Mitochondrial Behavior

Mitochondrial Behavior NIH VideoCast 41.2K subscribers Subscribe 121 Share Download Clip Save 6,308 views Jun 13, 2019 Mitochondrial Behavior Air date: Wednesday, June 5, 2019, 3:00:00 PM Category: WALS - Wednesday Afternoon Lectures Runtime: 01:05:57 Description: NIH Director's Wednesday Afternoon Lecture Series Dr. Nunnari is a Distinguished Professor and Chair of the Department of Molecular and Cellular Biology, College of Biological Sciences at the University of California, Davis. She is an elected member of the National Academy of Sciences, a member of the American Society for Cell Biology, and has served as its president and the first woman to serve as Editor-in-Chief of The Journal of Cell Biology. Jodi Nunnari is a pioneer in the field of mitochondrial biology. She was the first to describe the organelle as a dynamic network in homeostatic balance and decipher the mechanisms of the machines responsible for mitochondrial division and fusion, which are critical determinants of overall mitochondrial shape and distribution. Mitochondria are double membrane-bounded organelles that perform a myriad of diverse and essential functions in cells, dependent on the collective behavior of the organelle. Dr. Nunnari’s laboratory has uncovered contact sites that intimately link mitochondria with the ER and described their roles in mitochondrial positioning and dynamics and mtDNA segregation. They have addressed how mitochondrial membranes are sub-compartmentalized to reveal how the complex internal architecture of the organelle is generated. They are using system-based approaches to address how mitochondrial behavior is physiologically regulated within cells and organisms to shed light onto how mitochondrial dysfunction contributes to human disease. For more information go to https://oir.nih.gov/wals/2018-2019 Author: Jodi Nunnari, Ph.D., Distinguished Professor and Chair of the Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis Permanent link: https://videocast.nih.gov/launch.asp?... Key moments View all Transcript Follow along using the transcript. Show transcript Skip navigation Search 9+ Avatar image 32:50 / 1:05:57 Mitochondrial Behavior NIH VideoCast 41.2K subscribers Subscribe 121 Share Download Clip Save 6,309 views Jun 13, 2019 Mitochondrial Behavior Air date: Wednesday, June 5, 2019, 3:00:00 PM Category: WALS - Wednesday Afternoon Lectures Runtime: 01:05:57 Description: NIH Director's Wednesday Afternoon Lecture Series Dr. Nunnari is a Distinguished Professor and Chair of the Department of Molecular and Cellular Biology, College of Biological Sciences at the University of California, Davis. She is an elected member of the National Academy of Sciences, a member of the American Society for Cell Biology, and has served as its president and the first woman to serve as Editor-in-Chief of The Journal of Cell Biology. Jodi Nunnari is a pioneer in the field of mitochondrial biology. She was the first to describe the organelle as a dynamic network in homeostatic balance and decipher the mechanisms of the machines responsible for mitochondrial division and fusion, which are critical determinants of overall mitochondrial shape and distribution. Mitochondria are double membrane-bounded organelles that perform a myriad of diverse and essential functions in cells, dependent on the collective behavior of the organelle. Dr. Nunnari’s laboratory has uncovered contact sites that intimately link mitochondria with the ER and described their roles in mitochondrial positioning and dynamics and mtDNA segregation. They have addressed how mitochondrial membranes are sub-compartmentalized to reveal how the complex internal architecture of the organelle is generated. They are using system-based approaches to address how mitochondrial behavior is physiologically regulated within cells and organisms to shed light onto how mitochondrial dysfunction contributes to human disease. For more information go to https://oir.nih.gov/wals/2018-2019 Author: Jodi Nunnari, Ph.D., Distinguished Professor and Chair of the Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis Permanent link: https://videocast.nih.gov/launch.asp?... Key moments View all Transcript Follow along using the transcript. Show transcript NIH VideoCast 41.2K subscribers Videos About Comments are turned off. Learn more Transcript Search in video 0:05 >> GOOD AFTERNOON, EVERYBODY. I'M A PROGRAM DIRECTOR WITH THE 0:11 NATIONAL CANCER INSTITUTE. IT'S MY PLEASURE TO INTRODUCE JODI NUNNARI, TODAY'S WALS 0:17 LECTURE SPEAKER. JODI STUDIED PHARMACOLOGY AT 0:22 VANDERBILT. SHE STUDIED WITH PETER WALTER, WHERE SHE FELL IN LOVE WITH THE STRUCTURE AND FUNCTION OF 0:28 ORGANELLES. JODI HAS BEEN A PIONEER IN THE STUDY OF MITOCHONDRIAL -- AND 0:35 CONTINUES TO BE A CHAMPION OF CELL BIOLOGY. SHE IS THE CHAIR OF THE DEPARTMENT OF MOLECULAR AND 0:40 CELLULAR BIOLOGY AT THE UNIVERSITY OF CALIFORNIA AND DAVIS. SHE'S A MEMBER OF THE NATIONAL 0:45 ACADEMY OF SCIENCES. CONGRATULATIONS, JODI. AND THE PAST PRESIDENT OF THE 0:52 AMERICAN SOCIETY FOR CELL BIOLOGY. IN THIS CAPACITY, I FIRST MET 0:58 JODI AND WE COLLABORATED OVER THE LAST FIVE YEARS IN PUTTING IN THE ASCB ANNUAL MEETING AND 1:06 EMBEDDED SYMPOSIA ON EMERGING STUDIES ON MITOCHONDRIA AND 1:12 CANCER AND IT'S BEEN A REAL HIT WITH THE COMMUNITY, EMERGING THE FIELD OF CELL BIOLOGY AND CANCER 1:18 CELL BIOLOGY. FUN FACTS ABOUT JODI, SHE LOVES HORSES AND SHE LOVES WHISKEY. 1:23 SO WITHOUT FURTHER ADO, WE REALLY LOOK FORWARD TO JODI'S TALK TODAY ON "MITOCHONDRIAL 1:30 BEHAVIOR." [APPLAUSE] 1:38 >> YES, HE KNOWS ME WELL. I DO LOVE WHISKEY AND I HAVE A SLIGHT HORSE ADDICTION. 1:44 WE'RE CURRENTLY AT THREE HORSES IN MY FAMILY. THEY'RE THEY'RE A VERY EXPENSIVE HABIT. 1:53 SO I'M REALLY HAPPY TO BE HERE TODAY AND SHARE THE WORK WE DO IN MY LAB WITH YOU. WE'RE REALLY INTERESTED IN 2:00 MITOCHONDRIA. WHEN I WAS A POSTDOC IN PETER WALTER'S LAB, I WORKED ON 2:06 ENDOPLASMIC RETICULUM AND KIND OF HIT A ROADBLOCK IN MY STUDIES ON WHAT WAS CALLED THE RIBOSOME 2:12 RECEPTOR THEN AND IT TURNED OUT TO BE THE TRANSLOCON AND I JUST DI SIDED 2:18 DECIDED TO SWITCH DIRECTIONS AND I FELL IN LOVE WITH THIS ORGANELLE PRIMARILY BECAUSE I BECAME FASCINATED BY THE FACT 2:23 THAT IT HAS ITS OWN CHROMOSOME, AND HOW THE -- WHAT WERE THE RULES THERE IN TERMS OF 2:30 CHROMOSOMAL TRANSMISSION. AND THAT LED TO JUST DESCRIBING 2:36 THE COMPARTMENT AND ON THIS FIRST SLIDE HERE IS A HUMAN CELL IN CULTURE. 2:42 ONE OF THE MODEL SYSTEMS WE USE. WE'RE CARD CARRYING CELL BIOLOGISTS. I'M ALSO, BY THE WAY, 2:49 EDITOR-IN-CHIEF OF THE "JOURNAL OF CELL BIOLOGY," FOR ANYBODY WHO WANTS TO TELL ME ABOUT THEIR LATEST AND GREATEST STORY, I 2:56 ALWAYS HAVE OPEN AND EAGER EARS. AND WE USE CELLS AS A MODEL SYSTEM TO KIND OF UNCOVER 3:02 FUNDAMENTAL PRINCIPLES, SO I WHO ARE DOING MORE RELEVANT WORK 3:07 IN TISSUES. 