1 00:00:06,374 --> 00:00:08,342 >> HEY, EVERYONE. 2 00:00:08,342 --> 00:00:10,311 MY NAME IS YOMI. 3 00:00:10,311 --> 00:00:17,218 I'M A POST-BACC AT THE NIDIC, 4 00:00:17,218 --> 00:00:19,086 IT'S MY PLEASURE TO INTRODUCE 5 00:00:19,086 --> 00:00:21,923 TODAY'S SPEAKER, LING GANG WU, 6 00:00:21,923 --> 00:00:25,126 COMPLETED MD FROM SECOND CAN 7 00:00:25,126 --> 00:00:26,961 COLLEGE FROM SHANGHAI BEFORE 8 00:00:26,961 --> 00:00:30,298 MOVING TO TEXAS AND WAS AT 9 00:00:30,298 --> 00:00:32,567 BAYLOR UNIVERSITY OF MEDICINE, 10 00:00:32,567 --> 00:00:33,868 STUDYING PRESYNAPTIC CALCIUM 11 00:00:33,868 --> 00:00:35,603 CHANNELS AND NEUROTRANSMITTER 12 00:00:35,603 --> 00:00:39,206 RELEASE, WENT ON TO DO TWO 13 00:00:39,206 --> 00:00:40,841 POST-DOC, FIRST AT THE 14 00:00:40,841 --> 00:00:42,543 UNIVERSITY OF COLORADO SCHOOL OF 15 00:00:42,543 --> 00:00:44,011 MEDICINE, WHERE HE STUDIED 16 00:00:44,011 --> 00:00:46,647 KINETICS OF VARIOUS STEPS IN 17 00:00:46,647 --> 00:00:49,617 SYNAPTIC VESICLE RECYCLING. 18 00:00:49,617 --> 00:00:52,987 FIRST ONE IN HEIDELBERG GERMANY, 19 00:00:52,987 --> 00:00:54,221 COMBINED BACKGROUND TO 20 00:00:54,221 --> 00:00:56,390 INVESTIGATE MECHANISMS BEFORE 21 00:00:56,390 --> 00:00:58,192 VESICLE RELEASE AND SHORT-TERM 22 00:00:58,192 --> 00:00:59,026 SYNAPTIC PLASTICITY. 23 00:00:59,026 --> 00:01:02,229 AFTER FOUR YEARS AS AN ASSISTANT 24 00:01:02,229 --> 00:01:04,432 PROFESSOR AT W WASH U, STARTED 25 00:01:04,432 --> 00:01:07,702 HIS OWN LAB AT THE NINDS AND HAS 26 00:01:07,702 --> 00:01:11,205 BEEN HERE FOR 20 YEARS. 27 00:01:11,205 --> 00:01:14,041 ENDOAND EXOCYTOSIS AND UNCOVERED 28 00:01:14,041 --> 00:01:15,343 FUNDAMENTAL MECHANISMS BEHIND 29 00:01:15,343 --> 00:01:16,344 BUDDING AND FUSION AND THEIR 30 00:01:16,344 --> 00:01:18,012 RELATION TO VESICLE RELEASE. 31 00:01:18,012 --> 00:01:19,380 FURTHER HIS LAB USES STATE OF 32 00:01:19,380 --> 00:01:21,515 THE ART IMAGING APPROACHES TO 33 00:01:21,515 --> 00:01:23,684 STUDY THE FUSION PORE, A 34 00:01:23,684 --> 00:01:26,320 STRUCTURE FORMED DURING 35 00:01:26,320 --> 00:01:26,621 EXOCYTOSIS. 36 00:01:26,621 --> 00:01:28,522 JOIN ME IN WELCOMING DR. WU TO 37 00:01:28,522 --> 00:01:29,056 THE FRONT. 38 00:01:29,056 --> 00:01:31,459 THANK YOU. 39 00:01:31,459 --> 00:01:37,598 [APPLAUSE] 40 00:01:37,598 --> 00:01:41,002 >> THANK YOU FOR YOUR GREAT 41 00:01:41,002 --> 00:01:42,737 INTRODUCTION, YOMI. 42 00:01:42,737 --> 00:01:44,572 IT'S MY GREAT PLEASURE AND HONOR 43 00:01:44,572 --> 00:01:45,906 TO BE HERE. 44 00:01:45,906 --> 00:01:51,145 MY FIRST TIME, AS YOMI SAID, IN 45 00:01:51,145 --> 00:01:54,448 THE LAST 20 YEARS OF MY LIFE, 46 00:01:54,448 --> 00:01:59,587 AND AS YOU CAN SEE HERE, IN THIS 47 00:01:59,587 --> 00:02:03,758 CERTIFICATE AWARDED BY OUR 48 00:02:03,758 --> 00:02:08,796 INSTITUTE, 20 YEARS HERE. 49 00:02:08,796 --> 00:02:11,298 A GOOD LIFE HERE AS WELL. 50 00:02:11,298 --> 00:02:13,734 I'M GETTING OLD NOW. 51 00:02:13,734 --> 00:02:16,671 SO I'M GOING TO SHOW YOU IN THIS 52 00:02:16,671 --> 00:02:21,609 20 YEARS, OUR JOURNEY TOWARDS 53 00:02:21,609 --> 00:02:28,049 METHYLIZING THE VESICLE FUSION. 54 00:02:28,049 --> 00:02:29,383 VISUALIZATION, I CAN'T HELP BUT 55 00:02:29,383 --> 00:02:31,652 SHOW YOU SOME OF MY OWN DATD 56 00:02:31,652 --> 00:02:33,587 COLLECTIONS, NOT IN THE LAB, BUT 57 00:02:33,587 --> 00:02:34,922 IN THE NEIGHBORHOOD. 58 00:02:34,922 --> 00:02:36,590 JUST A BIT OF FUN BEFORE WE GET 59 00:02:36,590 --> 00:02:36,824 SERIOUS. 60 00:02:36,824 --> 00:02:37,792 SO HERE WE GO. 61 00:02:37,792 --> 00:02:42,596 I SEE A BIG VESICLE HERE WITH 62 00:02:42,596 --> 00:02:46,867 SOME STRUCTURES THAT I'M SURE 63 00:02:46,867 --> 00:02:49,937 YOU'RE FAMILIAR WITH, AND A 64 00:02:49,937 --> 00:02:53,841 SWOLLEN VESICLE AND A BEAUTIFUL 65 00:02:53,841 --> 00:02:56,577 EVENING ON THE HARBOR HERE, A 66 00:02:56,577 --> 00:02:59,547 DYNAMIC FLOW OF THE MEMBRANE IN 67 00:02:59,547 --> 00:03:02,283 THIS BEAUTIFUL PARK, 20 MINUTES 68 00:03:02,283 --> 00:03:03,851 AWAY FROM HERE. 69 00:03:03,851 --> 00:03:05,586 SO IF YOU'VE NEVER BEEN HERE IN 70 00:03:05,586 --> 00:03:07,154 THIS PARK, I WOULD SUGGEST YOU 71 00:03:07,154 --> 00:03:08,089 TO GO. 72 00:03:08,089 --> 00:03:09,190 BUT NOT RIGHT NOW. 73 00:03:09,190 --> 00:03:14,228 AFTER MY TALK, PLEASE. 74 00:03:14,228 --> 00:03:16,831 YES, MORE OF THE MEMBRANE 75 00:03:16,831 --> 00:03:18,165 DYNAMICS IN THE RESTING 76 00:03:18,165 --> 00:03:20,134 CONDITIONS BEFORE THE SUNRISE, 77 00:03:20,134 --> 00:03:23,771 AND ALSO IMAGES, LANDSCAPES AND 78 00:03:23,771 --> 00:03:24,004 ANIMALS. 79 00:03:24,004 --> 00:03:25,740 HERE ARE THE MOST BEAUTIFUL 80 00:03:25,740 --> 00:03:30,144 PICTURES I'VE EVER SEEN, 81 00:03:30,144 --> 00:03:35,382 BEAUTIFUL MOUNT AN BIRDS 82 00:03:35,382 --> 00:03:40,087 CROSSING THE RIVER BEFORE THE 83 00:03:40,087 --> 00:03:40,321 SUNRISE. 84 00:03:40,321 --> 00:03:42,823 THIS IS THE LAST BOVINE. 