE.
AND AS YOU MOVE DEEPER INTO THE
CELL, YOU GET A HIGHER
CONCENTRATION OF THOSE MYOSIN
TWO MOLECULES.
IF YOU DO A SCAN, WE SEE THESE
TWO HUMPS WHICH REPRESENT THE
TWO HEADS OF THE MOTOR OR MYOSIN
FILAMENT.
NOW PRIOR TO USING STRUCTURAL
ELIMINATION MICROSCOPY, WHEN
PEOPLE LOOKED AT THIS ALL THEY
WOULD SEE BECAUSE OF THE
TWO-FOLD LESS RESOLVABLE
APPROACH WITH JUST CONVENTIONAL
DEFRACTION LIMITED IMAGE, IS
JUST A BLUR.
WE CAN ACTUALLY DISTINGUISH THE
TWO HEADS OF THIS FILAMENT AND
IF WE USE LOCALIZATION
MICROSCOPY, WHICH HAS A 10-FOLD
INCREASE IN RESOLUTION, WE GET A
BETTER LOCALIZATION AND THE
DISTANCE BETWEEN THESE TWO
POINTS HERE FITS VERY NICELY
WITH THE DISTANCE KNOWN BETWEEN
THE TWO HEADS OF THAT MYOSIN
FILAMENT.
TO FURTHER CONFIRM WHAT WE ARE
LOOKING SAT INDIVIDUAL MYOSIN
FILAMENTS, WE THEN LABELED THE
TAIL DOMAIN OF MYOSIN TWO WITH A
DIFFERENT COLOR FLORESCENT
PROTEIN AND APPLE.
AND EXPRESS IT AND THE GREEN
LABELED AND EMERALD LABEL GOP
AND LOOKED AT HOW THEY
DISTRIBUTE ACROSS THOSE ACTIN
ARCS YOU CAN SEE A BEAUTIFUL
ARRANGEMENT OF THOSE FILAMENTS.
WE DO A LINE SCAN ACROSS ANY ONE
OF THESE, YOU CAN SEE A PATTERN
THAT FITS NICELY WITH THE
PATTERN BY WHICH THE HEAD, TAIL
AND HEAD IS ORGANIZED ON THESE
INDIVIDUAL ACTIN MYOSIN TWO
FILAMENTS.
WELL, CAN WE WATCH THIS?
WHAT ARE THESE FILAMENTS DOING
IN THE CONTEXT OF THE ACTIN ARCS
THAT ARE ON THE DORSAL SURFACE
OF THE CELL?
AND IF YOU DO LIVE CELL 3D
STRUCTURAL ELIMINATION AS SHOWN
HERE, WHAT CAN YOU SEE IS THAT
WHAT THESE MYOSIN 2 FILAMENTS
ARE DOING IS CONTRACTING THESE
ACTED IN ARCS.
IT'S VERY SIMILAR TO THE WAY
THAT THE MYOSIN TWO IS
CONTRACTING MUSCLE FIBERS.
IT IS WALKING ALONG THESE FIBERS
IN OPPOSITE DIRECTION THEY GET
PULLED TOGETHER.
THAT'S WHAT WE THINK IS
HAPPENING WHEN THESE MYOSIN 2
MOLECULES ARE ASSEMBLING ON
THOSE ARCS AT THE SURFACE OF THE
CELL.
THEY ARE ESSENTIALLY SQUEEZING
THE TOP SURFACE INWARD.
THIS IS LABEL THINK ACTIN WITH
ALPHA ATTEN ME, WHICH IS TAGGED
WITH A FLORESCENT PROTEIN IN
ORDER FOR YOU TO SEE WHAT IS
HAPPENING TO ACTIN URN THESE
CONDITIONS AND YOU CAN SEE THE
ACTIN IS NOT ONLY ON THESE ARCS,
PERIODICALLY PUNCTUATIONED WITH
A MYOSIN 2 YOU'RE NOT SEEING,
BUT YOU CAN SEE THE ACTIN ON
THESE STRESS FIBERS THAT RUN
FROM FOCAL ADHESIONS AT THE BASE
OF THE CELL TO THE TOP OF THE
CELL WHERE THIS CONTRACTING
ACTED IN ARCS IS ESSENTIALLY
CONTRACTING INWARDS.