3:12 THIS GIVES YOU AN IDEA OF PROBABLY WHAT HAPPENS THERE. THIS IS THE BIG PICTURE QUESTION THAT HAS DRIVEN THE WORK IN MY 3:19 LAB WHICH IS HOW IS MITOCHONDRIAL BEHAVIOR CONTROLLED IN SIDE CELLS AND 3:24 WHAT THAT BOILS DOWN TO IS FINDING THE MACHINES ON A MOLECULAR SCALE THAT FUNCTION AT THIS COMPARTMENT WHICH YOU SEE 3:30 HERE IN BLUE. TO GIVE IT THIS CHARACTERISTIC IN THIS PARTICULAR CELL TYPE, 3:38 THIS IS A -- THIS CHARACTERISTIC DISTRIBUTION AND OVERALL LOOK. IT'S VERY TUBULAR, VERY 3:44 CONNECTED. AND OVER THE YEARS IN MY LAB, WE HAVE DEFINED THE MACHINES THAT 3:50 WORK ON THIS ORGANELLE SUCH AS DYNAMICS, DIVISION AND FUSION, 3:56 THE PROTEINS THAT DO THAT JOB, ACTIVE MECHANISMS TO POSITION 4:02 THE MITOCHONDRIA AT CERTAIN PLACES WHERE THEY'RE NEEDED. WE'VE RECENTLY BEGUN TO WORK ON 4:08 HOW THEY MOVE AROUND INSIDE 4:13 CELLS, AND HAND IN HAND AS WE ALL KNOW STRUCTURE/FUNCTION RELATIONSHIPS, WE REALLY ARE 4:18 INTERESTED IN HOW THE COMPARTMENT IS PUT TOGETHER IN ITS VERY DETAIL, AND I'LL TALK 4:23 ABOUT THAT TOWARDS THE END. BUT REALLY IT BOILS DOWN TO HOW 4:30 ARE ALL THESE MACHINES THAT WE'VE IDENTIFIED AND KIND OF DELVED INTO MECHANISTICALLY, HOW DO THEY FUNCTION TOGETHER TO DO 4:36 THE JOB THAT YOU SEE HERE? AND THE BIAS THAT WE'VE HAD IN MY LAB IS THAT THEY DO SO WITH 4:43 THE GOAL HOPEFULLY OF TRANSMITTING THE CHROMOSOME IN THAT COMPARTMENT. 4:49 AND IF YOU NOTICE HERE IN RED, 4:56 THERE'S ANOTHER STAR OF THE SHOW, THE ENDOPLASMIC RETICULUM, AND ONE OF THE PUNCH LINES OF MY 5:02 TALK TODAY IS THAT THESE TWO FUNDAMENTAL EUKARYOTIC DEFINING 5:08 COMPARTMENTS, SO MY WORLD VIEW IS THAT EVERYTHING ELSE CAN BE MADE DE NOVO EXCEPT THESE TWO, 5:14 AND OF COURSE THE PLASMA MEMBRANE, HOW DO THEY 5:20 COLLABORATE TOGETHER TO GOVERN THE BEHAVIOR OF MITOCHONDRIA? AND AGAIN, HERE'S MY WORLD VIEW. 5:28 RIGHT HERE, HERE'S A VERY, VERY SIMPLE TREE OF LIFE ON THIS PLANET. 5:34 AND THERE'S THREE KINGDOMS, EVERYBODY KNOWS THIS, AND HERE WE ARE, EUKARYOTES, WE'RE PRETTY 5:41 SPECIAL, OUR CELLS CAN BECOME SPECIALIZED. WE CAN WALK AND TALK AND DO ALL 5:46 THOSE GREAT THINGS, AND THAT HAPPENED RIGHT HERE AT THIS BIFURCATED POINT HERE IN THE 5:52 TREE OF LIFE, AND THIS IS RIGHT WHERE WE ACQUIRED MITOCHONDRIA. 5:57 SO I LIKE ASKING PEOPLE HOW MANY PEOPLE BELIEVE IN 6:03 EXTRATERRESTRIAL LIFE OUT THERE. , RAISE YOUR HAND? 6:11 THAT'S SO HOPEFUL. [LAUGHTER] 6:18 I LOVE THAT IN PEOPLE, THAT HOPE. SO NICK LANE, WHO THINKS A LOT MORE ABOUT THE EVOLUTIN OF LIFE THAN I DO, IN PARTICULAR THE 6:25 ROLE OF MITOCHONDRIA, SAID IF YOU WERE TO MEET A MARTIAN, AN EXTRATERRESTRIAL BEING, THEY 6:30 WOULD HAVE MITOCHONDRIA. SO THAT'S KIND OF HOW 6:37 FUNDAMENTAL THEY ARE. AND WHY IS THAT? BECAUSE THEY'RE ANOTHER COMPARTMENT AND THEY DO THE WORK 6:42 OF A LOT OF METABOLISM, INCLUDING ENERGY PRODUCTION, AND HAVE ALLOWED US TO BECOME WAY 6:48 MORE EFFICIENT AND SPECIALIZED. HENCE, EUKARYOTES. BUT A LOT HAS HAPPENED SINCE WE 6:55 ACQUIRED MITOCHONDRIA, AND I THINK PEOPLE GENERALLY AGREE THAT MITOCHONDRIA CAME FROM A 7:01 BACTERIAL ANCESTOR THAT HAD PROBABLY ABOUT ON THE ORDER OF 7:06 4,000 GENES. IN HUMAN MITOCHONDRIA, THERE'S ONLY 37 OF THOSE GENES NOW, SO A 7:11 LOT HAS HAPPENED SINCE THEN. SO A LOT OF THESE GENES HAVE BEEN LOST TO THE NUCLEUS OR LOST 7:18 FOREVER. PROBABLY ALSO DRIVING EVOLUTION IN THAT WAY. BUT THE MITOCHONDRIAL PROTEOME 7:25 AS WE KNOW IS MUCH MORE COMPLEX THAN JUST 37 COMPONENTS. IT'S MADE UP OF AROUND 1100 7:32 PROTEINS. AND SO A VAST MAJORITY OF THE PROTEINS THAT COMPRISE 7:39 MITOCHONDRIA COME FROM NUCLEAR ENCODED GENES, AND SO DURING THIS PROCESS OF EVOLUTION, A LOT 7:45 HAS HAD TO HAPPEN FOR THESE COMPARTMENTS TO BECOME WHAT THEY 7:51 ARE NOW, INCLUDING THE ABILITY TO IMPORT PROTEINS, THE NUCLEAR-ENCODED COMPONENTS THAT 7:56 I JUST TALKED ABOUT, AND ALSO THEIR LIPID COMPONENTS. SO MITOCHONDRIA HAVE TWO MAJOR 8:04 BIOSYNTHETIC PATHWAYS FOR PHOSPHOLIPIDS, AND THE SIGNATURE 8:09 LIPID IN THIS COMPARTMENT AGAIN VERY SIMILAR TO BACTERIAL 8:17 ANCESTORS CARDIOLIPIN. THEY CAN'T MAKE THEM BY THEMSELVES. THEY NEED THE PRECURSORS FOR 8:22 THESE LIPIDS THAT COME FROM THE 8:27 ENDOPLASMIC RETICULUM. THAT'S THE FIRST HINT THAT THESE TWO COMPARTMENTS COLLABORATE EXTENSIVELY. AND ALL THOSE MACHINES THAT I 8:34 TALKED ABOUT BRIEFLY ON THAT FIRST SLIDE GIVE RISE TO THE 8:39 BEHAVIOR OF THIS COMPARTMENT. OUR NEW COMPARTMENT, 8:46 ARE NEW INVENTIONS BY AND LARGE. TOGETHER, THESE KINDS OF MACHINES ALLOW THE COMPARTMENT TO BE BUILT, OF COURSE NOT DE 8:53 NOVO BUT TO CONTINUE TO BE BUILT 8:59 AND ALSO INTEGRATE WITH OTHER PATHWAYS IN THE CELL THAT ARE EXTRA MITOCHONDRIAL. SIGNALING GOES BACK AND FORTH 9:05 BETWEEN THE MITOCHONDRIA NUCLEUS, FOR EXAMPLE. SO THE STORY I'M GOING TO TELL YOU TODAY IS HOW WE'VE LEARNED 9:14 HOW THESE MACHINES ARE CONNECTED, WE THINK, IT'S A WORKING MODEL OF HOW PATHWAYS THAT COUPLE THE VARIOUS 9:20 DISPARATE MACHINES ARE LINKED TOGETHER TO GIVE A MECHANISM TO 9:27 TRANSMIT MITOCHONDRIAL CHROMOSOME WITHIN THE CELL. THIS IS ANOTHER MODEL SYSTEM WE 9:32 USE. 9:39 WE LIKE IT BECAUSE IT'S AN IN VIVO SYSTEM, IT ALSO HAS FABULOUS GENETICS, BUT MOSTLY 9:45 BECAUSE IT'S IN VIVO BR WHERE WE BY AND LARGE ASK SIMILAR QUESTIONS, WE TEND TO GO BACK 9:50 AND FORTH IN MY LAB AND WHILE SOME OF THE COMPONENTS AREN'T NECESSARILY STRINGENTLY 9:56 ORTHOLOGS OF ONE ANOTHER, THE STRATEGIES SEEM TO BE COFFIN SERVED BE CON SERVED 10:02 WITH HOW THEY GET TRANSMISSION DONE. USING THIS SIMPLE SYSTEM, MY LAB AND A COUPLE OF OTHER LABS, MANY 10:10 YEARS AGO, DISCOVERED THE GENES THAT ENCODE THE PROTEINS THAT 10:15 REGULATE DIRECTLY MITOCHONDRIAL DIVISION AND FUSION AND THOSE ARE LISTED -- THE PROTEINS ARE LISTED ON EITHER SIDE OF THIS 10:21 EQUATION. AND SO MUCH LIKE IN THE HUMAN CELL THAT I SHOWED YOU IN YEAST, MITOCHONDRIA ARE ALSO VERY 10:27 CONNECTED INTO THIS TUBULAR NETWORK AND SO-CALLED WILD TYPE CELLS, BUT YOU CAN DISRUPT THIS 10:33 NETWORK BY DISRUPTING DIVISION INFUSION SELECTIVELY. SO IF YOU BLOCK FUSION, DIVISION 10:39 GOES ON IN THIS NETWORK DISCONNECTS AND ON THE OTHER SIDE OF THE EQUATION, IF YOU 10:45 BLOCK DIVISION, FUSION GOES ON AND THE CONNECTIONS EVEN GET 10:50 MORE CONNECTED INTO THIS NET-LIKE STRUCTURE THAT YOU SEE HERE, AND THAT WASN'T SURPRISING TO US BECAUSE WHEN WE DESCRIBED 10:56 THIS SYSTEM, IT TURNS OUT THE RATES OF THESE EVENTS OF DIVISION AND FUSION ARE ROUGHLY 11:02 EQUIVALENT IN THIS CELL TYPE. SO YOU CAN TIP THE MORPHOLOGY OF THE ORGANELLE, HOW IT LOOKS INSIDE CELLS, BY JUST ALTERING 11:09 THE RELATIVE RATES OF DIVISION AND FUSION. ONE OF THE MAJOR PHENOTYPES THAT 11:15 WE NOTICED IN THESE CELLS WAS THAT IF YOU DISRUPT DYNAMICS, 11:20 THE TRANSMISSION IN THE CHROMOSOME, THE FIDELITY OF THAT PROCESS GOES DOWN. YOU BLOCK FUSION, YOU GET 11:27 COMPLETE LOSS. DIVISION INCREASES THE RATE OF LOSS. AND TOGETHER, IT DESCRIBED A PICTURE OF WHAT THE FUNCTION OF 11:35 DYNAMICS REALLY IS INSIDE CELLS FUNDAMENTALLY. AND IT'S NOT THAT FUSION IS IMPORTANT OR DIVISION IS 11:41 IMPORTANT BECAUSE THEY ARE, BUT WHAT REALLY WE LEARNED IS THAT WHAT'S IMPORTANT IS THEY BOTH 11:46 EXIST TOGETHER. SO WE HAVE TO HAVE ONE AND THE 11:52 OTHER. AND TOGETHER, THESE PROCESSES MAINTAIN THE FUNCTION, YOU NEED 11:58 MITOCHONDRIAL DNA, THEY ENCODE PROTEINS INVOLVED IN OXIDATIVE 12:05 PHOSPHORYLATION, RESPIRATORY CHAIN, AND DISTRIBUTION, AND THAT'S MOST APPARENT IN THESE CELLS HERE, WHERE IF FOR EXAMPLE 12:11 YOU FOCUSED ON THIS CELL ON THE TOP, THIS IS A FULL CELL PROJECTION, IT WOULD BE BARE. THERE WOULD BE NO MITOCHONDRIA. 