85 00:03:42,823 --> 00:03:46,761 WHILE I'M HAVING FUN IMAGING THE 86 00:03:46,761 --> 00:03:49,196 ANIMALS IN MY LAB IS IMAGING 87 00:03:49,196 --> 00:03:52,800 CELLS FROM ANIMALS, PARTICULARLY 88 00:03:52,800 --> 00:03:53,033 BOVINE. 89 00:03:53,033 --> 00:03:57,505 THIS IS A CELL, BOVINE CELLS 90 00:03:57,505 --> 00:03:59,240 CONTAINING MULTIPLE VESICLES 91 00:03:59,240 --> 00:03:59,573 HERE. 92 00:03:59,573 --> 00:04:02,109 THESE VESICLES UNDERGO FUSION 93 00:04:02,109 --> 00:04:05,613 AND BUTTON, SO THAT'S THE -- AND 94 00:04:05,613 --> 00:04:05,946 BUDDING. 95 00:04:05,946 --> 00:04:07,815 THAT'S THE TOPIC OF THE DAY. 96 00:04:07,815 --> 00:04:10,551 AS YOU KNOW, VESICLE FUSION AND 97 00:04:10,551 --> 00:04:12,386 BUDDING IS INVOLVED IN MANY 98 00:04:12,386 --> 00:04:14,488 PROCESS VERY FUNDAMENTAL FOR 99 00:04:14,488 --> 00:04:18,292 EVERY CELL LIFE, FROM THE ESPN 100 00:04:18,292 --> 00:04:22,363 DOUGH PALACE MECHANIC RETICULUM 101 00:04:22,363 --> 00:04:26,333 AND BACK TO THE ENDOSOMES AND 102 00:04:26,333 --> 00:04:26,600 LYSOSOMES. 103 00:04:26,600 --> 00:04:29,837 THEY ARE INVOLVED IN THE GLUCOSE 104 00:04:29,837 --> 00:04:31,405 LEVEL RELATED TO DIABETES, THEY 105 00:04:31,405 --> 00:04:34,842 ARE INVOLVED IN MEDIATING VIRAL 106 00:04:34,842 --> 00:04:36,277 INFECTION INCLUDING THE 107 00:04:36,277 --> 00:04:38,012 CORONAVIRUS INFECTION THAT 108 00:04:38,012 --> 00:04:40,147 CAUSED THE COVID-19 PANDEMIC. 109 00:04:40,147 --> 00:04:42,383 THEY ARE ALSO INVOLVED IN 110 00:04:42,383 --> 00:04:44,885 MEDIATING SYNAPTIC TRANSMISSION 111 00:04:44,885 --> 00:04:48,189 BY EXOCYTOSIS AND ENDOCYTOSIS. 112 00:04:48,189 --> 00:04:49,957 EXOCYTOSIS RELEASES TRANSMITTER 113 00:04:49,957 --> 00:04:52,660 TO ACTIVATE THE TRANGS MITTER 114 00:04:52,660 --> 00:04:55,596 REACCEPT -- TRANSMITTER 115 00:04:55,596 --> 00:04:58,232 RECEPTORS, AND AFTER THE 116 00:04:58,232 --> 00:04:59,567 EXOCYTOSIS, ENDOCYTOSIS, 117 00:04:59,567 --> 00:05:01,435 RECYCLES VESICLE TO SUSTAIN 118 00:05:01,435 --> 00:05:02,503 SYNAPTIC TRANSMISSION. 119 00:05:02,503 --> 00:05:05,239 THIS IS IMPORTANT FOR OUR BRAIN 120 00:05:05,239 --> 00:05:07,007 FUNCTIONS LIKE MY TALK RIGHT 121 00:05:07,007 --> 00:05:07,341 NOW. 122 00:05:07,341 --> 00:05:11,378 THE SYNAPSE IS THE MOST USED FOR 123 00:05:11,378 --> 00:05:13,447 THE STUDY OF FUSION AND BUDDING. 124 00:05:13,447 --> 00:05:21,121 BACK IN 1970S AND 1980S, JIM AND 125 00:05:21,121 --> 00:05:24,725 TOM FROM NIH DISCOVERED THESE 126 00:05:24,725 --> 00:05:28,362 STRUCTURES HERE, THEREFORE THEY 127 00:05:28,362 --> 00:05:33,267 PROPOSED FRR FUSIONS INVOLVED IN 128 00:05:33,267 --> 00:05:34,802 DILATION OF THE FUSION PORE, AND 129 00:05:34,802 --> 00:05:38,072 HERE THE VESICLE IS FLATTENED AT 130 00:05:38,072 --> 00:05:39,073 THE MEMBRANE. 131 00:05:39,073 --> 00:05:42,977 AFTER THIS THEY FOUND THIS 132 00:05:42,977 --> 00:05:44,545 INVOLVING THE ENDOCYTOSIS THAT 133 00:05:44,545 --> 00:05:48,349 REFORMS THE VESICLES FORESEE 134 00:05:48,349 --> 00:05:50,317 CYCLING VESICLES, CLASSICAL 135 00:05:50,317 --> 00:05:53,821 PROCESS, INVOLVING ALMOST THE 136 00:05:53,821 --> 00:05:57,992 REVERSE PROCESS. 137 00:05:57,992 --> 00:06:01,829 AT ABOUT THE SAME TIME, 138 00:06:01,829 --> 00:06:05,099 PERFORMED THE FORM OF EXOCYTOSIS 139 00:06:05,099 --> 00:06:07,067 AND ENDOCYTOSIS BASED ON 140 00:06:07,067 --> 00:06:09,670 OBSERVATION ONLY OF THE PROFILE 141 00:06:09,670 --> 00:06:10,204 THERE. 142 00:06:10,204 --> 00:06:12,973 SO THE FUSION FOR OPTIMUM CLOSE 143 00:06:12,973 --> 00:06:17,578 VERY RAPIDLY, PORE VERY OFTEN 144 00:06:17,578 --> 00:06:19,647 INTERPRETED AS SMALL PORE, 145 00:06:19,647 --> 00:06:24,718 RECYCLES VESICLES REALLY FAST. 146 00:06:24,718 --> 00:06:27,021 SO THAT WAS IN THE 1970S, AND 147 00:06:27,021 --> 00:06:30,791 TWO DECADES PASSED, FUSION HAD 148 00:06:30,791 --> 00:06:34,561 BEEN DEBATED FOR A LONG TIME, 149 00:06:34,561 --> 00:06:37,731 BUT IT WAS FOLLOWED BY 150 00:06:37,731 --> 00:06:43,203 ENDOCYTOSIS HAS BEEN VERY WELL 151 00:06:43,203 --> 00:06:43,570 RESEPTEMBERRED. 152 00:06:43,570 --> 00:06:46,807 AT THE END OF THE 1990S, WE LIKE 153 00:06:46,807 --> 00:06:48,409 TO STUDIED, DETERMINE WHETHER WE 154 00:06:48,409 --> 00:06:51,278 CAN SEE THEM IN THE LIVE CELLS, 155 00:06:51,278 --> 00:06:53,814 AND IF SO, THEIR UNDERLYING 156 00:06:53,814 --> 00:06:54,682 MECHANISMS AND FUNCTIONS. 157 00:06:54,682 --> 00:06:56,016 SO THESE ARE THE THREE QUESTIONS 158 00:06:56,016 --> 00:06:59,353 THAT WE WOULD LIKE TO ADDRESS. 159 00:06:59,353 --> 00:07:02,256 AT THE END OF THE 19 THE 0S, THE 160 00:07:02,256 --> 00:07:03,657 SUPER MICROSCOPE HAS NOT BEEN 161 00:07:03,657 --> 00:07:09,363 BORN YET, SO WE USE THE 162 00:07:09,363 --> 00:07:13,767 ELECTROSET UP, WE MEASURE THE 163 00:07:13,767 --> 00:07:14,535 CAPACITORS PROPORTION TO 164 00:07:14,535 --> 00:07:18,806 MEMBRANE AREA. 165 00:07:18,806 --> 00:07:22,076 THE MEMBRANE AREAS INCREASE, 166 00:07:22,076 --> 00:07:26,146 THEREFORE CAPACITANCE INCREASES. 