NOW, ONCE WE COLLECTED THESE
MOVIES, WE HAD A BIG DISCUSSION
ABOUT WHAT THIS MEANT.
AND WHAT CLEARLY STARTED COMING
FORWARD IN OUR THINKING WAS THAT
WHAT THE SYSTEM REALLY LOOKS
LIKE IS A TENTED.
IF YOU -- IT'S HARD TO SEE IT
BUT IN 3D, IF YOU ROTATE THIS
SYSTEM IS THAT CONTRACTILE
SYSTEM IS ACTUALLY WORKING LIKE
A TENT.
NOW LET ME DESCRIBE WHAT I MEAN.
WHAT I'M EMPHASIZING IS THE
FORCE DISTRIBUTION THAT ALLOWS A
TENT LIKE THIS TO STAND UP SO
WHAT WE THINK IS THE POLES OF A
INTERPRET HERE, REP SEPTEMBER
THE STRESS FIBERS THAT GO FROM
THE BASE OF THE CELL UP TO THE
TOP OF THE CELL.
AND THEY DON'T HAVE ANY TYPE OF
CONTRACTILE ABILITY.
BUT THEY CAN BE AFFECTED.
THEY CAN BE IMPACTED BY THIS
ACTIN ARC MESH WORK WHICH IS A
CONTRACTILE SYSTEM.
NOW IN THE TENT ANALOGY, THAT
ACTIN ARK MESH WORK IS THE
CANVASS.
SO IF I SQUEEZE ON THIS CANVASS,
YOU CAN SEE I CAN CHANGE THE
SHAPE OF THE CELL.
AND IN PARTICULAR, IF I SQUEEZE
HARD ENOUGH, I CAN ACTUALLY
THANK YOU'S TENT TO CONVERT ITS
SHAPE INTO ONE THAT LOOKS LIKE A
CRAWLING CELL WHERE HAVE YOU
THIS FLAT FRONT EDGE.
AND ALL I'M DOING IS ESSENTIALLY
SQUEEZING THIS CANVASS.
THAT LEADS TO THE POLLS
ESSENTIALLY BEING PUSHED
DOWNAWARDS.
AS THEY GET PUSHED DOWNWARDS BY
THE CONTRACTING CANVASS, THE
WHOLE CELL GETS FLAT.
SO THAT'S A COOL MODEL.
BUT HOW CAN WE TEST IT?
WE HAVE TWO WAYS WE TESTED THIS.
AND THE FIRST RELATES TO THE
IMPACT OF CONTRACTING THE
CANVASS ON THE TENT POLES.
SO WE PREDICT THAT AS THE
CANVASS CONTRACTS AND AS THESE
ARCS MOVE UP THE SURFACE OF THE
CELL AND CONTRACT IT, IT PUTS A
FORCE ON THESE STRESS FIBERS AND
CAUSES THE STRESS FIBE TOYS MOVE
DOWNWARDS.
AS THAT HAPPENS -- FIBERS TO
MOVE DOWNWARDS.
THESE POLLS GET PULLED UP AND IT
HAS IMPACT ON THE FOCAL
ADHESIONS AND BUT IT IS ALSO
GOING TO TOTALLY IMPACT THESE
STRESS FIBERS.
IT WILL CHANGE THE WAY THE
STRESS FIBERS ARE BEHAVING.
AND SO IN ORDER TO TEST THAT,
WHAT DYLAN DID WAS A SPECKAL
ANALYSIS TO LOOK AT THE BEHAVIOR
OF ACTIN ON THESE STRESS FIBERS
TO SEE WHETHER THERE IS A FORCE
THAT IS ACTUALLY BEING EXERTOD
THESE STRESS FIBERS AS THE ARCS
ARE MOVING UPAWARDS.