12:17 THE WAY YEAST WORK IS THEY POSITION THEIR MITOCHONDRIA AT THE CORTEX BECAUSE THEY DON'T REALLY PROBABLY KNOW WHERE THE 12:23 BUD IS GOING TO FORM, WHERE THEY HAVE TO DISTRIBUTE. SO YOU NEED DYNAMICS TO MAINTAIN 12:28 FUNCTION AND DISTRIBUTION AND NOT SURPRISINGLY, IF YOU DISRUPT THESE PROCESSES IN HUMAN CELLS, 12:35 YOU CAN -- IT RESULTS IN A LOT OF DIFFERENT TYPES OF HUMAN 12:40 DISEASES. 12:47 SO WHEN THESE GENES WERE CLONED FOR DIVISION AND FUSION, THE HEART OF THE GENES WE DISCOVERED 12:56 CALL TO A PARTICULAR FAMILY, I HAD THE OPPORTUNITY TO COLLABORATE WITH JENNY HINSHAW 13:02 HERE IN THE AUDIENCE, AND THIS IS A MICROGRAPH THAT SHE TOOK I DON'T KNOW, HOW LONG AGO, JENNY? 13:07 A WHILE AGO, OF THE PRO TOE TYPIC MEMBER OF THIS FAMILY CALLED DYNAMIN 1. 13:18 THESE ARE LARGE GTP ACES, THEY DON'T FUNCTION LIKE SWITCHES BUT THEIR MECHANIC ENZYMES THAT SELF 13:26 ASSEMBLE INTO STRUCTURES LIKE THIS ASSOCIATED WITH MEMBRANES, SO THEY CAN DO THE WORK OF MEMBRANE DYNAMICS THROUGH 13:32 ASSEMBLY AND ASSEMBLY STIMULATED CONFORMATIONAL CHANGES THAT ARE MEDIATED BY HYDROLYSIS OF GTP. 13:39 SO, SO CALLED POWER STROKE. FOR MITOCHONDRIAL DIVISION, THERE'S A SINGLE ONE THAT LIVES IN THE CYTOPLASM, I'LL TALK 13:50 ABOUT THAT IN A SECOND, IN DIFFUSION THE DYNAMINS ARE 13:56 DISTRIBUTED ONE ON EACH MEMBRANE AND WE WERE ABLE TO SHOW BY RECONSTITUTING FUSION IN VITRO THAT THEY EACH CONTROL THE 14:01 FUSION BEHAVIOR OF THE MEMBRANE THAT THEY INHABIT AND YOU CAN 14:07 SEPARATE MITOCHONDRIAL OUTER AND INNER FUSION, INTERESTINGLY. SO GIVEN THAT THE DIVISION 14:13 SYSTEM WAS PRETTY SIMPLE, WE DID A LOT OF WORK OVER A NUMBER OF YEARS RECONSTITUTING THE PROCESS 14:18 OF DIVISION AND LEARNING A LOT ABOUT ITS MECHANISM. AND THIS IS JUST A SUMMARY OF SOME OF THE WORK WE DID, AGAIN, 14:24 A LOT OF IT IN COLLABORATION WITH JENNY, WHERE THE TWO 14:31 NON-DYNA MIN MEMBERS REQUIRED ARE RESPONSIBLE FOR TARGETING TO 14:37 THE MITOCHONDRIAL MEMBRANE AND ONE OF THOSE PLAYERS, THIS PROTEIN CALLED MVV1, THEN 14:42 FUNCTIONS AS A POSITIVE EFFECTOR OF DYNAMIN ASSEMBLY, SO IT'S LIKE OKAY, YOU'RE HERE, YOU'RE 14:48 AT THE RIGHT SPOT, ASSEMBLE INTO A HELICAL STRUCTURE THAT IN A 14:54 GTP-DEPENDENT HYDROLYSIS MANNER, MEDIATES A POWER STROKE PRECISION. AND THE THEN POSTDOC IN MY LAB 15:02 SOLVED THE CRYSTAL STRUCTURE OF DIANA MIN WHICH TURNED OUT TO BE A REALLY GOOD MODEL FOR HOW THE 15:09 MITOCHONDRIAL DIVISION DYNAMIN WORK WHERE YOU GET G DOMAINS INTERACTING ACROSS HELICAL 15:16 WRUNGS TO PRODUCE THE POWER STROKE. I'M NOT GOING TO GO INTO THE DETAILS, BUT WE DID A LOT OF WORK ON THIS AND IT WAS REALLY 15:23 COOL. I ENJOY THE MECHANISTIC WORK AND IT WAS GREAT, BUT SOMETHING ALWAYS BOTHERED US, AND THAT WAS 15:29 THAT ALL THE MACHINERY WHETHER YOU'RE IN A YEAST CELL HERE WHICH I JUST TOLD YOU ABOUT OR 15:36 IN A MAMMALIAN CELL WHICH, BY AND LARGE, DOESN'T HAVE 15:43 CONSERVED RECEPTORS OR EFFECTORS ON THE OUTER MEMBRANE, NO MATTER WHAT, THEY ALL ARE ON THE OUTER 15:49 MEMBRANE. SO WE COULD NEVER FIND ANYTHING INSIDE THE CELL -- I MEAN INSIDE MITOCHONDRIA THAT HELPED 15:56 DIVISION. DROVE US CRAZY. SO WE OFTEN WONDERED THEN, WHAT IN THE WORLD IS GOING ON IN 16:01 TERMS OF DIVISION SITE PLACEMENT? SO LET'S GO BACK NOW IN EV 16:08 LUKE'S EVOLUTION TO THE BACTERIAL PROGENITOR OF MITOCHONDRIA AND TALK A LITTLE BIT ABOUT HOW THEY 16:14 DIVIDE. THEY USE A GTP ACE ALSO, BUT IT'S MORE RELATED TO TUBULIN 16:19 CALLED FDSE, AND THEY USE A MECHANISM CALLED THE MEN SYSTEM 16:26 AND AN OCCLUSION SYSTEM THAT SAYS -- IT SAYS DON'T ASSEMBLE 16:35 AT THE POLE THIS MACHINERY, AND ONLY ASSEMBLE IN THE MIDDLE. AND REASON FOR THAT, OF COURSE, 16:40 IS THAT YOU'RE SEGREGATING THE CHROMOSOMES HERE AND YOU WANT EACH OF THE DAUGHTERS TO GET ONE. THAT'S THE WHOLE POINT. 16:47 YOU FAST FORWARD HERE, I TOLD YOU ALREADY THERE'S -- NOTHING ON THE INSIDE, BUT THERE ARE IN 16:53 YOU'RE CARE YOTS, CERTAIN EUKARYOTES, A HYBRID SYSTEM, AND IF YOU LOOK AT THIS SYSTEM THAT 17:00 OFTEN RETAINS THE MIN SYSTEM, 17:08 FTSZ, IT'S OFTEN ACQUIRED AN 17:14 OUTSIDE DYNAMIN. IF YOU LOOK AT THE MORPHOLOGY IN THESE SYSTEMS, THESE 17:19 MITOCHONDRIA ARE PRETTY DISCRETE, BACTERIAL-LIKE IN TERMS OF THEIR MORPHOLOGY. 17:27 AND SO A MECHANISM TO PLACE THE DIVISION APPARATUS THAT RELIES ON THE ENDS KIND OF MAKES SENSE, 17:34 BUT IN THE MITOCHONDRIA I SHOWED YOU IN YEAST AND IN HUMAN CELLS, THE ENDS HAVE LOST THEIR MEANING 17:40 ESSENTIALLY. IT'S SO HETEROMORPHIC, THEY'RE NOT A VERY GOOD REFERENCE POINT. AND SO WHAT THEN DECIDES WHERE 17:48 DIVISION OCCURS? 17:54 THE ANSWER CAME IN THE FORM OF A 17:59 COLLABORATION, MY WHERE HERE'S A MOVIE THAT LAURA TOOK, NOW 18:06 YOU'RE LOOKING IN GREEN AT THE ENDOPLASMIC RETICULUM, RED ARE THE MITOCHONDRIA, AND IF YOU 18:12 FOCUS HERE ON THIS LITTLE ER TUBULE THAT SEEMS TO BE CLOSE BY THE MITOCHONDRIA HERE, AND YOU 18:18 WATCH IT FOR A WHILE, THAT'S WHERE DIVISION IS GOING TO TAKE PLACE. THIS WAS BLUE -- THIS BLEW MY 18:25 MIND. HERE'S VIDEO FROM GIA VOELTZ AND 18:31 HER THEN GRADUATE STUDENT JONATHAN FRIEDMAN WHO NOW IS A 18:37 POSTDOC FOR ME. THE SAME IS TRUE HERE, WHERE E.R. AND MITOCHONDRIA COME CLOSER TOGETHER IS WHERE A 18:43 FUTURE DIVISION SITE WILL HAPPEN. SO IT'S NOT AN INSIDE MECHANISM. 18:49 MECHANISM. AND SO OF COURSE, BEING THE MECHANISTIC TYPES, WE WANTED TO KNOW WHAT THE MOLECULAR BASIS 18:54 WAS AND AT THE TIME, WORK 19:00 IDENTIFIED THE FIRST REAL PRO TOE TYPIC TETHER BETWEEN 19:07 MITOCHONDRIA IN THE E.R., AND IT'S A SO CALLED COMPLEX AND 19:13 WHAT'S IMPORTANT HERE ON THIS SLIDE IS TO KNOW THAT THERE ARE INTEGRAL ER MEMBRANE PROTEINS AS 19:20 A CONSTITUENT OF THIS COMPLEX AND INTEGRAL OUTER MEMBRANE PROTEINS AND INTRINSIC TO THIS 19:26 COMPLEX IS THE ABILITY TO TRANSPORT LIPIDS. AND IN FACT, IT'S BECOMING CLEAR 19:34 THAT A MORE GENERALIZED FUNCTION OF CONTACT SITES BETWEEN ORGANELLES IS TO BE A HUB FOR 19:41 LIPID TRANSPORT. WILL PRINCE DOES A LOT OF THAT WORK. 19:47 HE'S HERE, WE'VE COLLABORATED WITH HIM, HE'S AWESOME. ALSO A COMPONENT OF THIS, THIS 19:52 ALSO MIGHT BE A THEME EMERGING, IS THIS PROTEIN CALLED MBM10, WHICH ALSO MOONLIGHTS IN THE 20:00 BIOGENESIS OF MY TOE MITOCHONDRIAL, SO HERE YOU HAVE A CASSETTE THAT 20:07 DOT BOTH LIPID AND PROTEIN BIOGENESIS, QUITE INTERESTING. THEN AN ANCILLARY BUT REALLY 20:13 TIGHTLY ASSOCIATED COMPONENT OF THIS IS THE YEAST ORTHOLOG OF A PROTEIN CALLED MIRO WHICH IS 20:20 REALLY IMPORTANT FOR MOVING MITOCHONDRIA AROUND IN MAMMALIAN 20:26 CELLS. IT HOOKS UP TO ADAPTERS THAT IN TURN HOOK UP TO THE CYTOSKELETON, BUT HERE IN YEAST, 20:33 MITOCHONDRIAL TRANSPORT IS ALL ACTIN BASED AND WE REALLY DON'T KNOW WHAT THE HOOKUP HERE IS. 20:40 WHAT'S INTERESTING IS THIS IS AN INTEGRAL COMPONENT OF THIS CONTACT SITE. SO I WOULDN'T HAVE TALKED A LOT 20:46 ABOUT THIS COMPLEX IF I COULDN'T TELL YOU THAT WHEN YOU LOOK AT WHERE THIS COMPLEX RESIDES, IT 20:52 HAS A VERY CHARACTERISTIC FOCAL APPEARANCE SO NOW WE'RE LOOKING AT MVM34, I BELIEVE, AS A 20:59 COMPONENT OF THE AIR MAZE COMPLEX, THEY ALL LOOK LIKE THIS. THIS IS WHERE THE ER AND MITOs 21:06 COME IN CONTACT WITH EACH OTHER. SO IF I HAD ANOTHER COLOR TO SHOW YOU, ER, THIS IS PUBLISHED, WHICH WE DID, IT WOULD BE RIGHT 21:12 HERE. AND IF YOU DO A LINE SCAN ACROSS 21:18 MITOCHONDRIA, THIS IS A MATRIX MARKER, THESE MITOCHONDRIA ARE ALREADY CONSTRICTED AT THAT 21:23 POINT OF CONTACT. THIS IS PRIOR, AGAIN, IN OTHER EXPERIMENTS TO THE RECRUITMENT OF THE MITOCHONDRIAL DIVISION 21:29 DYNAMIN. SO YOU FIRST GET SOMEHOW THIS ER LINK MITOCHONDRIAL CONSTRICTION, 21:35 WHICH WE STILL DON'T UNDERSTAND THE MECHANISM. MAYBE ACTIN-RELATED BASED ON OTHERS' WORK. 21:40 YOU THEN GET DRP TARGETING HERE ONCE YOU GET SOME CONSTRUCTION 21:46 OF THE MITOCHONDRIA, YOU GET SITION OF THE MITOCHONDRIA AND THEN THE TIPS MOVE APART. 