167 00:07:26,146 --> 00:07:27,781 SOS DECAY MEANING ENDOCYTOSIS, 168 00:07:27,781 --> 00:07:30,050 WE CAN LOOK AT THE DECAY TIME TO 169 00:07:30,050 --> 00:07:33,153 EXAMINE THE TIME COURSE OF THE 170 00:07:33,153 --> 00:07:33,454 ENDOCYTOSIS. 171 00:07:33,454 --> 00:07:35,889 TO DO THE CAPACITANCE 172 00:07:35,889 --> 00:07:41,028 MEASUREMENTS, WE USE VERY LARGE 173 00:07:41,028 --> 00:07:44,732 PREPARATION IN THE BRAINSTEM, 174 00:07:44,732 --> 00:07:46,467 THE PRESYNAPTIC CELLS AND 175 00:07:46,467 --> 00:07:47,368 POSTSYNAPTIC CELLS. 176 00:07:47,368 --> 00:07:50,337 MY LAB'S PROJECT IS TO ESTABLISH 177 00:07:50,337 --> 00:07:51,305 THE CENTRAL SYNAPSE. 178 00:07:51,305 --> 00:07:53,207 WHAT WE SEE HERE IS THE 179 00:07:53,207 --> 00:07:54,475 CAPACITANCE INCREASE THAT 180 00:07:54,475 --> 00:07:57,344 CORRESPONDS TO THE RECORDED 181 00:07:57,344 --> 00:08:00,547 SIMULTANEOUSLY AT THE SANE 182 00:08:00,547 --> 00:08:00,848 SYNAPSE. 183 00:08:00,848 --> 00:08:04,151 THIS MEANS ARTIFACTS REFLECTS 184 00:08:04,151 --> 00:08:04,551 EXOCYTOSIS. 185 00:08:04,551 --> 00:08:05,986 THEN WE CAN EXAMINE THE TIME 186 00:08:05,986 --> 00:08:08,522 COURSE OF THE ENDOCYTOSIS 187 00:08:08,522 --> 00:08:10,157 BECAUSE INCREASE MEANS 188 00:08:10,157 --> 00:08:10,524 EXOCYTOSIS. 189 00:08:10,524 --> 00:08:12,459 WE SEE THE SLOW ONES AND FAST 190 00:08:12,459 --> 00:08:14,328 ONES TAKING A FEW SECONDS, ONE, 191 00:08:14,328 --> 00:08:17,064 TWO, THREE SECONDS HERE TO HAVE 192 00:08:17,064 --> 00:08:17,364 ENDOCYTOSIS. 193 00:08:17,364 --> 00:08:18,399 AT THE TIME WE COULD INTERPRET 194 00:08:18,399 --> 00:08:21,468 THAT AS THE KISS AND RUN, 195 00:08:21,468 --> 00:08:22,336 BECAUSE IT'S THOUGHT TO BE 196 00:08:22,336 --> 00:08:23,670 REALLY FAST. 197 00:08:23,670 --> 00:08:24,838 ALTERNATIVELY, MOST PEOPLE WOULD 198 00:08:24,838 --> 00:08:26,807 ARGUE THAT THIS COULD STILL BE 199 00:08:26,807 --> 00:08:28,375 VERY FAST TO RUN TRANSITIONS 200 00:08:28,375 --> 00:08:28,675 HERE. 201 00:08:28,675 --> 00:08:29,910 SO WE DON'T KNOW. 202 00:08:29,910 --> 00:08:33,514 SO HERE, IT'S A WHOLE CELL 203 00:08:33,514 --> 00:08:37,684 RECORDING, FROM 10,000 OR 20,000 204 00:08:37,684 --> 00:08:38,852 VESICLE EXOCYTOSIS. 205 00:08:38,852 --> 00:08:41,622 IN ORDER TO SEE THE SINGLE 206 00:08:41,622 --> 00:08:44,224 VESICLE FUSION, WE RECORD THE 207 00:08:44,224 --> 00:08:47,094 MEDIATOR FROM POSTSYNAPTIC CELL, 208 00:08:47,094 --> 00:08:50,464 AND CALL THE POSTSYNAPTIC 209 00:08:50,464 --> 00:08:51,665 CAPACITANCE AND HOPE TO SEE THIS 210 00:08:51,665 --> 00:08:53,534 INCREASE IN DECAY, BUT THE 211 00:08:53,534 --> 00:08:55,002 SIGNAL NOISE IS TOO SMALL TO WE 212 00:08:55,002 --> 00:08:59,440 HAVE TO AVERAGE ABOUT 320,000, 213 00:08:59,440 --> 00:09:02,276 THIS FORMAL POST-DOC, HE'S 214 00:09:02,276 --> 00:09:05,779 PROBABLY HERE, RUNNING ALSO TO 215 00:09:05,779 --> 00:09:09,383 GET TO THIS, WE SEE VERY TINY 216 00:09:09,383 --> 00:09:13,320 SIGNAL, THE CAPACITANCE 217 00:09:13,320 --> 00:09:14,421 INCREASE, 10,000 TIMES LESS THAN 218 00:09:14,421 --> 00:09:19,026 THE SCALE HERE, TO INDICATE 219 00:09:19,026 --> 00:09:20,327 EXO AND ENDOCYTOSIS, BUT THIS IS 220 00:09:20,327 --> 00:09:21,428 TOO LABORIOUS. 221 00:09:21,428 --> 00:09:22,796 IF WE WANT TO CONTINUE FURTHER, 222 00:09:22,796 --> 00:09:24,598 WE HAVE TO RECORD FROM SINGLE 223 00:09:24,598 --> 00:09:26,133 TRACE, AND TO DO THAT, WE HAVE 224 00:09:26,133 --> 00:09:28,435 TO DO THE CELL TEST RECORDINGS 225 00:09:28,435 --> 00:09:30,170 AND ASKING THE VESICLES TO FUSE 226 00:09:30,170 --> 00:09:31,538 ON THE TEST MEMBRANE. 227 00:09:31,538 --> 00:09:35,008 TO DO THAT, WE THEN USE A LARGE 228 00:09:35,008 --> 00:09:36,643 PIPETTE TO PULL AWAY THE 229 00:09:36,643 --> 00:09:37,945 POSTSYNAPTIC CELLS AND THEN USE 230 00:09:37,945 --> 00:09:40,814 A SMALLER PIPETTE TO PASS ON THE 231 00:09:40,814 --> 00:09:44,418 RELEASE PHASE OF THE NERVE 232 00:09:44,418 --> 00:09:44,818 TERMINAL. 233 00:09:44,818 --> 00:09:46,587 UNDER THIS CONDITION WE CAN SEE 234 00:09:46,587 --> 00:09:50,424 SINGLE CHANNEL CALCIUM CODONS IN 235 00:09:50,424 --> 00:09:52,326 THIS CASE AT THE TERMINALS AND 236 00:09:52,326 --> 00:09:54,495 WE CAN ALSO SEE THE SINGLE 237 00:09:54,495 --> 00:09:55,896 VESICLE FUSION THAT'S SHOWN HERE 238 00:09:55,896 --> 00:09:58,198 IN THIS CAPACITANCE INCREASE AND 239 00:09:58,198 --> 00:10:01,268 DECREASE, THAT IS ACCOMPANIED BY 240 00:10:01,268 --> 00:10:04,004 THE FUSION CONDUCTOR INCREASE IN 241 00:10:04,004 --> 00:10:04,438 DECAY. 242 00:10:04,438 --> 00:10:05,606 THIS PROVIDES STRONG EVIDENCE 243 00:10:05,606 --> 00:10:10,077 FOR THE KISS AND RUN, AT THE 244 00:10:10,077 --> 00:10:10,310 SYNAPSE. 245 00:10:10,310 --> 00:10:11,311 AT THE SAME TIME WE ALSO SEE 246 00:10:11,311 --> 00:10:13,447 VERY OFTEN THE CONDUCTANCE 247 00:10:13,447 --> 00:10:15,549 INCREASE TO BEYOND OUR 248 00:10:15,549 --> 00:10:16,617 DETECTION. 249 00:10:16,617 --> 00:10:17,518 THAT'S CONSISTENT. 