SO LET'S FIRST JUST LOOK AT
THE -- USING SPECKEL ANALYSIS AT
THE MOVEMENT OF ACTIN WITHIN
THESE ARCS AND YOU CAN SEE THEY
ARE MOVING INWARD TO THE TOP OF
THE CELL AND IF WE ADD THE
MYOSIN 2 INHIBITOR, WE STOP IT.
IT MAKES SENSE.
IF WE DO THE SAME TYPE OF
ANALYSIS BUT NOW LOOKING AT THE
STRESS FIBER, WHAT WE SEE IS AN
IDENTICAL MOVEMENT OF ACTIN ON
THAT STRESS FIBER BUT
IMPORTANTLY, IF WE INHIBIT
MYOSIN TWO, WE STOP THE
MOVEMENT.
WHEN THE ACTIN MESH WORK, WHICH
IS THE CANVASS OF THE TENT
CONTRACTS, IT CAUSED THE DORSAL
STRESS FIBERS THAT THE STRESS
FIBE TOURS ACTUALLY GET SHOVED
DOWN TO BE PUSHED DOWN, PULLED
ON IN A SENSE.
AS YOU PULL ON THESE STRESS
FIBERS, THE WHOLE SURFACE OF THE
CELL DROPS DOWN.
OKAY.
A SECOND PREDICTION OF THIS
MODEL IS THAT IN A CELL, THAT IS
CRAWLING, THAT HAS THIS FLAT
LAMELLA WHICH WE BELIEVE IS DUE
TO THIS SYSTEM OF DORSAL ACTIN
ARCS CONTRACTING THE TOP SURFACE
OF THE CELL DOWN, THE PREDICTION
IS THAT IF WE INHIBIT THE
CONTRACTILE CAPABILITY OF THOSE
ARCS, WE WILL LOSE THE FLATTENED
FRONT EDGE.
SO TO TEST THAT, WE USED
STRUCTURAL ELIMINATION
MICROSCOPY TO LOOK AT THE SHAPE
OF THE CELL WHEN WE INHIBIT
MYOSIN TWO USING BIEBBISTATIN.
WE LOSE THIS FLATTENED FRONT
ONLY OF THE CELL THAT IS SO
CRUCIAL FOR THE CELL TO BE ABLE
TO MIGRATE INTO DIFFERENT
REGIONS.
AND MOVE ACROSS A SUBSTRATE.
FURTHERMORE, IF WE INHIBITED
MYOSIN TWO WITH siRNA,
ESSENTIALLY WE REMOVED IT, WE
KNOCKED IT DOWN.
AGAIN, WE FOUND THE CELL LOST
ITS SHAPE.
WE NO LONGER HAVE THAT FLATTENED
FRONT EDGE.
FURTHERMORE, IF WE TAKE CELLS,
IN THIS CASE, CAST SEVEN CELLS
THAT DON'T HAVE MYOSIN 2A OR ARE
NOT MOW TILE AND DON'T HAVE THAT
FRONT EDGE, AND ADD MYOSIN 2 TO
THIS SYSTEM, SO HERE IS THE CELL
WE ARE EXOGENOUSLY EXPRESSING
MYOSIN 2A YOU CAN SEE GAINED THE
ACTIN ARCS AS WELL AS
REORGANIZING THIS STRESS FIBERS
BUT IMPORTANTLY YOU GAIN THAT
FRONT EDGE OF THE CELL.
SO THIS IS THE MODEL WE THINK
CAN EXPLAIN HOW THE CELL GETS
ITS SHAPE AND HOW A CELL
INTERROGATING ITS ENVIRONMENT
CAN CREATE ITS SHAPE.