21:52 THIS IS A VERY CHARACTERISTIC PROCESS AND IT ALLOWED MY THEN 21:57 STUDENT ANDY TO ORDER FUNCTIONALLY THE COMPONENTS I JUST TOLD YOU ABOUT IN THIS PATHWAY NOW. 22:09 AIR MAZE MEDIATES THE CONTACT AND PERHAPS SOME OF THE CONSTRICTION, WE'RE TESTING THAT. YOU GET TARGETING OF THE 22:15 DIVISION DYNAMIN BY A MECHANISM I DESCRIBED, AND THEN THIS MOTILITY STEP, THE SEPARATION OF 22:23 THE DAUGHTER TIPS FROM ONE ANOTHER IS DEPENDENT ON GEM1, SO 22:28 NOW WE'VE COUPLED MITOCHONDRIAL DIVISION TO MOTILITY. 22:41 BACTERIA IS TO SEGREGATE THE CHROME ZONE AND TRANSMIT IT SO WE ASKED A SIMILAR QUESTION. 22:53 IT'S NOT IMPORTANT WHAT IT DOES, IT JUST MARKS ALL THE MITOCHONDRIAL CHROMOSOMES IN YEAST WHICH WE CALL KNEW CLEE 22:59 OIDS, AND NOW THE LITTLE DOTS THAT YOU'RE SEEING ARE THE CHROMOSOMES, NOT CONTACT SITES. 23:04 AND IF YOU FOLLOW THEM, THE 23:10 NUCLEOIDS THEMSELVES, YOU SEE DIVISION OCCURS IN A MANNER THAT'S SPATIALLY LINKED TO WHERE THEY ARE, IN ABOUT 80% OF THE 23:15 CASES. NOT 100 BUT 80%. AND SO THIS HAS LED IN MY LAB TO A VERY SIMPLE MODEL WHERE THE 23:23 CONTACTS, THE WHOLE FUNCTION OF THE CONTACTS MARKING DIVISION IS TO LINK THE DISTRIBUTION OF 23:30 MITOCHONDRIA AND MITOCHONDRIAL DNA. AND SO WE WANTED TO TEST WHETHER 23:35 THIS WAS TRUE IN HUMAN CELLS, WHETHER THIS WHOLE MODE OF DISTRIBUTION IS CONSERVED, AND 23:41 USING -- THIS WAS DONE BY MY POSTDOC SAMANTHA LEWIS, WHO'S AWESOME. AND THE REASON IS NOT LIKE A 23:50 ME-TOO, YOU KNOW, FOR THE SAKE OF ACADEMICS. RATHER, THERE ARE MANY COMPONENTS HERE THAT AREN'T 23:55 CONSERVED. ERMES IS NOT CONSERVED, NUCLEOID 24:05 ARE DIFFERENT, YEAST ARE DIFFERENT THAN HUMAN CELLS. SO WE THOUGHT THIS WAS REALLY IMPORTANT, AND WHAT I'M GOING TO TELL YOU IS VERY ROBUST IN THE 24:11 SENSE THAT WE HAVE SEEN THIS PROCESS OCCUR IN A NUMBER OF 24:17 DIFFERENT CELL LINES IN CULTURE. AND IT WAS VERY CHALLENGING BECAUSE MITOCHONDRIA, UNLIKE 24:23 YEAST, ARE MORE HETEROMORPHIC IN HUMAN CELLS. YOU CAN'T KEEP TRACK OF ALL OF THEM AT ONCE WHICH DRIVES ME 24:30 CRAZY. THE ER IS MUCH MORE PERVASIVE AND THERE ARE MANY, MANY, MANY 24:37 MORE COPIES OF MITOCHONDRIAL DNA IN THE ORDER OF TWO ORDERS OF 24:45 MAGNITUDE, SOMETIMES MORE. 24:50 THE FRS THING SAM DID WAS TO DESCRIBE THE SYSTEM AND WE DO THIS A LOT IN A QUANTITATIVE WAY. SHE LOOKED AT WHERE THE KNEW 24:56 CLEE OIDS ARE, HERE IN GREEN, MARKED BY A PROTEIN CALLED T FAMILIAR, WHICH IS BASICALLY THE 25:02 CHROMATIN OF MITOCHONDRIAL DNA. 25:11 WHERE THE ENDOPLASMIC RETICULUM COME IN CLOSE CONTACT WITH 25:17 MITOCHONDRIA POTENTIALLY. I'LL JUST DESCRIBE THE QUANTITATIVE ANALYSIS SHE DID WHICH WAS BY CROSS CORRELATION 25:23 COEFFICIENT, THERE WAS AN EXTREMELY HIGH PROBABILITY THAT IF YOU'RE A KNEW CLEE OID, 25:29 YOU'RE GOING TO HAVE SOME ER TUBULE VERY CLOSE TO YOU. SO MAYBE IT WASN'T THAT 25:35 SURPRISING THAT WHEN SHE LOOKED AT MITOCHONDRIAL DIVISION RELATIVE TO NUCLEOIDS AND 25:41 RELATIVE TO THE ER, SHE OBSERVED VERY SIMILAR TO THE YEAST SYSTEM IN ABOUT 80% OF THE CASES, 25:48 MITOCHONDRIAL DIVISION LINKED TO WHERE THE ER CONTACTS ARE IS GOING TO HAPPEN IN 80% OF THE 25:53 CASES NEXT TO A NUCLEOID OR IN CLOSE PROXIMITY. 25:58 I'LL JUST REMIND PEOPLE THAT METEOROLOGIST HAVE TWO MEMBRANES, OUTER AND INNER, AND THE NUCLEOIDS IN THE MOST INNER 26:07 COMPARTMENT. ALSO VERY SIMILAR TO YEAST, ALTHOUGH I DIDN'T TELL YOU THIS, 26:13 IS THAT SOMETIMES YOU GET NUCLEOIDS CLOSE TO BOTH ENDS FROM DIVISION, AND SOMETIMES ONLY ONE END, AND WHEN YOU ONLY 26:21 HAVE IT LINKED TO ONE END IN SEGREGATION, THAT'S THE END THAT GOES WITH THE ER. 26:28 SO IT'S A REAL STABLE CONNECTION THERE THAT WE THINK. THE OTHER THING SHE NOTICED, THIS WAS DONE INDEPENDENTLY, IF YOU JUST LOOK AT NUCLEOIDS, 26:36 HUMAN CELLS IN CULTURE OVER TIME, A LOT OF THEM HAVE A LOT OF SHORT RANGE MOTILITY, BUT 26:43 THERE'S A SUBSET THAT MOVED QUITE LONG DISTANCES, RELATIVELY SPEAKING, AND RETROSPECTIVELY, 26:49 IF SAM WENT BACK AND TOOK A LOOK AT WHAT THOSE WERE, THEY WERE PRODUCTS OF MITOCHONDRIAL DIVISION. AND SO NOW, THIS SHOULD FEEL 26:59 KIND OF SIMILAR TO WHAT I TOLD YOU IN YEAST, AGAIN THE STRATEGIES BEING CONSERVED, THAT THE ER IS SERVING TO LINK THE 27:05 DIVISION AND MOTILITY MACHINES TOGETHER, WE THINK, PROBABLY BY 27:10 CREATING A MICROENVIRONMENT THAT CLUSTERS, FOR EXAMPLE, THE 27:17 MOTORS OR ALLOWS THE TARGETING OF THE DIVISION MACHINERY RIGHT 27:23 THERE. SO ONE THING THAT KIND OF STARTED DRIVING US CRAZY, THOUGH, WAS, OKAY, THAT'S GREAT, 27:30 BUT HOW MANY CONTACTS, ER MITOCHONDRIAL CONTACTS ARE THERE IN A CELL? 27:38 SO IF THESE REALLY MARK DIVISION SITES, ARE THEY ALL DIVIDING? WHAT'S GOING ON? SO SAM TOOK A STEP BACK AND 27:50 MARKED REGIONS WHERE THEY COLOCALIZED AS POTENTIAL REGIONS OF CONTACT BETWEEN THE TWO 27:56 ORGANELLES AND THEN USED THE GREAT EQUALIZER OF TIME TO DETERMINE WHICH WERE STABLE AND WE DEFINE THOSE AS REAL CONTACT 28:02 SITES, AND I CAN TELL YOU THAT A LOT OF THESE COLOCALIZATIONS 28:10 WERE UNSTABLE, SO FOR PEOPLE WHO WANT TO STUDY MEMBRANE CONTACT SITES, IT'S NOT COOL TO USE 28:16 FIXED DATA IN COLOCALIZATION TO QUANTIFY REGIONS OF CONTACT 28:21 BECAUSE A LOT OF THEM AREN'T IN CONTACT, THEY'RE PROBABLY JUST CLOSE BY, OKAY? THIS IS THE PROBLEM WITH LIGHT 28:27 MICROSCOPY. SO THERE'S A LOT, THERE'S OVER 100 PER CELL THAT ARE STABLE -- 28:34 REGIONS OF STABLE COLOCALIZATION. IF SHE LOOKED AT THOSE REGIONS, SHE REALLY COULDN'T SEE ANYTHING 28:39 HAPPENING TO A MAJORITY OF THOSE, AND BY WHAT I MEAN, EVENTS LINKED TO DIVISION. 