250 00:10:17,518 --> 00:10:20,787 IF IT'S NOT ACCOMPANIED BY THE 251 00:10:20,787 --> 00:10:22,055 CAPACITANCE DECAY HERE. 252 00:10:22,055 --> 00:10:25,159 AND SOMETIMES, WE ALSO SEE THIS 253 00:10:25,159 --> 00:10:29,563 GIANT VESICLE FUSIONS WHICH 254 00:10:29,563 --> 00:10:31,732 CAUSES THE LAST CAPACITANCE 255 00:10:31,732 --> 00:10:34,902 INCREASE SHOWN HERE, THE SCALE 256 00:10:34,902 --> 00:10:37,437 IS ABOUT 20 TIMES LARGER THAN 257 00:10:37,437 --> 00:10:39,606 HERE, THAT'S BECAUSE OF THE 258 00:10:39,606 --> 00:10:40,607 VESICLE-VESICLE FUSION THAT 259 00:10:40,607 --> 00:10:43,243 CAUSES A COMPOUND VESICLE, AND 260 00:10:43,243 --> 00:10:45,712 THIS UNDERGOES COMPOUND 261 00:10:45,712 --> 00:10:46,013 EXOCYTOSIS. 262 00:10:46,013 --> 00:10:48,682 WE CAN SEE THOSE IN THE ELECTRON 263 00:10:48,682 --> 00:10:49,683 MICROSCOPE. 264 00:10:49,683 --> 00:10:53,187 ACTUALLY, EARLY IN TIME, ALSO 265 00:10:53,187 --> 00:10:56,456 SEES THIS GIANT VESICLE AFTER 266 00:10:56,456 --> 00:11:02,396 THE INTENSIVE STIMULATION. 267 00:11:02,396 --> 00:11:03,130 ALL RIGHT. 268 00:11:03,130 --> 00:11:04,798 SO I SPENT TEN YEARS, MORE THAN 269 00:11:04,798 --> 00:11:07,534 TEN YEARS IN DOING MOST 270 00:11:07,534 --> 00:11:08,835 DIFFICULT PHYSIOLOGICAL 271 00:11:08,835 --> 00:11:09,603 EXPERIMENTS, 272 00:11:09,603 --> 00:11:10,037 ELECTROPHYSIOLOGICAL 273 00:11:10,037 --> 00:11:13,674 EXPERIMENTS, IN MY OPINION. 274 00:11:13,674 --> 00:11:16,877 STILL IT'S AN ESTIMATE BASED ON 275 00:11:16,877 --> 00:11:19,947 THE FUSION CONDUCTANCE WHICH IS 276 00:11:19,947 --> 00:11:23,350 LIMITED TO ABOUT FIVE NANOMETERS 277 00:11:23,350 --> 00:11:25,419 SO STILL THE ASPECT HERE, 278 00:11:25,419 --> 00:11:27,921 ANYTHING BEYOND FIVE NANOMETERS 279 00:11:27,921 --> 00:11:30,457 BELONGS TO SPECULATION. 280 00:11:30,457 --> 00:11:35,395 AT THE TIME THE STEP MICROSCOPE, 281 00:11:35,395 --> 00:11:37,764 ABOUT 60 TO 80 NANOMETERS EVERY 282 00:11:37,764 --> 00:11:41,602 100 SECONDS OR SO, BECOMING 283 00:11:41,602 --> 00:11:41,868 AVAILABLE. 284 00:11:41,868 --> 00:11:43,937 THIS IS STILL INSUFFICIENT TO 285 00:11:43,937 --> 00:11:46,273 RESOLVE THE VESICLE FUSIONS, 286 00:11:46,273 --> 00:11:48,775 SYNAPTIC VESICLE, BECAUSE THE 287 00:11:48,775 --> 00:11:50,310 SYNAPTIC VESICLE IS ABOUT 288 00:11:50,310 --> 00:11:51,178 30 NANOMETERS OR SO. 289 00:11:51,178 --> 00:11:55,449 SO WE HAD TO USE DIFFERENT, 290 00:11:55,449 --> 00:11:58,385 BOVINE CELL, AND THIS IS A 291 00:11:58,385 --> 00:11:59,720 NEUROENDOCRINE CELL SIMILAR TO 292 00:11:59,720 --> 00:12:03,991 THE NEURONS IN MANY PROPERTIES. 293 00:12:03,991 --> 00:12:06,293 MORE IMPORTANTLY, IT HAS VERY 294 00:12:06,293 --> 00:12:09,329 LARGE VESICLES, 300-NANOMETER 295 00:12:09,329 --> 00:12:10,764 VESICLES THAT WE CAN SEE IF THEY 296 00:12:10,764 --> 00:12:13,166 ARE UNDERGOING THE KISS AND RUN 297 00:12:13,166 --> 00:12:17,771 OR THE FULL CLASS FUSIONS. 298 00:12:17,771 --> 00:12:19,973 SO THE CELLS ON THE COVER SLIPS 299 00:12:19,973 --> 00:12:24,444 AND ADD DYE ON 647 OR 532 IN THE 300 00:12:24,444 --> 00:12:27,648 BATH SOLUTION, AND YOU CAN SEE 301 00:12:27,648 --> 00:12:30,017 THE DARK AREA HERE. 302 00:12:30,017 --> 00:12:34,655 THIS IS THE CELL FOOTPRINT, THE 303 00:12:34,655 --> 00:12:36,056 FROZEN SOLUTION, IT'S BETWEEN 304 00:12:36,056 --> 00:12:40,227 THE CELL MEMBRANE AND THE COVER 305 00:12:40,227 --> 00:12:40,527 SLIPS. 306 00:12:40,527 --> 00:12:43,297 WHEN VESICLE FUSION OCCURS, THIS 307 00:12:43,297 --> 00:12:47,467 FLUORESCENT DYE WILL DIFFUSE 308 00:12:47,467 --> 00:12:48,769 INTO THE FUSION SPOTS. 309 00:12:48,769 --> 00:12:49,836 YOU'LL SEE SPOTS. 310 00:12:49,836 --> 00:12:52,806 WE GIVE ONE SECOND 311 00:12:52,806 --> 00:12:54,808 DEPOLARIZATION THAT DECAY IS 312 00:12:54,808 --> 00:12:57,611 INDICATED IN EXOCYTOSIS AND 313 00:12:57,611 --> 00:12:58,078 ENDOCYTOSIS. 314 00:12:58,078 --> 00:12:59,746 I'LL PLAY A VIDEO OF THE PROCESS 315 00:12:59,746 --> 00:13:01,481 TO SEE AFTER THE DEPOLARIZATION 316 00:13:01,481 --> 00:13:03,750 YOU'LL SEE AN APPEARANCE OF THE 317 00:13:03,750 --> 00:13:07,888 SPOTS HERE. 318 00:13:07,888 --> 00:13:08,789 DEPOLARIZATION RIGHT NOW. 319 00:13:08,789 --> 00:13:10,657 SO YOU SEE THE SPOTS COMING 320 00:13:10,657 --> 00:13:12,059 HERE, AND THAT MEANS THAT WE CAN 321 00:13:12,059 --> 00:13:13,694 SEE THE FUSION IN THE BOTTOM OF 322 00:13:13,694 --> 00:13:14,695 THE CELL. 323 00:13:14,695 --> 00:13:17,097 NOW, TO SEE THE MEMBRANE PROFILE 324 00:13:17,097 --> 00:13:24,304 CHANGES, WE OVEREXPRESS THE PS 325 00:13:24,304 --> 00:13:29,242 DOMAIN, AATTACHED -- THIS WAS 326 00:13:29,242 --> 00:13:31,812 DEVELOPED BY THOMAS HERE AT NIH. 327 00:13:31,812 --> 00:13:33,947 IT BINDS TO THE INNER LEAFLET OF 328 00:13:33,947 --> 00:13:38,618 THE PLASMA MEMBRANE. 