WE THINK IT IS A PROCESS THAT
INVOLVES EYE CONTACTILE ACTIVITY
OF THE CELL WHEREBY ACTIN ARCS
BEING GENERATED THROUGH THE
ACTIVITY OF THE FRONT EDGE HERE
CREATES ACTIN ARCS THAT ARE, AS
THEY MOVE UP THE SURFACE OF THE
CELL, CONTRACT AND CREATE A
FORCE ON STRESS FIBERS THAT LEAD
THE STRESS FIBERS TO BE PUSHED
DOWN.
YOU CAN SEE THE BOTTOM OF THE
PEGS AT THE BOTTOM OF THE TENT
ARE FLIPPED UP AND WE THINK THAT
PLAYS A HUGE ROLE IN ALLOWING
THE CELL TO SENSE ITS SUBSTRATE
ESSENTIALLY PULLING AND PUSHING
TO MODULATE ITS SUBSTRATE.
ZERO THAT, I WANT TO END AND SAY
WE THINK THAT THIS MODEL FOR HOW
THE CELL IS SHAPING ITSELF,
BUILT ON THIS CONTRACTILE
ACTIVITY OF THE CYTOPLASM, HAS A
LOT OF POTENTIAL FOR NOT ONLY
EXPLAINING HOW CELLS SHAPE
THEMSELVES IN COMPLEX
ENVIRONMENTS AND SURFACES, BUT
COULD BE EXTREMELY IMPORTANT FOR
HOW CELLS PROTRUDE INTO TIGHT
SPACES AND HOW THEY GENERATE 3D
ROTATIONAL FORCES ON THE GROWTH
SUBSTRATE WHERE THESE FOCAL
ADHESIONS ARE ATTACHED TO THESE
STRESS FIBER THAT MOVE TO THE
TOP OF THE CELL.
I WANT TO THANK THE PEOPLE
INVOLVED.
DYLAN BURNETTE WHO IS IN THE
AUDIENCE OVER THERE, IS REALLY
THE MAJOR DRIVER FOR THE WORK
I'M TALKING ABOUT INCREDIBLY
TALENTED AND HELPED BY PEOPLE IN
THE LAB AND IN ALL OF THESE
TECHNIQUES I TALKED ABOUT.
WE HAVE ALSO BEEN VERY FORTUNATE
TO HAVE A LOT OF COLLABORATORS.
I ALSOMENT TO THANK ERIC AND
LYNN SHOW WHO WAS A POSTDOC WITH
THE LATE -- WHO BUILT A LIVE
CELL SIM SYSTEM WITH THAT SYSTEM
THAT WE WERE ABLE TO DO THE LIVE
CELL IMAGING I TALKED TO YOU
ABOUT, ALTHOUGH THE REST OF THE
WORK WAS DONE WITH OUR OWN
COMMERCIAL SIM SYSTEM HERE AT
NIH.
AND FINALLY I WANT TO THANK MIKE
DAVIDSON AT FLORIDA STATE
>> LET'S TAKE SOME QUESTIONS.
>> THIS IS PRETTY FANTASTIC
WORK.
MY QUESTION IS DOT CELL SURFACE
COATED WITH SUGARS AND HOW IS
THOSE SUGARS -- BECAUSE THEY ARE
THE MOST FLEXIBLE MOLECULES
COMPARED TO PROTEIN CELL.
DO THEY ENHANCE THIS MOTION?
SO IF YOU CHANGE THE SUGARS THEY
ARE DIFFERENT?
>> SO THE QUESTION IS WE KNOW
THE CELL SURFACE IS LOADED WITH
GLYCOPROTEINS, AND LOTS OF SUGAR
TEXT OF THIS SYSTEM I TALK TO
YOU ABOUT.
OF COURSE THEY ARE DOING ALL
KINDS OF THINGS IN TERMS OF
ALLOWING THE CELL TO ATTACH TO
THE MATRIX.
IT COULD PLAY A VERY IMPORTANT
ROLE IN ALLOWING SORT OF FOCAL
ADHESIONS AND ADHESIVE ELEMENTS
ON THIS CELL TO BE OR TO TOUCH
THIS PARTICULAR SUBSTRATE.