28:45 SO ABOUT 10% OF THEM HAD -- OF THESE CONTACTS HAD REGIONS WHERE 28:52 THE MITOCHONDRIA WERE CONSTRICTED LINK TO THEM THEN DURING A FIVE MINUTE PERIOD, 28:58 ABOUT 1% OF THOSE CONVERTED INTO DECISION EVENTS JUST AS THE 29:04 PROCESS I DESCRIBED TO YOU BEFORE. SO WHAT THIS TOLD US IS THAT, YES, ER MITOCHONDRIA CONTACTS 29:11 MARK FUTURE SITES OF DIVISION, BUT THEY'RE NOT RATE-LIMITING FOR DIVISION, SO I LOVE KICKING THE CAN DOWN THE ROAD. 29:18 WE DO THAT A LOT IN THIS PROJECT, SO THE QUESTION THEN BECOMES WHAT DETERMINES DIVISION SITE PLACEMENT BEYOND THE 29:24 CONTEXT. SO OUR LOGIC WAS, WELL, THE CONTACTS, WE THINK, ARE 29:30 SPATIALLY LINKED TO NUCLEOIDS. I TOLD YOU THAT ALREADY. SO IS THERE SOMETHING INSIDE AND 29:35 ARE ALL THE NUCLEOIDS THE SAME, ARE THEY PART OF THE PROCESS, AND SO WE LOOKED SPECIFICALLY AT 29:42 THE FATE OF NUCLEOIDS THAT WERE UNDERGOING REPLICATION, I GUESS THAT'S HOW YOU KNOW YOU'RE 29:47 ALIVE. YOU REPLICATE. SO TO DO THAT, WE HAD TO DEVELOP 29:55 A REAL ACCURATE IN-CELL MARK FOR REPLICATING NUCLEOIDS FOR LIVE CELL IMAGING, AND WE TURNED TO 30:03 AN ACCESSORY SUBUNIT OF THE MITOCHONDRIAL DNA POLYMERASE 30:09 BASED ON WORK FROM A COLLEAGUE WHO SHOWED THIS GFP FUSION WAS FUNCTIONAL AT A BIOCHEMICAL 30:14 LEVEL, EVEN. AND HERE WE'RE USING THE ANALOG EDU TO MONITOR INCORPORATION OF 30:21 NUCLEOTIDES INTO MITOCHONDRIAL DNA, AND I'LL JUST BLOW THIS UP FOR YOU AND SHOW YOU THAT 30:26 NUCLEOIDS THAT ARE INCORPORATING THAT ARE REPLICATING ARE ALSO MARKED WITH THE ACCESSORY 30:33 SUBUNIT OF POLG2. SO WE THOUGHT WE HAD A GOOD MARKER AND IN FACT IF YOU DO A 30:38 COMPARISON OF EDU LABELING OF NUCLEOIDS VERSUS POLG2, THEY'RE THE SAME, AND THAT'S, OF COURSE, 30:44 IN CONTRAST TO THE TOTAL POPULATION WHICH I TOLD YOU ALREADY WE COULD MARK WITH TFAM OR THERE'S A DYE THAT YOU CAN 30:52 USE, SO AT ANY SNAPSHOT IN TIME, ONLY A SMALL FRACTION OF THE 30:59 TOTAL MITOCHONDRIAL DNA GENOMES IN A CELL ARE REPLICATING, ABOUT 31:04 10%. AND THIS MAKES IT A REALLY HARD SYSTEM BUT FASCINATING TO STUDY, BECAUSE IT'S NOT -- IT DOESN'T 31:09 REALLY NEED -- IT'S NOT A VERY STRINGENT TRANSMISSION SYSTEM LIKE THE NUCLEUS, WHICH YOU KNOW 31:16 THE WHOLE CELL CYCLE IS DEVOTED TO. SO ABOUT 10% AT ONCE, IT'S 31:23 ASYNCHRONOUS WITH EACH OTHER, IT'S ASYNCHRONOUS WITH THE CELL CYCLE, IT IS WHAT IT IS. SO WE USE THIS MARKER AND THIS 31:29 WAS LIKE ONE OF THE MOST -- THIS IS ONE OF MY FAVORITE OUTCOMES OF AN EXPERIMENT IN MY CAREER. 31:34 IT TURNS OUT THAT REPLICATING NUCLEOIDS ARE THE BEST MARKER WE HAVE FOR FUTURE SITES OF 31:40 DIVISION. NOT THE CONTACT SITES THEMSELVES, BUT THE REPLICATING 31:47 NUCLEOIDS LINKED TO THE CONTACT SITE ARE. AND THE NUMBERS ARE HERE, IF WE LOOK AT MITOCHONDRIAL DIVISIONS 31:53 STRINGENTLY MARKED BY THE DIVISION DYNAMIC DRP1, OUT OF THE TOTAL NUMBER ABOUT 80% OF 32:01 THESE ARE LINKED TO THIS PO LG2. IF YOU RECALL, THAT 80% NUMBER 32:06 IS WHAT I TOLD YOU WE GOT FROM LOOKING AT TFAM LABELED 32:12 NUCLEOIDS OF THE TOTAL POPULATION. SO I GUESS IN OTHER WORDS, IF YOU'RE GOING TO DIVIDE IN A NUCLEOID, YOU'RE DIVIDING IN A 32:19 NUCLEOID THAT'S UNDERGOING REPLICATION, AND THAT ACCOUNTS FOR 80% OF THE DIVISION EVENTS IN THESE CELLS IN CULTURE. 32:25 SO THERE THEY'RE A REAL DETERMINE NAPT OF FUTURE DIVISION SITES. 32:30 SO THIS COMBINED IS A REAL 32:35 PATHWAY WE THINK THAT'S ORCHESTRATED BY THE MEMBRANE CONTACT SITE, WHERE THE CONTACT SITE IS THERE, THE NUCLEOID 32:44 FIRES FOR REPLICATION INSIDE THE MITOCHONDRIA. THERE'S ABOUT AN HOUR THAT 32:49 HAPPENS HERE, AN HOUR LAPSED, AND THEN YOU GET THE SUCCESSIVE EVENTS OF CONSTRICTION, 32:55 DIVISION, AND MOTILITY. SO IT'S THIS PATHWAY WE THINK 33:01 FOR MITOCHONDRIAL DNA TRANSMISSION. AND SO WHAT REALLY BLEW US AWAY WAS ADDRESSING THE ROLE OF THE 33:06 ER IN ALL OF THIS. BECAUSE THE CONTACT ARE THERE. AND SO WE DID THIS BY MA ANYBODY 33:12 LATING THE STEADY STATE STRUCTURE OF ENDOPLASMIC RETICULUM WHICH IS PRESENT IN 33:18 BOTH MEMBRANE TUBULE-LIKE STRUCTURES AND SHEET-LIKE STRUCTURES. AND THESE HAVE SEPARATE 33:24 PROTEOMES. THIS IS WORK BY JIN JI HU, 33:32 THREAR FUNCTIONALLY SPECIALIZED REGIONS OF THE ER, AND MUCH LIKE IN MITOCHONDRIA TIPPING THE 33:37 BALLS OF DYNAMICS, YOU CAN TIP THE BALANCE OF THE STEADY STATE DISTRIBUTION OF THE ER MEMBRANES IN SHEETS AND TUBULES. 33:43 SO IF YOU OVEREXPRESS A PROTEIN 33:50 CALLED CLEM63, YOU DEPLETE THE 33:56 TUBULES, THIS IS A TUBULAR SHAPING PROTEIN, RTM4A, YOU 34:02 DEPLETE THE SHEETS AND PROLIFERATE TUBULES AND IF YOU COULD EXPRESS THEM, YOU COME UP 34:08 WITH A CELL THAT LOOKS KIND OF WILD TYPE IN A NICE DX AND THIS SERVES AS A NICE CONTROL. 34:13 SO WE DID THIS AND ASK, OKAY, WHAT'S THE STATUS OF MITOCHONDRIAL DNA REPLICATION? 34:19 AMAZINGLY ENOUGH, IF YOU DEPLATE THE ER TUBULES BY OVEREXPRESSING 34:27 CLIMP63, MITOCHONDRIAL REPLICATION PLUMENTS. AND WE THINK THAT IT'S MORE -- IT'S MORE CORRELATED WITH 34:33 TUBULAR ER FOR A COUPLE OF WREEN REASONS. DOUBLE OVEREXPRESSION DOESN'T DO 34:38 THAT. SO IT'S NOT CLIM -- THESE ARE 34:46 TWO MEASURES OF MITOCHONDRIAL DNA REPLICATION. AND THE OTHER COOL THING ABOUT THESE EXPERIMENTS IS THEY WERE 34:54 MOSAIC ON AN EXTRACELLULAR BASIS WHERE WHEN YOU OVEREXPRESS CL 35:00 IMP63, YOU CAN NEVER QUITE DEPLETE ALL OF THE TUBULAR ER. IN FACT, THE MITOCHONDRIAL DNA 35:06 GENOMES THAT WE'RE REPLICATING THAT WE CAN SEE IN THESE CELLS WERE ALWAYS ASSOCIATED WITH 35:15 ER2 -- AND NO PLACE ELSE. SO AGAIN, THIS REALLY TIGHT CORRELATION BETWEEN THAT. 35:22 SO THERE'S SOMETHING ABOUT THESE CONTACT SITES THAT ARE REALLY IMPORTANT FOR LICENSING THE REPLICATION OF MITOCHONDRIAL 35:27 DNA. SO YOU NEED THE ER TO GET REPLICATION, WHICH I THINK IS PRETTY PROFOUND, HAS A LOT OF 35:36 IMPLICATIONS IN TISSUES AND THINGS LIKE THAT. 35:43 INTERESTING THINGS TO TEST. SO THERE'S A LOT OF THINGS WE'RE ADDRESSING. AS I SAID, THESE CONTACT SITES 35:49 SEEM TO INTEGRATE MACHINES INTO FUNCTIONAL PATHWAYS. HOW DOES THAT HAPPEN? 35:55 AND HOW DOES THIS TUBULAR ER LICENSE MITOCHONDRIAL DUPLICATION, AND WE THINK THIS 36:02 IS INTEGRALLY RELATED TO THE QUESTION OF HOW DO CELLS KNOW AND COUNT THE NUMBER OF 36:08 MITOCHONDRIAL DNA GENOMES THEY HAVE? IT'S A RELAXED SYSTEM. REPLICATION IS, YOU KNOW -- 36:17 WOOO. HOW DOES THE CELL KEEP TRACK OF IT? BECAUSE THEY'RE NOT WILDLY VARYING IN TERMS OF COPY NUMBER, 36:23 SO HOW DO THE CELLS COUNT? SO I'M GOING TO TELL YOU A LITTLE BIT OF OUR UNPUBLISHED DATA ON THIS. WHERE WE'VE BEEN ASKING TO 36:29 ADDRESS THIS QUESTION ABOUT THE CROSSTALK BETWEEN THE ER AND THE 36:35 KNOWN COMPONENTS INVOLVED IN MITOCHONDRIAL DNA REPLICATION. SO THE MITOCHONDRIAL DNA 36:43 REPLISOME, BEAUTIFUL WORK HAS BEEN RECONSTITUTED IN VITRO, AT LEAST THE MINIMAL SYSTEM, AND IT 36:52 IT'S COMPRISED OF A MOL POLYMERASE, A TRIMER OF TWO ACCESSORY STUB 36:58 UNITS AND THE POLYMERASE, SINGLE STRANDED BINDING PROTEIN, AND 37:04 THIS HELICASE CALLED TWINKLE THAT'S DERIVED FROM PHA TB. E,GE, SO 37:13 EVOLUTION IS WILD. WE'RE ASKING ABOUT THESE COMPONENTS SMEFL AND THEIR RELATIONSHIP TO ER MITO CONTACT 37:20 SITES AND TRYING NOW TO ORDER THEIR ASSEMBLY. I CAN TELL YOU IN OUR HANDS, TWINKLE AND THIS IS WORK ALSO 37:27 DONE BY ANU IN MICE, TWINKLE& 37:33 SEEMS TO BE THE RATE LIMITING COMPONENT FOR DNA REPLICATION, IT'S THE ONLY COMPONENT THAT WE 37:39 CAN OVEREXPRESS THAT INCREASES THE NUMBER OF MITOCHONDRIAL GENOMES UNDERGOING REPLICATION. 37:46 SO JUST HAVE THAT IN YOUR HEAD. WE LOOK THEN AT TWINKLE, AND LO AND BEHOLD, TWINKLE MARKS 37:54 REPLICATING NUCLEOIDS, THAT'S REALLY GREAT, BUT WE NOTICED THAT A MAJORITY OF THE TWINKLE 38:01 FOCI DON'T LOCALIZE WITH NUCLEOIDS THAT ARE REPLICATING AS MARKED BY EDU AND POLG2, SO 38:07 THERE'S A FRACTION OF MORE, BUT IF WE LOOK AT ALL THE TWINKLE FOCI, WE NOTICE THAT LIKE THE 38:13 NUCLEOIDS, BECAUSE THEY'RE LOCALIZED TO NUCLEOIDS, THEY'RE 38:19 AT MITOCHONDRIAL CONTACTS ALSO, AND SO WE KIND OF SURMISE AND NOW WE'RE DOING LIVE CELL 38:26 EXPERIMENTS THAT TWINKLE PROBABLY LOADS ON TO THE NUCLEOIDS BEFORE THE POLYMERASE 38:32 DOES, WHICH WOULD MAKE SENSE IF IT'S RATE LIMITING. BUT WHAT WE REALLY NOTICED THAT KIND OF PUZZLED US AS FIRST WAS 38:38 THAT THERE WAS A SIGNIFICANT NUMBER OF TWINKLE FOCI THAT DIDN'T COLOCALIZE WITH 38:45 MITOCHONDRIAL DNA. IT WAS STILL AT ER CONTACTS BUT 38:51 IT DIDN'T COLOCALLIZE -- THEY WERE JUST KIND OF OUT THERE, THESE TWINKLE FOCI, THESE 38:56 STRUCTURES. AND THIS IS JUST WITH ANTIDNA ANTIBODY AND THIS IS ENDOGENOUS TWINKLE. 