329 00:13:38,618 --> 00:13:41,621 THEN THE XY PLANE, WHICH MEANS 330 00:13:41,621 --> 00:13:43,123 IT'S PARALLEL TO THE SCREEN 331 00:13:43,123 --> 00:13:47,427 HERE, SO YOU SEE HERE, THE 332 00:13:47,427 --> 00:13:49,629 STRETCH OF THE MEMBRANE AND IF 333 00:13:49,629 --> 00:13:54,968 YOU LABEL THE VESICLE, 334 00:13:54,968 --> 00:13:56,269 NEUROTRANSMITTER, WE SEE THE 335 00:13:56,269 --> 00:13:59,673 SPOTS ABOUT TO DOCK AT THE 336 00:13:59,673 --> 00:14:00,474 PLASMA MEMBRANE. 337 00:14:00,474 --> 00:14:04,277 WHEN THESE VESICLES FUSE, THE 338 00:14:04,277 --> 00:14:07,114 GREEN CAN FUSE INTO THE FUSION 339 00:14:07,114 --> 00:14:08,215 VESICLE AND THEREFORE TO LABEL 340 00:14:08,215 --> 00:14:09,316 THE FUSION VESICLE. 341 00:14:09,316 --> 00:14:11,284 IN OUR EXPERIMENTS, NORMALLY WE 342 00:14:11,284 --> 00:14:14,654 WOULD ADD A DYE OUTSIDE, IN THIS 343 00:14:14,654 --> 00:14:17,958 CASE 532 AND WE HAD THE GREEN 344 00:14:17,958 --> 00:14:19,926 MEMBRANE LABEL, SO HERE IT'S A 345 00:14:19,926 --> 00:14:22,562 STRETCH OF MEMBRANE HERE, 346 00:14:22,562 --> 00:14:23,630 UNDERNEATH THIS MEMBRANE IS A 347 00:14:23,630 --> 00:14:29,870 THIN LAYER OF THE DYE HERE IN 348 00:14:29,870 --> 00:14:34,474 THE XZ PLANE OF THE MICROSCOPE, 349 00:14:34,474 --> 00:14:36,877 AND YOU'LL SEE THE IMAGING UNDER 350 00:14:36,877 --> 00:14:39,279 THE COVER SLIP. 351 00:14:39,279 --> 00:14:42,149 SO DEPOLARIZATION CAUSES EXO AND 352 00:14:42,149 --> 00:14:44,751 ENDOCYTOSIS, AND WE SEE THE 353 00:14:44,751 --> 00:14:46,586 DIFFUSION OF THE GREEN INTO THE 354 00:14:46,586 --> 00:14:48,021 FUSION VESICLES AS WELL AS THE 355 00:14:48,021 --> 00:14:50,557 DYE THAT GETS INTO THE CAVITY OF 356 00:14:50,557 --> 00:14:52,626 THE VESICLES. 357 00:14:52,626 --> 00:14:54,895 SO HERE IS A REALTIME VIDEO OF 358 00:14:54,895 --> 00:14:56,897 THIS PROCESS. 359 00:14:56,897 --> 00:14:58,331 AFTER DEPOLARIZATION, YOU'LL SEE 360 00:14:58,331 --> 00:15:00,734 THIS APPEARANCE HERE, WE SEE 361 00:15:00,734 --> 00:15:02,369 THESE THREE FUSION EVENTS. 362 00:15:02,369 --> 00:15:04,237 SOMETIMES WE SEE THE GREEN 363 00:15:04,237 --> 00:15:05,539 SIGNALS FOLLOWED BY THE RED 364 00:15:05,539 --> 00:15:06,973 SIGNAL AS SHOWN HERE IN THIS 365 00:15:06,973 --> 00:15:08,275 REALTIME VIDEO. 366 00:15:08,275 --> 00:15:13,914 GREEN FIRST. 367 00:15:13,914 --> 00:15:16,049 AND HAD RED COMING LATER. 368 00:15:16,049 --> 00:15:17,651 THAT'S BECAUSE OF THE HEMIFUSION 369 00:15:17,651 --> 00:15:19,553 THAT ALLOWS FOR THE GREEN 370 00:15:19,553 --> 00:15:20,520 SIGNALS, MEMBRANES YOU SHOULD 371 00:15:20,520 --> 00:15:22,255 NEET THE MEMBRANES TO DIFFUSE IN 372 00:15:22,255 --> 00:15:24,591 AND YET BECAUSE THE PORE HAS NOT 373 00:15:24,591 --> 00:15:26,326 BEEN OPENED SO THE DYE CANNOT 374 00:15:26,326 --> 00:15:28,728 PENETRATE INTO THIS HEMIFUSION 375 00:15:28,728 --> 00:15:29,262 VESICLES. 376 00:15:29,262 --> 00:15:30,797 AFTER THE FULL FUSION, THEN THE 377 00:15:30,797 --> 00:15:32,999 DYE CAN PENETRATE, YOU SEE THE 378 00:15:32,999 --> 00:15:35,602 RED DYE APPEAR IN THE VESICLE. 379 00:15:35,602 --> 00:15:37,137 SOMETIMES WE'LL SEE THE GREENER 380 00:15:37,137 --> 00:15:38,772 SIGNALS FIRST AND WITHOUT SEEING 381 00:15:38,772 --> 00:15:40,340 THE RED SIGNALS, AS SHOWN HERE 382 00:15:40,340 --> 00:15:41,541 IN THIS VIDEO. 383 00:15:41,541 --> 00:15:44,444 GREEN SIGNALS FIRST, HEMIFUSION 384 00:15:44,444 --> 00:15:45,579 WITHOUT GOING THROUGH THE FULL 385 00:15:45,579 --> 00:15:46,546 FUSION PROCESS. 386 00:15:46,546 --> 00:15:50,183 SO WITH THIS, THE HEMIFUSION 387 00:15:50,183 --> 00:15:51,818 PATHWAY IN LIVE CELLS. 388 00:15:51,818 --> 00:15:54,120 AFTER THE HEMI TO FULL FUSION, 389 00:15:54,120 --> 00:15:55,288 WE SEE SOMETIMES THE FUSION AS 390 00:15:55,288 --> 00:15:56,823 YOU CAN SEE HERE, THIS EXAMPLE, 391 00:15:56,823 --> 00:16:02,963 AND SOMETIMES THE FUSION IS SO 392 00:16:02,963 --> 00:16:06,132 FAST, FIRST WE'LL SEE THE FUSION 393 00:16:06,132 --> 00:16:08,869 HERE, .1 SECOND AND 26 394 00:16:08,869 --> 00:16:10,170 MILLISECONDS, THAT'S BEFORE THE 395 00:16:10,170 --> 00:16:13,473 DIFFUSION OF THE GREEN PROBE WE 396 00:16:13,473 --> 00:16:16,877 SEE HERE, AND THIS IS THE XY 397 00:16:16,877 --> 00:16:18,211 IMAGES ACROSS THE SCREEN TO SHOW 398 00:16:18,211 --> 00:16:25,418 YOU THAT THE FUSION PORE. 399 00:16:25,418 --> 00:16:28,488 AFTER THE VESICLE FUSION 400 00:16:28,488 --> 00:16:30,790 OPENING, WE NOTICE THAT THESE 401 00:16:30,790 --> 00:16:32,993 VERY LAST PORES CAN CLOSE VERY 402 00:16:32,993 --> 00:16:34,494 QUICKLY, AS SHOWN HERE IN THIS 403 00:16:34,494 --> 00:16:34,728 EXAMPLE. 