YOU CAN IMAGINE SOME OF THESE
GLYCOPROTEINS COULD -- BECAUSE
THEY ARE VERY LONG, THEY COULD
INTERFERE IN SOME AREAS VERSUS
ABLE TO ATTACH TO THE SUBSTRATES
THROUGH THESE ADHESIONS.
IT'S REALLY EXCITING POSSIBILITY
THAT THE GLYCOPROTEINS MIGHT BE
PLAYING THAT ROLE.
>> [ INDISCERNIBLE ]
>> VERY GOOD POINT.
>> I HAVE A QUESTION.
OBVIOUSLY, YOU HAVE BEEN
INVESTIGATING THE ROLE OF THE
CYTOSKELETON AND THE CELL SHAPE
DETERMINATION AND IN MOVEMENT,
BUT YOU'RE ALSO INTERESTED IN
THE WAY IN WHICH MOLECULES MOVE
WITHIN CELLS.
USING THE SAME SUBSTRATE AS IT
TRACKS, SO DOES THE MOTION OF
THE CELL EFFECT METABOLISM?
>> OH, MY GOSH, YES.
>> THAT IS SOMETHING WE ARE
INCREDIBLY INTERESTED IN AND ONE
OF THE THINGS YOU MAY HAVE
NOTICED WHEN THE CELL -- THE
CELL BECOMES LESS -- THE VOLUME
OF THE CELL DROPS.
SO WHEN WE GO FROM THIS KIND OF
STRUCTURE TO THIS KIND OF
STRUCTURE, THERE IS A DECREES IN
CELL VOLUME.
AND WE THINK WATER IS ACTUALLY
BEING EXTRUDED OUT OF THE CELL
WHEN THE CELL IS UNDERGOING THIS
TYPE OF HIGHLY CONTRACTILE
MOTION AND THAT WILL HAVE A HUGE
IMPACT ON THE CROWDING OF
PROTEINS WITHIN THE CELL THAT
MIGHT IMPACT METABOLISM AND
OTHER PHYSIOLOGICAL PROCESSES
AND THIS IS SOMETHING THAT WE
ARE EXTREMELY INTERESTED IN
PURSUING.
>> WHAT YOU DID IS YOU ANALYZED
EVERYTHING IN A DISH AND HAVE
YOU LOOKED AT CELLS THAT YOU
FORCE IN BETWEEN TWO VERY NARROW
GLASS SLIDES AND HOW THE
SITUATION CHANGES WITH RESPECT
TO THE --
>> THERE ARE LOTS OF PEOPLE IN
THE FIELD WHO HAVE BEEN LOOKING
AT THESE SORTS OF THINGS.
IF YOU PUT CELLS TO -- CELLS CAN
USE OTHER TYPES OF MOW TILE
PROCESSES TO SORT OF PUSH
THEMSELVES THROUGH NARROW SPACES
WHEREAS, ESSENTIALLY ALL THAT
IS, IS A BREAK IN THE CYTOPLASM
AND AN ACTUAL CYTOLAMB AND
CHANGE IN TENSION.
WE THINK THAT IS NOT WHAT IS
OPERATING IN CELLS ON THESE
SUBSTRATES WHERE THEY ARE
CRAGGING ACROSS FIBER IN EFFECT
IN OR COLLAGEN ENRICHED AREAS OF
THE BODY.
YOUR POINT IS EXTREMELY WELL
TAKEN.
I THINK THE NEXT PHASE OF THIS
WORK WILL HAVE TO BE IN THESE
DIFFERENT CONTEXT.
>> IF THERE ARE NO FURTHER
QUESTIONS, WE WILL INVITE YOU TO
A RECEPTION AT THE FAS
SPONSORING IN THE LIBRARY AND
JENNIFER WILL BE THERE FOR A
WHILE AND I'M SURE WILL DEAL
WITH INDIVIDUAL QUESTIONS.
THANK YOU VERY MUCH.
[ APPLAUSE ]