39:01 SO WE GO BACK AND FORTH WITH SLIGHT OVEREXPRESSION THAT DOESN'T ALTER COPY NUMBER AND 39:07 LOOKING AT NATIVE PROTEINS TOO. SO THAT PROMPTED US TO ASK THE QUESTION, WELL, WHAT IS THE ROLE, THEN, IN TERMS OF THE 39:15 REPLY SOME ASSEMBLY OF MITOCHONDRIAL DNA AT ALL, IS THERE A ROLE? SO TO DO THIS, WE CREATED CELLS 39:22 THAT LACK MITOCHONDRIAL DNA AND CO-CULTURED THEM AND LOOKED AT 39:28 THESE COMPONENTS AGAIN, AND MAYBE NOT SURPRISINGLY, TFAM, 39:34 POLG2, MITOCHONDRIAL SSB LOST THAT FOCAL APPEARANCE THAT'S CHARACTERISTIC OF THE NUCLEOID 39:39 BECAUSE THAT'S WHERE THE DNA IS PACKAGED, SO THEY CAN NO LONGER BUY THE DNA BECAUSE THE DNA IS 39:46 NOT THERE, NOW THEY'RE DIFFUSE IN THE MATRIX. FINE, THAT MAKES SENSE. BUT LOOK HERE AT WHAT TWINKLE 39:51 DOES. TWINKLE REMAINS PUNCTATE, AND THIS IS JUST A BLOWUP WITHIN THE 39:58 MITOCHONDRION, AND THAT'S JUST THE QUANTIFICATION OF THAT. AND IN FACT, ENDOGENOUS BEHAVES THE SAME WAY, AND THE NUMBER OF 40:07 TWINKLE FOCI DON'T VARY TOO MUCH IN THE PRESENCE OR ABSENCE OF MITOCHONDRIAL DNA, SO THE NUMBERS ARE ROUGHLY EQUIVALENT. 40:13 SO WHAT ARE THESE STRUCTURES? WELL, I CAN TELL YOU THAT 40:19 THEY'RE ALSO AT MITOCHONDRIAL ER CONTACT SITES, THEY'RE ALL ENRICHED THERE, AND IF WE DO THE 40:25 SAME TYPE OF MANIPULATION OF THE ER, THEY REALLY REQUIRE THE TUBULAR ER FOR THEIR STABLE 40:31 FORMATION. SO THEY REALLY -- THEY'RE AT MITOCHONDRIAL CONTACTS AND THEY REQUIRE THESE CONTACT TO FORM. 40:43 AND THAT'S JUST REMINISCENT OF THE BEHAVIOR OF REPLICATING NUCLEOIDS SO THERE'S VERY TIGHT 40:49 CORRELATION HERE. AND THIS JUST STRUCK US BECAUSE GOING BACK TO THE YEAST SYSTEM, 40:55 AS I TOLD YOU, THEY REPLICATE 41:01 DIFFERENTLY, HUMAN MITOCHONDRIAL DNA REALLY DON'T COMBINE. THERE'S A COMPONENT CALLED 41:07 MGM101 THAT IN ROW ZERO CELLS IS ALSO FOCAL, MAINTAINS ITS FOCAL DISTRIBUTION, AND LOCK AT HOW 41:14 NICELY ALIGNED IT IS WITH THE ERMES COMPLEX. AGAIN, MGM1 ASSEMBLIES, ON THE 41:21 VERY INSIDE, ERMES IS ON THE VERY OUTSIDE OF THE MITOCHONDRIA, SO THIS IS CLEARLY 41:28 SPATIALLY LINKED WITH ONE ANOTHER. AND OUR THINKING IS SHAPED A LOT ON WHAT WE THINK CONTACT SITES 41:33 DO. THEY TRANSPORT LIPIDS AS I SAID, 41:39 AND WE KNOW BASED ON LOTS OF PUBLISHED WORK THAT THEY HAVE THEIR OWN PROTEOME KIND OF 41:44 THEMSELVES, SO BASED ON THIS, WE THINK THAT THEY ARE MICRO 41:51 ENVIRONMENTS TO MEDIATE THE ASSEMBLY THINGS LIKE -- OF TWINKLE, FOR EXAMPLE, INSIDE THE 41:57 MATRIX SOMEHOW, BECAUSE OF THEIR SPECIALIZED COMPOSITION OF LIPIDS AND PROTEINS. SO BASED ON THAT, WE BEGAN TO 42:04 ASK WHETHER ANY OF THE REPLY SOME COMPONENTS CARED ABOUT LIPIDS AT ALL. 42:10 AND THIS IS JUST OUR PRELIMINARY WORK SHOWING THAT TWINKLE -- AND 42:15 THIS IS IN COLLABORATION WITH MARIA FALKENBERG, WHO IS THE 42:20 EXPERT IN MAKING THESE PROTEINS, DOES HAVE AN AFFINITY FOR ACIDIC LIPIDS LIKE CARDIOLIPIN AND PA. 42:30 THAT'S SURPRISING THESE LIPIDS ARE PRECURSORS THAT COME FROM THE ER AND GET FUNNELED, SO WE 42:36 ASKED WHETHER IN THE CONTEXT OF A MEMBRANE, A LIPOSOME THAT WE MADE IN VITRO, WHETHER THIS IS 42:42 ALSO TRUE, AND TWINKLE, YES, DOES INTERACT SLEK SELECTIVELY WITH 42:48 LIPOSOMES THAT CONTAIN ABOUT TWO TIMES THE AMOUNT OF CARDIOLIE PEN THAT IS NORMALLY IN THE 42:55 MEMBRANE, PERHAPS WITH CONCENTRATION IN THE INNER DOMAIN. TWINKLE ITSELF IS A VERY 43:02 INTERESTING CASE, AT STEADY STATE, IT'S ALREADY ASSEMBLED 43:08 INTO THE CHARACTERISTIC DONUT SHAPE HELIXES THEY HAVE, BUT IT HAS A -- SIMILAR -- IT DOESN'T 43:15 FUNCTION IN MAKING -- IN DOING PRIMATE AS' WORK, RATHER IT'S A DOMAIN THAT KINE OF GETS 43:22 ASSEMBLED ON TOP OF IT VIA THIS REGION. AND SO WE'VE DONE A LOT OF STRUCTURE FUNCTION ANALYSIS TO 43:28 DETERMINE WHAT ARE THE IMPORTANT DETERMINANTS IN TWINKLE THAT 43:34 ALLOW TO INTERACT WITH THE MEMBRANE AND ALL OUR WORK IS CONSISTENT WITH THIS REGION HERE 43:40 THAT SOMEHOW HELPS THAT PRIME 43:46 ACEDOMAIN LOAD ON AND GET IT GOING. THESE ARE MUTATIONS THAT ABROGATE THE BINDING THAT ARE IN 43:52 THIS REACH. OTHER EU MUTATIONS IN THE L -- 44:00 THERE'S A VERY SHORT BUT PRETTY CLASSIC ALPHA HELIX HERE, KNOWN 44:06 FOR INTERACTING WITH MEMBRANES AND EVEN IN SOME CASES SHAPING THEM, AND CONSISTENTLY, THIS 44:12 LITTLE PIECE OF TWINKLE IS BOTH NECESSARILY OBVIOUSLY AND SUFFICIENT TO ALLOW TO INTERACT 44:18 WITH MEMBRANES IN VITRO. 44:24 I IMPLIED THIS BUT THIS IS JUST SHOWING YOU DATA THAT AMONG THE KNOWN REPLY SOME COMPONENTS, 44:32 ONLY TWINKLE HAS THE ABILITY TO BUY LIPIDS. SO WE DON'T SEE THIS FOR SSB, 44:37 FOR TFAM, FOR POLG, AND NOW 44:42 TWINKLE IS THE ONLY ONE THAT BINDS TO THESE LIPIDS. SO WE'RE LEFT NOW WITH A SIMPLE 44:49 MODEL OF COPY NUMBER CONTROL, WHERE AGAIN THE CONTACT SITE ARE MICRO DOMAINS THAT ALLOW THE 44:57 ASSEMBLY OF THIS TWINKLE-LIKE REPLICATION PLATFORM THAT'S 45:04 RATE-LIMITING FOR THE REPLICATION OF MITOCHONDRIA DNA AND IT'S PRESENT IN A FINITE AMOUNT. 45:11 SO THE COPY NUMBER THEN IS NOT IMAGINELY CONTROLLED. IT'S CONTROLLED BY THE NUMBER OF 45:16 ER MITOCHONDRIAL CONTACT SITES, THESE DOMAINS, AND THE AMOUNT OF TWINKLE THAT'S EXPRESSED INSIDE 45:23 MITOCHONDRIA AND THE ASSEMBLY OF THE STRUCTURE. THE DEPENDENCE OF THAT ON THE ASSEMBLY OF THE STRUCTURE. 45:28 SO THIS IS A MODEL THAT WE'RE TESTING AND OVERARCHINGLY IT 45:35 REALLY TELLS YOU THAT THE ER IN THIS CONTEXT IS A SYSTEM REGULATOR THAT ALLOWS ALL THESE 45:44 MACHINES TO COORDINATE INCLUDING REPLICATION OF MITOCHONDRIAL DNA AND ITS TRANSMISSION, SO IT'S 45:51 SEEN AS A SYSTEM REGULATOR. SO I WANT TO SWITCH GEARS A LITTLE BIT, I HOPE I'M NOT GOING TO OVERSTAY MY WELCOME, AND TELL 45:57 YOU A VERY SHORT STORY BECAUSE I THINK IT'S PROFOUND IN REGARDS TO THIS. 46:02 SO I TOLD YOU THAT WE'RE REALLY INTERESTED IN MITOCHONDRIAL 46:11 INFRASTRUCTURE. THIS IS A TOMOGRAM THAT TERRY 46:16 FRYE TOOK AND RECONSTRUCTED AND YOU SEE THIS IS THE INNER MEMBRANE, AND IT'S VERY ARTICULATED, EVERYBODY KNOWS 46:22 THAT THEY'RE CALLED CHRIS TAI, AND THEY PEN TRAY INTO THE 46:28 MATRIX, AND THAT'S WHAT IS REALLY GOING ON HERE. THERE'S DOMAINS WITHIN INNER 46:34 MEMBRANE, SO YOU HAVE AN INNER MEMBRANE THAT'S CONTINUOUS, AND THAT HAS TO LATERALLY 46:41 DIFFERENTIATE MUCH LIKE THE ER STRUCTURE TUBES AND SHEETS. 46:46 THIS ALSO IS LATERALLY DIFFERENTIATED INTO WHAT WE CALL A BOUNDARY REGION, WHICH IS THE 46:51 MEMBRANE CLOSE TO OUTER MEMBRANE AND WHAT THIS HAS STARTED US 47:01 THINKING ABOUT IS NOW BACK TO THE TREE OF LIFE, HOW ME METABOLISM 47:07 DRIVES THE SELF ORGANIZATION OF THIS MEMBRANE. AND WE'VE WORKED ON HOW THIS MEMBRANE GETS PUT TOGETHER. 47:16 WE KNOW PHOSPHOLIPIDS ARE REALLY IMPORTANT, IN PARTICULAR, THE SIGNATURE ONES I TOLD YOU ABOUT 47:23 TO DIFFERENTIATE THIS MEMBRANE. THE RESPIRATORY COMPLEXES THEMSELVES PLAY A ROLE AND IS 47:29 ENRICHED AND ON THE CURVED EDGES OF THESE CRYSTAE. 