404 00:16:34,728 --> 00:16:37,163 THIS IS A REALTIME SAMPLE AFTER 405 00:16:37,163 --> 00:16:38,665 DEPOLARIZATION WE SEE THE FUSION 406 00:16:38,665 --> 00:16:40,901 FOR OPEN AND CLOSE AND HAD THIS 407 00:16:40,901 --> 00:16:44,070 PORE IS 200, 300 NANOMETERS OR 408 00:16:44,070 --> 00:16:45,839 SO, WITHIN 100 MILLISECONDS OR 409 00:16:45,839 --> 00:16:46,006 SO. 410 00:16:46,006 --> 00:16:48,008 THIS IS FOLLOWED BY THE 411 00:16:48,008 --> 00:16:49,309 BLEACHING OF THE RED DYE BECAUSE 412 00:16:49,309 --> 00:16:52,212 WE GIVE A STRE STRONG EXCITATION 413 00:16:52,212 --> 00:16:54,814 OF THE RED DYE AND WHEN THE PORE 414 00:16:54,814 --> 00:16:59,953 IS CLOSED, THEN THE DYE CAN ALL 415 00:16:59,953 --> 00:17:02,589 BE BLEACHED BECAUSE THERE'S ONLY 416 00:17:02,589 --> 00:17:03,557 A LIMITED NUMBER OF THE DYE 417 00:17:03,557 --> 00:17:05,225 INSIDE THIS CLOSED CAVITY, SO IT 418 00:17:05,225 --> 00:17:09,262 CAN -- CAN ALL BE CLOSE AND HAD 419 00:17:09,262 --> 00:17:10,997 THIS BLEACHING ALSO CONFIRMS OUR 420 00:17:10,997 --> 00:17:14,401 INDICATION THAT THE PORE IS 421 00:17:14,401 --> 00:17:15,068 CLOSED. 422 00:17:15,068 --> 00:17:16,736 CL WHEN THE PORE IS NOT 423 00:17:16,736 --> 00:17:18,672 CLOSESSED, THEN WE SEE NO 424 00:17:18,672 --> 00:17:19,773 BLEACHING OF THE DYE AS YOU CAN 425 00:17:19,773 --> 00:17:21,875 SEE HERE IN THIS EXAMPLE. 426 00:17:21,875 --> 00:17:28,515 HERE FUSION PORE STAYS LONG 427 00:17:28,515 --> 00:17:28,949 TIME. 428 00:17:28,949 --> 00:17:30,917 NOW WE WOULD LIKE TO KNOW 429 00:17:30,917 --> 00:17:32,686 WHETHER THEY DO THE FOCAL 430 00:17:32,686 --> 00:17:35,655 FUSIONS HERE, AND WE DO NOT SEE 431 00:17:35,655 --> 00:17:36,957 THE FOCALIZED. 432 00:17:36,957 --> 00:17:38,592 INSTEAD IT'S THE SAME FUSIONS AS 433 00:17:38,592 --> 00:17:41,227 SHOWN HERE IN THIS EXAMPLE IS IN 434 00:17:41,227 --> 00:17:42,395 THIS VIDEO. 435 00:17:42,395 --> 00:17:44,497 MEANING THAT THE VESICLE'S BODY 436 00:17:44,497 --> 00:17:47,567 IS SHRINKING INSTEAD OF THE PORE 437 00:17:47,567 --> 00:17:47,934 DILUTION. 438 00:17:47,934 --> 00:17:48,768 AS SHOWN HERE. 439 00:17:48,768 --> 00:17:50,437 HERE IS THE VESICLE FUSION AND 440 00:17:50,437 --> 00:17:54,040 THEN IT SHRINKS IT, AGAIN, NICE 441 00:17:54,040 --> 00:17:57,644 REPEATED AGAIN, SO THE VIDEO 442 00:17:57,644 --> 00:18:04,551 REPEATS AGAIN. 443 00:18:04,551 --> 00:18:06,686 HAD. 444 00:18:06,686 --> 00:18:08,154 I WOULD LIKE TO SHOW YOU ONE 445 00:18:08,154 --> 00:18:10,757 MORE TIME THE SHRINKING OF THE 446 00:18:10,757 --> 00:18:12,525 VESICLES SHOWN HERE. 447 00:18:12,525 --> 00:18:18,431 SO ONCE AGAIN, IT DISERNT 448 00:18:18,431 --> 00:18:20,233 DILATE, INSTEAD IT SHRINKS. 449 00:18:20,233 --> 00:18:22,002 SOMETIMES WE SEE THE ENLARGEMENT 450 00:18:22,002 --> 00:18:23,903 OF THE VESICLES AS SHOWN HERE IN 451 00:18:23,903 --> 00:18:25,972 THIS EXAMPLE AND IN THIS VIDEO. 452 00:18:25,972 --> 00:18:28,608 VESICLES FUSION AND THEN IT 453 00:18:28,608 --> 00:18:31,444 ENLARGES IN ABOUT FIVE TO TEN 454 00:18:31,444 --> 00:18:31,678 SECONDS. 455 00:18:31,678 --> 00:18:37,484 THIS IS REPEATED AGAIN. 456 00:18:37,484 --> 00:18:39,352 SOMETIMES WE SEE THE SEQUENTIAL 457 00:18:39,352 --> 00:18:40,520 COMPOUND FUSION, WHICH MEANS 458 00:18:40,520 --> 00:18:44,591 THAT THE VESICLE FUSES, RECENTLY 459 00:18:44,591 --> 00:18:46,993 FUSED VESICLES, AND IT GENERATES 460 00:18:46,993 --> 00:18:49,295 OR COL LAPS INTO FORMING A LARGE 461 00:18:49,295 --> 00:18:51,297 VESICLE, AS SHOWN HERE IN THIS 462 00:18:51,297 --> 00:18:53,767 EXAMPLE AND ALSO IN THIS 463 00:18:53,767 --> 00:18:54,768 REALTIME VIDEO. 464 00:18:54,768 --> 00:18:56,302 SO VESICLE FUSION OCCURS HERE 465 00:18:56,302 --> 00:18:57,837 AND ON TOP OF THIS FUSION, THEN 466 00:18:57,837 --> 00:19:03,543 THERE WILL BE ANOTHER VESICLE 467 00:19:03,543 --> 00:19:04,177 FUSION. 468 00:19:04,177 --> 00:19:07,247 SO SEQUENTIAL COMPOUND FUSION. 469 00:19:07,247 --> 00:19:08,548 SO I'VE SHOWN YOU DIFFERENT 470 00:19:08,548 --> 00:19:10,483 FORMS OF THE EXOCYTOSIS THAT WE 471 00:19:10,483 --> 00:19:12,385 DID NOT EXPECT FROM LIVE CELLS, 472 00:19:12,385 --> 00:19:15,822 AND NEXT I WILL SHOW YOU THE 473 00:19:15,822 --> 00:19:17,657 ENDOCYTIC MEMBRANE 474 00:19:17,657 --> 00:19:18,024 TRANSFORMATIONS. 475 00:19:18,024 --> 00:19:19,526 THESE ARE SOME OF THE FIRST 476 00:19:19,526 --> 00:19:21,928 VERSIONS OF THE FLAT TO LONG 477 00:19:21,928 --> 00:19:23,329 TRANSITIONS, AND HERE I'LL SHOW 478 00:19:23,329 --> 00:19:27,701 YOU A VIDEO OF THIS PROCESS. 479 00:19:27,701 --> 00:19:30,136 FLAT MEMBRANES AND AFTER THE 480 00:19:30,136 --> 00:19:32,972 DEPOLARIZATION INDICATED HERE AS 481 00:19:32,972 --> 00:19:35,375 A GRAY TRIANGLE HERE, THEN THE 482 00:19:35,375 --> 00:19:37,677 MEMBRANE BECOMES CURVED AND 483 00:19:37,677 --> 00:19:39,312 EVENTUALLY FORMS A VESICLE 484 00:19:39,312 --> 00:19:41,181 WITHIN ABOUT 10, 20 SECONDS. 485 00:19:41,181 --> 00:19:44,684 SO I'LL SHOW YOU THIS PROCESS. 486 00:19:44,684 --> 00:19:48,621 SO NOW YOU SEE SOME SPIKES 487 00:19:48,621 --> 00:19:54,294 COMING HERE. 488 00:19:54,294 --> 00:19:56,262 IT FORCES ON THE SIDE THAT 489 00:19:56,262 --> 00:19:58,431 FORCES THE VESICLE TO BECOME A 490 00:19:58,431 --> 00:19:58,665 VESICLE. 491 00:19:58,665 --> 00:19:59,866 THIS IS FOLLOWED BY BLEACHING OF 492 00:19:59,866 --> 00:20:02,135 THE RED DYE, CONFIRMING THAT THE 493 00:20:02,135 --> 00:20:05,271 PORE IS CLOSED. 494 00:20:05,271 --> 00:20:07,140 SO THIS PROCESS CAN BE DIVIDED 495 00:20:07,140 --> 00:20:09,109 INTO THREE MULTIPLE STEPS. 