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 ]