47:34 THERE'S A COMPLEX THAT WE THINK DETERMINES THE COPY NUMBER OF 47:43 CHRSTE AND HOW PROTEINS STORE INTO THESE DOMAINS. SO TO APPROACH THIS, WE SIMPLY 47:50 INVENTORIED THE DOMAIN LOCALIZATION OF A BUNCH OF INNER MEMBRANE FUNCTIONS AND WE TURNED 47:56 BACK TO OUR YEAST SYSTEM TO DO THIS. SO THERE'S A PROBLEM, EVERYBODY HAS HIT IT AT ONE POINT WHO DOES 48:02 MICROSCOPY, THIS IS A KNOWN BOUNDARY PROTEIN AND A KNOWN 48:10 CRYSTAE -- IF YOU LOOK AT THIS IN AN -- YOU CAN SEE THEY'RE NOT RESOLVED AT ALL, RIGHT? 48:16 BUT WE KNOW THAT THEY ARE BY EM. WE KNOW THIS. SO YOU CAN DO SUPER RESOLUTION 48:22 IN OEM, PRETTY LOW THROUGHPUT, 48:27 SO WE DID A RMENTD UMENTD FMENTD RIFF ON E XPANSION 48:33 MICROSCOPY WHERE YOU CAN TAKE THIS ERMES COMPLEX AND THE 48:40 MORPHOLOGICAL CHANGE THAT HAPPENS AS A RESULT OF LOSS OF THIS COULD BE TACT IS THAT THE TUBULAR ER COLLAPSES INTO A 48:47 PRETTY LARGE SPHERE. IF YOU TAKE A LOOK AT THAT 48:53 SPHERE NOW, A BOUNDARY AND A CRYSTE MARKER, WE CAN DO THOSE 49:00 BECAUSE IT'S JUST MAKING -- THIS 49:06 IS BEAUTIFUL WORK AND YEETS HAS 49:13 BEEN NICELY ANNOTATED AND CHARACTERIZED BASED ON MASS SPEC. IT'S VERY DENSE. 49:18 OUT OF THOSE PROTEINS, WE CAN ATTORNEY ABOUT 300 OF THEM. 49:24 THEY'RE FUNCTIONAL GFP FUSIONS THAT ARE NICELY EXPRESSED. AND THIS IS THE DISTRIBUTION WE 49:29 GET. A MA SCWHRORT MAJORITY ARE IN THE CRYSTE, THERE ARE SOME IN THE BOUNDARY. 49:35 THIS OTHER CATEGORY IS THE ONE, OF COURSE, THAT MY LAB BECAME INTERESTED IN BECAUSE THEY'RE KIND OF PERVERSE LIKE THAT. 49:43 THEY REPRESENT PROTEINS THAT ARE TARGETED TO MULTIPLE ORGANELLES, THIS IS INTERESTING, SOME THAT 49:48 ARE NOT ENRICHED IN EITHER COMPARTMENT. REALLY EMPHASIZING THERE MUST BE 49:55 ACTIVE MECHANISMS FOR THIS. AND THEN THE OTHER CATEGORY IS 50:01 PROTEINS THAT FORM THEIR OWN DOMAINS, NOT BOUNDARY, NOT 50:07 CRYSTAE BUT SOMETHING ELSE. THEY INCLUDED A CLUSTER OF 50:13 PROTEINS THAT ARE RESPONSIBLE FOR THE BIOSYNTHESIS OF THIS REALLY ANCIENT LIPID COQ. 50:22 FOR THIS, WE TEAMED UP WITH EXPERTS IN THIS FIELD, DAVE AND 50:27 JOSH, SO WHAT DID WE SEE? HERE ARE THE CLUSTER OF COQ PROTEINS WE SAW. SO AGAIN, THAT BOUNDARY, NOT 50:34 CHRIS TAI, BUT RATHER THEY FORM INCREASED DOMAINS, WE KNOW 50:41 THEY'RE INNER MEMBRANE ASSOCIATE, AND COENZYME Q IS AN 50:46 EXTREMELY GREECEY LIPID. LOOK AT THE MA MAIL YAP VERSION. 50:54 MAMMALIAN TABLES. THAT NECESSARY TO MOVE ELECTRONS WITHIN THE ELECTRON TRANSPOT CHAIN AND PUMP PROTONS. 51:00 IT'S ESSENTIAL FOR RESPIRATION. 51:06 SO HOW DOES IT GET MADE? IT USES - WHICH IS ALSO 51:11 RESPONSIBLE FOR SYNTHESIZING STERILES, AND THESE ISOPRINE 51:17 UNITS ALONG WITH THE PRECURSOR HEAD GROUP TYROSINE OR 4HB GET 51:24 TRANSPORTED INTO MITOCHONDRIA, STILL MYSTERIOUS HOW THAT HAPPENS. THE FIRST THING THAT HAPPENS IS THESE TAILS GET POLYMERIZED BY 51:31 COQ1 THAT CAN SOMEHOW COUNT THEM, DO 6 IN YEAST AND 10 IN MAMMALIAN CELLS, AND THEN THE HEAD IMETS ATTACHED GETS ATTACHED TO THE TAIL 51:39 AND THE REST OF THIS PATHWAY, ALL THESE OTHER THINGS SUCCESSIVELY MODIFY THE HEAD TO 51:45 DO THE BIOCHEMISTRY. SO IMAGINE THIS, A LIPID GETS MADE AND IT'S IN THE MEMBRANE, 51:51 AND THIS MACHINERY HAS TO SOMEHOW FIND THAT LIPID AND 51:57 MODIFY THE HEAD GROUP OF IT. AND SOME OF THESE PROTEINS ARE 52:02 ENZYMATIC AND HAVE ENZYMATIC ROLES. OTHERS DO NOT. FOR EXAMPLE, THIS COQ8 IS THOUGHT TO BE A LIPID CHAPERONE 52:09 THAT KIND OF PULLS ON THE HEAD TO ALLOW ACCESSIBILITY. 52:14 ANYWAY, SO WE ASKED MANY QUESTIONS. ONE OF WHICH IS, WHAT DO THESE PROTEINS LOOK LIKE IN NORMAL 52:20 LOOKING MITOCHONDRIA, AND WE SAW THAT NEITHER THE TAIL POLYMERASE 52:25 NOR THE HEAD TO TAIL ATTACHMENT HAD ANY SPECIAL DOMAIN 52:33 LOCALIZATION, AND THESE ARE ALL FUNCTIONAL BUT STRIKINGLY WHAT WE SAW WAS ALL THE HEAD MODIFIERS LOCALIZED TO VERY 52:39 DISCRETE REGIONS THAT WE CAN IDENTIFY. I HOPE YOU CAN SEE THESE HERE. EVEN WITHIN A NORMAL TUBULAR 52:44 MITOCHONDRIA, WE HAD THE RESOLUTION TO DO THAT. 52:55 NON-ENZYMATIC, IT'S PROBABLY SURVEYING THE ENTIRE MEMBRANE FOR HEAD TO TAIL, FOR COQ 53:05 INTERMEDIATES. SO WHEN THEY FORM THESE DOMAINS, THEY'RE ALL TOGETHER IN THESE DOMAINS, SO THE DOMAINS AS FAR AS OUR ANALYSIS GOES ALL HAVE 53:12 THE SAME COMPOSITION OF THESE HEAD-MODIFYING ENZYMES. AND WE CAN DETERMINE THE COPY 53:17 NUMBER RELATIVE TO ONE ANOTHER, FOR EXAMPLE. WE'RE DETERMINING RELATIVE STRIKOMETRY USING A STANDARD. 53:24 THIS IS A CLUSTERED PROTEIN OF NOPE KNOWN NUMBERS AND THESE ARE QUITE BIG. 53:29 WE ESTIMATE THERE'S LIKE 180 COPIES OF COQ9 IN THESE CLUSTERS. THESE ARE BIG. 53:35 AND OF COURSE THE MACHINERY, BECAUSE IT'S AN ANCIENT LIPID, IS STRICTLY CONSERVED BETWEEN 53:40 YEAST IN HUMANS, AND SO WE LOOKED IN HUMAN CELLS AND BY AND LARGE, THIS IS A VAL DAYTIVE 53:47 ANTIBODY AGAINST NATIVE COQ9 WHICH IS A HEAD MODIFIER. YOU CAN SEE AS IN YEAST, THEY 53:53 FORM THESE DOMAINS. SO WHAT'S REQUIRED FOR DOMAIN 53:59 ASSEMBLY? EVEN THOUGH COQ1 AND COQ2 ARE 54:04 NOT THEMSELVES COMPONENTS OF THESE DOMAINS, THEY'RE REQUIRED AND IN FACT ANY ESSENTIAL COMPONENT IN THIS PATHWAY TO 54:09 MAKE THIS LIPID IS REQUIRED. IT'S AN ALL OR NONE THING. IT DOESN'T MATTER WHERE YOU ARE, WHAT STEP YOU MEDIATE IN THE 54:15 PATHWAY, IF YOU'RE ESSENTIAL AND YOU DELETE THE GENE FOR THAT ENZYME OR THAT PROTEIN, YOU DON'T GET DOMAINS. 54:24 THE NORTHERN ESSENTIAL ONES, YOU STILL GET DOMAINS. SO WHAT WE KNOW IS THAT THESE 54:29 COQ DOMAINS ARE TIGHTLY CORRELATED WITH A FUNCTIONAL PATHWAY. AND IT'S ALL OR NONE. 54:36 EXTREMELY HIGHLY COOPERATIVE. YOU EITHER GET THEM OR YOU DON'T, AND THAT, AGAIN, CORRELATES WITH PRODUCTION OF PRODUCT. AND THAT REALLY SUGGESTS THIS 54:42 ROLE FOR THE LIPID ITSELF. THE INTERMEDIATE LIPID. AGAIN THIS IDEA THAT THE 54:48 CHEMICALS AND THEIR FLUXES AND THEIR INTERACTIONS THROUGH THE 54:55 METABOLIC PATHWAYS IS DRIVING SELF ORGANIZATION. AND THIS KIND OF GOES WITH DATA 55:01 FROM CATHY CLARKE'S LAB, WHO'S ALSO A PIONEER IN THIS FIELD, 55:06 WHERE IT DOESN'T AGAIN MATTER WHERE YOU DELETE. EVEN IF YOU DELETE A COMPONENT LIKE, FOR EXAMPLE, 5, WHICH IS 55:13 DOWNSTREAM, YOU ONLY ACCUMULATE THIS VERY FIRST PRODUCT OF HEAD TO TAIL ATTACHMENT, ALL OR NONE, 55:19 HIGHLY COOPERATIVE. SO TO ASK WHETHER THE SUBSTRATE ITSELF, THE COQ INTERMEDIATE 55:24 LIPID IS REQUIRED, WE JUST DID A LITTLE DEAD BODY EXPERIMENT WHERE THIS ALLELE OF COQ6 IS 55:31 INACTIVE, AND YOU CAN BYPASS ITS FUNCTION BY ADDING THIS COMPONENT CALLED VANILIC ACID. 55:44 THE BODY IS REQUIRED TO GET THIS TO WORK. SO HERE ARE YEAST CELLS THAT HARBOR THIS ALLELE OF COQ6 IN 55:50 THE ABSENCE OF VANILIC ACID, THERE ARE NO DOMAINS. IF YOU ADD VANILIC ACID, DOMAINS 55:59 FORM. WE'VE ALSO DONE THE REVERSE, TO SHUT OFF FLUX FOR THIS PATHWAY AND THE DOMAINS DISAPPEAR. 