496 00:20:09,109 --> 00:20:11,177 ONE IS TO THE LUMBAR 497 00:20:11,177 --> 00:20:17,317 TRANSITIONS, SECOND IS LUMBAR TO 498 00:20:17,317 --> 00:20:18,418 OMEGA TRANSITIONS, HERE IS THE 499 00:20:18,418 --> 00:20:20,487 FROM THE TIME IT LAWM BAR 500 00:20:20,487 --> 00:20:24,758 TRANSITIONS SHOWN FLAT TO LAMBDA 501 00:20:24,758 --> 00:20:26,626 TRANSITIONS, NOTICE SOME SPIKES 502 00:20:26,626 --> 00:20:28,928 PULL THE MEMBRANE FORWARD TO 503 00:20:28,928 --> 00:20:30,797 FORM THIS LAMBDA SHAPED 504 00:20:30,797 --> 00:20:31,664 MEMBRANE. 505 00:20:31,664 --> 00:20:38,338 AFTER DEPOLARIZATION WE SEE. 506 00:20:38,338 --> 00:20:40,507 LET ME SEE IF I CAN PLAY IT 507 00:20:40,507 --> 00:20:44,010 AGAIN. 508 00:20:44,010 --> 00:20:44,277 OKAY. 509 00:20:44,277 --> 00:20:50,350 SO YOU SEE THE SPIKES COMING 510 00:20:50,350 --> 00:20:51,484 HERE. 511 00:20:51,484 --> 00:20:52,986 THIS MEMBRANE PROFILE DOES NOT 512 00:20:52,986 --> 00:20:56,322 PROCEED INTO BECOMING AN OMEGA 513 00:20:56,322 --> 00:20:58,424 SHAPED PROFILE. 514 00:20:58,424 --> 00:20:59,826 SO IT STOPS HERE. 515 00:20:59,826 --> 00:21:03,363 AND BECAUSE OF THIS REASON, YOU 516 00:21:03,363 --> 00:21:08,134 SEE THE PREFORMED IF LAMBDA 517 00:21:08,134 --> 00:21:10,436 EXPHAIPPED OMEGA SHAPED 518 00:21:10,436 --> 00:21:13,173 PROFILE -- SHAPED AND THE OMEGA 519 00:21:13,173 --> 00:21:14,407 SHAPED PROFILE CONDITIONS, THEY 520 00:21:14,407 --> 00:21:16,576 ARE PREFORMED. 521 00:21:16,576 --> 00:21:18,411 ELECTRON MICROSCOPES. 522 00:21:18,411 --> 00:21:20,079 THESE STRUCTURES CAN FURTHER 523 00:21:20,079 --> 00:21:22,582 BECOME A VESICLE AFTER THE 524 00:21:22,582 --> 00:21:23,583 DEPOLARIZATION AS SHOWN IN THIS 525 00:21:23,583 --> 00:21:24,350 EXAMPLE HERE. 526 00:21:24,350 --> 00:21:27,520 THIS IS LAMBDA SHAPE GROWS UP TO 527 00:21:27,520 --> 00:21:29,355 DEPOLARIZATION AND THEN BECOMES 528 00:21:29,355 --> 00:21:30,356 AN OMEGA SHAPE. 529 00:21:30,356 --> 00:21:31,791 HERE SHOWS YOU AN EXAMPLE OF 530 00:21:31,791 --> 00:21:33,960 THIS PROCESS. 531 00:21:33,960 --> 00:21:35,628 SO THIS IS LAMBDA AND FORCES ARE 532 00:21:35,628 --> 00:21:39,899 PUSHING HERE, SO IT BECOMES 533 00:21:39,899 --> 00:21:44,504 OMEGA SHAPE. 534 00:21:44,504 --> 00:21:47,040 THE SMALLER ONES ARE GENERALLY 535 00:21:47,040 --> 00:21:48,975 SMALLER OMEGA PROFILES. 536 00:21:48,975 --> 00:21:51,811 SO THIS SHOWS YOU A PREFORMED 537 00:21:51,811 --> 00:21:54,881 OMEGA SHAPED MEMBRANE CAN CAUSE 538 00:21:54,881 --> 00:21:56,916 THE PORES UP TO DEPOLARIZATION 539 00:21:56,916 --> 00:22:00,386 SHOWN HERE IN THIS EXAMPLE. 540 00:22:00,386 --> 00:22:03,890 SO HERE CLOSES THE PORES REALLY 541 00:22:03,890 --> 00:22:14,400 QUICKLY, FOLLOWED BY THE DYE. 542 00:22:15,134 --> 00:22:19,005 SO THESE ARE THE PREFORMED OMEGA 543 00:22:19,005 --> 00:22:19,272 CLOSURES. 544 00:22:19,272 --> 00:22:20,773 I HAVE SHOWN YOU DIFFERENT 545 00:22:20,773 --> 00:22:23,409 TRANSITIONS FROM ARE FLATD TO 546 00:22:23,409 --> 00:22:25,912 LAMBDA TO OMEGA TO O'S AND 547 00:22:25,912 --> 00:22:27,547 DIFFERENT LAMBDA SHAPES CAN 548 00:22:27,547 --> 00:22:29,382 GENERATE DIFFERENT SIZE OF THE 549 00:22:29,382 --> 00:22:29,649 VESICLES. 550 00:22:29,649 --> 00:22:30,283 ALL RIGHT. 551 00:22:30,283 --> 00:22:32,619 SO I HAVE SHOWN YOU THE LIVE 552 00:22:32,619 --> 00:22:36,556 CELL OBSERVATIONS OF THE FUSION 553 00:22:36,556 --> 00:22:40,360 AND BUDDING, I ALSO INCLUDED THE 554 00:22:40,360 --> 00:22:43,363 COMPOUND FUSION AS SYNAPSE HERE. 555 00:22:43,363 --> 00:22:45,665 NEXT, I WOULD LIKE TO TALK ABOUT 556 00:22:45,665 --> 00:22:48,735 THE MECHANISM, THE MOLECULAR 557 00:22:48,735 --> 00:22:49,602 MECHANICS UNDERLYING THIS 558 00:22:49,602 --> 00:22:50,303 DIFFERENT TRANSITION THAT WE 559 00:22:50,303 --> 00:22:52,572 HAVE STUDIED SO FAR. 560 00:22:52,572 --> 00:22:54,540 THE FIRST IS ENDOCYTOSIS. 561 00:22:54,540 --> 00:23:00,914 FLAT TO LAMBDA TRANSITION, THE 562 00:23:00,914 --> 00:23:04,417 ACTION, BECAUSE IF YOU BLOCK THE 563 00:23:04,417 --> 00:23:06,719 ACTIN, WE SEE THE REDUCTION OR 564 00:23:06,719 --> 00:23:09,622 THE FLAT TO ROUND TRANSITIONS 565 00:23:09,622 --> 00:23:14,494 THEY MATT CLI. 566 00:23:14,494 --> 00:23:19,632 AND WITH K44A WHICH IS NEGATIVE, 567 00:23:19,632 --> 00:23:24,871 IT BLOCKS THE FLAT TRANSITION. 568 00:23:24,871 --> 00:23:30,710 IF YOU LABEL THE ACT INWITH -- 569 00:23:30,710 --> 00:23:33,780 ACTIN, YOU SEE THE ACTINS THAT 570 00:23:33,780 --> 00:23:36,049 APPEARS TO PULL THE MEMBRANE 571 00:23:36,049 --> 00:23:37,517 INWARD, IN THIS EXAMPLE. 572 00:23:37,517 --> 00:23:39,686 AS YOU CAN SEE HERE IN THIS 573 00:23:39,686 --> 00:23:39,953 REALTIME. 574 00:23:39,953 --> 00:23:43,523 SO HERE IS ACTEDIN FILAMENTS 575 00:23:43,523 --> 00:23:45,925 HERE AND IT'S VERY THIN RIGHT 576 00:23:45,925 --> 00:23:48,861 NOW, AND AFTER DEPOLARIZATION IT 577 00:23:48,861 --> 00:23:51,397 BECOMES STRONGER AND STRONGER 578 00:23:51,397 --> 00:23:52,732 AND FORMS FILAMENT THAT APPEARS 579 00:23:52,732 --> 00:23:59,605 TO PULL THE MEMBRANE INWARD. 