56:05 SO IT'S VERY CLEAR THAT DOMAIN FUNCTION AND PRODUCTION OBVIOUSLY ARE TIGHTLY CORRELATED 56:11 AND WE BELIEVE THAT THE COQ SUBSTRATE ITSELF IS REQUIRED TO 56:16 FORM THESE REGIONS OF 56:24 CONCENTRATED HEAD MODIFYING ENZYMES. WE DON'T THINK IT'S DE NOVO SYNTHESIS OR ANYTHING FANCY LIKE 56:29 THAT BECAUSE IF YOU LOOK AT TOTAL FOR ENS PRESENCE, THE 56:34 PROTEIN LEVELS DON'T CHANGE IN THESE COMPONENTS, RATHER THEY TAKE WHAT'S THERE AND GET 56:39 REORGANIZED INTO THESE ASSEMBLIES. WHICH IS PRETTY COOL. 56:44 SO THIS IS PRETTY INTERESTING. WE THINK DOMAIN FORMATION AND 56:50 THE NUMBER ARE REALLY SENSITIVE BIOMARKERS FOR FLUX THROUGH THIS PATHWAY, WHICH COULD BE USEFUL 56:58 FOR OTHER PEOPLE'S WORK. THEY'RE NOT DEPENDENT ON MITOCHONDRIAL DNA. WE'RE LOOKING GENOME-WIDE AT WHAT THEY ARE DEPENDENT ON 57:05 BEYOND WHAT I'VE TOLD YOU. SO THEY'RE STANDALONE STRUCTURES VERY SIMILAR TO THE TWINKLE THAT I TOLD YOU ABOUT PREVIOUSLY FOR 57:13 MITOCHONDRIAL DNA REPLICATION, WHICH IS WHY WHEN WE LOOKED AT WHERE THESE DOMAINS WERE 57:19 POSITIONED, I GUESS WE WERE NOT THAT SURPRISED, BUT THEY'RE POSITIONED BY ER MITOCHONDRIAL 57:25 CONTACT SITES. SO WE HAVE IN YEAST A VERY GOOD MARKER FOR ALL THE ER 57:30 MITOCHONDRIAL CONTACTS. IT'S A PROTEIN CALLED LTC1, THE 57:35 TRANSPORT STERILES AT THESE SITES. IT'S NOT IMPORTANT WHAT IT DOES REALLY FOR THE PURPOSE OF THIS. AND YOU CAN SEE THAT WHERE THE 57:42 CONTACTS ARE, WE GET VERY GOOD 57:48 COLOCALIZATION OF THE COQ DOMAINS, IT'S ANOTHER 57:57 ORGANIZATIONAL CENTER, IN THIS CASE IT'S A METABOLIC PATHWAY, AND IF WE DISRUPT THE TWO TYPES OF CONTEXTS PRESENT IN YEAST, 58:05 THIS ERMES CONTEXT I TOLD YOU ABOUT OR THIS L TC1, THE DOUGH MARINES ARE INCREDIBLY 58:12 DISRUPTED, THEY DECREASE IN THEIR NUMBERS AND IN FACT THEY 58:17 TEND TO COALESCE SO THEIR DISTRIBUTION IS COMPLETELY SCREWED UP. 58:23 SO THE DOMAINS ARE -- CONTACT SITES ALSO FOR THEIR FORMATION. 58:32 SO THE MAIN THING IS TO MAINTAIN THE SUBSTRATE ACCESSIBILITY. SO AGAIN GOING BACK TO THE FACT THAT THE LIPID IS SUCH A 58:38 GREECEBALL, IF YOU 58:44 GREASEBALL, IF YOU LET GO OF THE HEAD, HOW ARE YOU GOING TO FIND IT AGAIN. SO YOU HAVE A REALLY PROGRESSIVE SYSTEM AND THAT'S WHAT I THINK 58:49 THESE DOMAINS ARE FOR, YOU WOULDN'T WANT ANY OLD COQ INTERMEDIATE HANGING AROUND, AND 58:56 THIS HAS A NAME. OTHER PEOPLE CALL THEM 59:05 METABALONS. THE ENZYMES FORM THESE CLUSTERS 59:11 OF PROTEIN SOMETIMES IN PROXIMITY TO MITOCHONDRIA BECAUSE THEY SHARE SUBSTRATES 59:16 THROUGH THIS PATHWAY. SO THESE ME TAB LONS ARE REALLY IMPORTANT, AND THEY'RE NOT JUST 59:25 FOR COQ BIOSYNTHESIS, FOR THE TCA CYCLE, FOR PYRUVATE DEHIGH 59:35 JOJ NAIS AND WE'RE NOW PUTTING THEM TOGETHER IN THIS FRAMEWORK OF HOW THEY ALL RELATE TO ONE ANOTHER. SO I GUESS THAT REITERATES THE 59:41 POINT THAT ER MITOCHONDRIAL CONTACTS ARE REALLY SYSTEM 59:47 REGULATORS FOR ASSEMBLY, AND FOR DISTRIBUTION AND TO CREATE PATHWAYS INSIDE CELLS. 59:55 SO I'LL END THE WORK ON THE NUCLEOID I TOLD YOU ABOUT IN HUMAN CELLS IS DONE BY SAMANTHA LEWIS, THE WORK ONCOQ IS DONE BY 1:00:02 KELLY AND SAM WAS HELPED OUT BY A POSTDOC MARINA AND FORMER UNDERGRAD IN COLLABORATION WITH 1:00:09 MARIA FALKENBERG AND DAVE AND JOSH. SO HAPPY TO TAKE QUESTIONS. THANK YOU FOR YOUR ATTENTION. 1:00:14 [APPLAUSE] 1:00:28 >> THE FAES IS GOING TO HAVE A RECEPTION IN THE LIBRARY AFTER 1:00:33 THIS, BUT WE HAVE TIME FOR A FEW QUESTIONS. INTO URBAN 1:00:38 >> VERY, VERY FASCINATING TALK. >> THANK YOU. >> THANK YOU. 1:00:45 >> IT'S VERY CLOSE TO WHAT I'M DOING AS FAR AS BIOLOGY OF MITOCHONDRIA IN RELATION TO 1:00:54 IMMUNOBIOLOGY OF CANCER. MY QUESTION IS, OR MAYBE IT'S NOT FAIR TO ASK, BUT TO PROPOSE, 1:01:04 FOR EXAMPLE, IF THERE IS AN ACTIVATED CELL WHICH HAS A LOT 1:01:10 OF MITOCHONDRIA-LIKE MACROPHAGES, DO YOU SEE SOME 1:01:15 DIFFERENCES WHEN YOU ACTIVATE OR IN ARRESTING STATUS OF THE 1:01:24 IMMUNE CELLS LIKE MICROPHAGE M1M2, WHETHER THERE IS THE 1:01:32 CONNECTION BETWEEN ER AND MITOCHONDRIA WOULD CHANGE AND/OR 1:01:42 WHETHER THE CHRONIC STIMULATION WOULD DISTURB EVEN THE 1:01:47 REPLICATION OF MITOCHONDRIA? >> THAT'S A GREAT QUESTION. WE'VE ONLY WORKED UNDER THESE 1:01:53 HOMEOSTATIC CONDITIONS THAT I'VE DESCRIBED. I'LL JUST MAKE ONE SHORT COMMENT 1:01:59 ON THAT WHICH IS THAT THIS SYSTEM MIGHT BE TWEAKED FROM CELL TYPE TO CELL TYPE. 1:02:06 IF DIFFERENT THINGS BECOME RATE-LIMITING, THAT'S DEFINITELY A POSSIBILITY, BUT CERTAINLY ANY 1:02:13 PERTURBATION OF THE ERMITO CONTEXT I WOULD PREDICT WOULD HAVE PRETTY DRAMATIC EFFECTS ON 1:02:20 MITOCHONDRIAL DNA REPLICATION. >> VERY, VERY GOOD, GOOD SUBJECT. >> THANKS. >> THAT WAS BEAUTIFUL. 1:02:30 YOU SHOW ELIMINATING ER TUBULES WITH A CLIMP63 KNOCKOUT -- 1:02:35 >> OVER EXPRESSION. >> -- OVEREXPRESSION CAUSED -- WHAT DOES IT CAUSE, A DECREASE 1:02:41 IN MITOCHONDRIAL DNA REPLICATION. >> YES. >> SO IS IT UNIDIRECTIONAL FROM THE ER AND WHAT HAPPENS TO 1:02:47 DIVISION OR CONTACT SITES IN ROW ZERO CELLS? OR IN TWINKLE KNOCKOUT CELLS. 1:02:54 >> SO DON'T KNOW ABOUT TWINKLE 1:03:01 KNOCKOUTS YET. >> THAT MIGHT BE THE KEY, RIGHT? >> YES, WE'RE DOING THOSE EXPERIMENTS NOW. I CAN TELL YOU OUR PRELIMINARY 1:03:07 DATA SUGGESTS THE CONTACTS ARE STILL THERE ON THE TWINKLE CELLS. SOL I THINK IT'S, YOU KNOW, 1:03:13 KICKING THE CAN A LITTLE BIT, 1:03:18 BUT THERE'S SOMETHING AT THE VERY TOP OF THE CHAIN. >> LAST QUICK QUESTION BECAUSE IT'S CHANGED OVER THE YEARS. 1:03:25 CURRENTLY WHAT'S THE IDEA ON THE NUMBER OF DNA COPY NUMBERS PER NUCLEOID? >> SO I THINK, YOU KNOW, WE DID 1:03:33 SOME STEAD MICROSCOPY OURSELVES, WORK FROM AT LEAST TWO OTHER LABS, DAVID CLAYTON AND NILS 1:03:43 LARSON CONSISTENT WITH THAT, THESE CELLS IN CULTURE, THEY'RE SOLITARY. >> OKAY. 1:03:50 >> YEAH. THEY'RE SINGLE COPY. >> GREAT WORK. I WAS WONDERING IF WE COULD GET 1:03:56 YOUR THOUGHTS ON THE POTENTIAL CONTRIBUTION OF MECHANICAL STRAIN OR TENSION AT THE 1:04:01 MITOCHONDRIAL MEMBRANE THAT MIGHT BE DRIVING FISS ION, THIS 1:04:08 WOULD BE CONSISTENT WITH THE ER MODEL AND ACTIN MODEL AND POTENTIAL -- THE DENSITY OF 1:04:13 NUCLEOIDS ALSO CHANGING THE STREAM AT THE MEMBRANE THAT MIGHT DRIVE THAT, AND ALSO IS 1:04:19 THERE ANY EVIDENCE THAT A SIMILAR TYPE OF MODEL MIGHT BE 1:04:25 CONTRIBUTING TO THE PROTEIN -- FORMATION WHERE THERE'S POTENTIAL MEMBRANE SENSING 1:04:33 CAPABILITIES OF PROTEINS THAT MIGHT DRIVE -- FORMATION. >> WOW. 1:04:38 THE MOST RECENT WORK DOING THAT 1:04:44 BEAUTIFUL HIGH RESOLUTION ACTIN ASSEMBLIES, IT'S LIKE -- PURSE 1:04:51 STRINGS, RIGHT? THAT THAT MUST BE INVOSMED IN THAT 1:04:56 INITIAL CONSTRICTION STEP. WE HAVEN'T DONE ANY WORK OURSELVES RELATED TO THAT. BUT I DO LIKE THE IDEA -- I LIKE 1:05:03 SIMPLE MODELS BASED ON REALITY. I TELL MY STUDENTS AND POSTDOCS, I JUST WENT TO THE -- I'M A BIG 1:05:13 ASBC PERSON AND I SAID OH, MY GOD, I CAME BACK FROM THIS MEETING, YOU GO TO ASB, YOU 1:05:20 LEARN HOW THINGS WORK, YOU GO TO THE -- MEETINGS AND YOU LESH HOW THEY REALLY WORK AND THAT'S BECAUSE OF ALL THE PHYSICAL 1:05:26 PARAMETERS. WE'RE NOT DOING ANY OF THAT, BUT I THINK IT MUST PLAY A ROLE. LIKE, FOR EXAMPLE, WHY DOES 1:05:33 MOTILITY HAPPEN AFTER DIVISIN? IT MUST BE CLUSTERS OF MOTORS THAT ARE NEEDED TO GENERATE THE 1:05:38 FORCE, THINGS LIKE THAT. SO I CAN'T REALLY -- >> GREAT. 1:05:43 THANK YOU. >> SO I'M AFRAID WE'RE GOING TO 1:05:48 HAVE TO CLOSE THE SESSION, AND TAKE UP THE QUESTIONS ONE ON ONE IN THE LIBRARY. 1:05:53 THANK YOU. NIH VideoCast 41.2K subscribers Videos About