580 00:23:59,605 --> 00:24:01,908 SO IT EVENTUALLY FORMS A 581 00:24:01,908 --> 00:24:03,876 FILAMENT THAT PULLS THE 582 00:24:03,876 --> 00:24:06,746 MEMBRANES INWARD HERE. 583 00:24:06,746 --> 00:24:09,816 WE ALSO LABEL IN RED AND YOU CAN 584 00:24:09,816 --> 00:24:12,952 SEE THE DYNAMIN IN THE BASE OF 585 00:24:12,952 --> 00:24:19,058 THE LAMBDA ANDS HERE IS THE 586 00:24:19,058 --> 00:24:23,629 DYNAMIN IN RED RAE CRUTD TO THIS 587 00:24:23,629 --> 00:24:25,365 GROWING PROFILE -- RECRUITED TO 588 00:24:25,365 --> 00:24:27,100 THIS GROWING PROFILE. 589 00:24:27,100 --> 00:24:28,668 SO WITH THESE RESULTS, WE 590 00:24:28,668 --> 00:24:33,473 PROPOSE THAT THE ACTIN AND 591 00:24:33,473 --> 00:24:35,341 DYNAMIN FORMS BUNDLES TO PULL 592 00:24:35,341 --> 00:24:41,381 THE MEMBRANES INWARD AND FORM 593 00:24:41,381 --> 00:24:41,781 THIS. 594 00:24:41,781 --> 00:24:43,783 SO NEXT QUESTION IS HOW THIS 595 00:24:43,783 --> 00:24:45,284 LAMBDA SHAPE IS CONVERTED INTO 596 00:24:45,284 --> 00:24:46,152 THE OMEGA SHAPE. 597 00:24:46,152 --> 00:24:50,189 WE NOTICE THAT THE DYNAMIN FORMS 598 00:24:50,189 --> 00:24:52,592 THE BASE OF THE LAMBDA SHAPE AND 599 00:24:52,592 --> 00:24:54,260 ALSO THE BASE OF THIS OMEGA 600 00:24:54,260 --> 00:24:54,827 SHAPE. 601 00:24:54,827 --> 00:24:57,997 AS YOU CAN SEE HERE, AND IS THE 602 00:24:57,997 --> 00:25:01,167 XY IMAGES OF THE DYNAMIN 603 00:25:01,167 --> 00:25:03,136 SURROUNDING THE POLAR REGIONS. 604 00:25:03,136 --> 00:25:05,872 SO IT APPEARS THE DYNAMIN 605 00:25:05,872 --> 00:25:07,173 CONSTRICTS THE BASE OF THE 606 00:25:07,173 --> 00:25:08,941 LAMBDA AND THEREFORE TO 607 00:25:08,941 --> 00:25:10,476 GENERATED AN OMEGA BASE. 608 00:25:10,476 --> 00:25:12,412 THIS WILL SHOW YOU A VIDEO OF 609 00:25:12,412 --> 00:25:14,847 THE PROCESS. 610 00:25:14,847 --> 00:25:16,716 DYNAMIN IS IN RED AND 611 00:25:16,716 --> 00:25:17,450 SURROUNDING ALWAYS ON THE BASE 612 00:25:17,450 --> 00:25:19,052 AND THE SIDE OF THE LAMBDA AND 613 00:25:19,052 --> 00:25:20,987 WHY THE LAMBDA IS GOING TO 614 00:25:20,987 --> 00:25:21,988 BECOME OMEGA SHAPE. 615 00:25:21,988 --> 00:25:26,359 SO HERE IS THE DYNAMIN IN RED 616 00:25:26,359 --> 00:25:28,127 THAT SURROUNDS THE BASE AND 617 00:25:28,127 --> 00:25:29,028 BECOMES OMEGA BASE. 618 00:25:29,028 --> 00:25:35,968 IT STOPS HERE SOMEHOW. 619 00:25:35,968 --> 00:25:46,312 SO WE'LL PLAY AGAIN. 620 00:25:52,251 --> 00:25:54,687 SO DYNAMIN IS SURROUNDING AND 621 00:25:54,687 --> 00:25:58,191 APPEARS TO CONSTRICT THE 622 00:25:58,191 --> 00:25:58,624 MEMBRANES. 623 00:25:58,624 --> 00:26:01,594 IF IT BLOCKS WE SEE THE LAMBDA 624 00:26:01,594 --> 00:26:03,763 TO OMEGA TRANSITIONS REDUCTIONS 625 00:26:03,763 --> 00:26:04,097 DRAMATICALLY. 626 00:26:04,097 --> 00:26:07,066 THIS MEANS THE DYNAMIN IS ABLE 627 00:26:07,066 --> 00:26:09,702 TO CONSTRICTED THIS VERY LAST 628 00:26:09,702 --> 00:26:13,072 LAMBDA SHAPE WHICH IS ABOUT 800, 629 00:26:13,072 --> 00:26:14,073 SOMETIMES 800 NANOMETERS. 630 00:26:14,073 --> 00:26:19,011 WHAT WE KNOW ABOUT DYNAMIN IS IT 631 00:26:19,011 --> 00:26:21,414 CAN CONSTRICT ABOUT 10-NANOMETER 632 00:26:21,414 --> 00:26:21,614 PORES. 633 00:26:21,614 --> 00:26:24,117 WHY THIS IS A SURPRISE TO US. 634 00:26:24,117 --> 00:26:29,956 IN CORD WITH NIDHI AND JENNY 635 00:26:29,956 --> 00:26:33,259 HINSHAW, THEY PUT DYNAMIN ALONG 636 00:26:33,259 --> 00:26:35,661 IN VITRO AND CAN CONSTRICT 637 00:26:35,661 --> 00:26:36,996 LYSOSOMES TO BECOMES VERY SMALL, 638 00:26:36,996 --> 00:26:38,397 FROM HUNDREDS OF NANOMETERS TO 639 00:26:38,397 --> 00:26:40,366 ABOUT TENS OF NANOMETERS. 640 00:26:40,366 --> 00:26:44,637 AND HERE IT SHOWS THE 641 00:26:44,637 --> 00:26:47,206 RECONSTRUCTION OF THE DYNAMIN 642 00:26:47,206 --> 00:26:51,744 THAT APPEAR TO SURROUND HELICES, 643 00:26:51,744 --> 00:26:56,749 PROPOSING THAT THE DYNAMIN FORMS 644 00:26:56,749 --> 00:26:58,751 HELICES AROUND THE BASE OF THE 645 00:26:58,751 --> 00:27:00,620 LAMBDA AND CONSTRICTION OF THE 646 00:27:00,620 --> 00:27:01,521 OMEGA PROFILES. 647 00:27:01,521 --> 00:27:05,558 AFTER THAT DYNAMIN WILL CONTINUE 648 00:27:05,558 --> 00:27:08,227 ON CONSTRICTED AND FORCE THE 649 00:27:08,227 --> 00:27:10,062 PORE TO BECOME A VESICLE AS 650 00:27:10,062 --> 00:27:13,032 SHOWN THIS EXAMPLE HERE WITH 651 00:27:13,032 --> 00:27:15,468 DYNAMIN IN RED SURROUNDING THE 652 00:27:15,468 --> 00:27:17,303 PORE REGION AND COME TOGETHER TO 653 00:27:17,303 --> 00:27:18,738 FORCE THE MEMBRANE TO CLOSE THE 654 00:27:18,738 --> 00:27:22,241 PORE, SHOWN HERE IS A REALTIME 655 00:27:22,241 --> 00:27:23,409 VIDEO. 656 00:27:23,409 --> 00:27:25,978 DYNAMIN IS IN RED AND SURROUNDS 657 00:27:25,978 --> 00:27:29,815 THE BASE PORE REGIONS. 658 00:27:29,815 --> 00:27:31,984 MY FILE I GUESS IS TOO LARGE. 659 00:27:31,984 --> 00:27:42,028 SO WE'LL PLAY IT AGAIN. 660 00:27:42,028 --> 00:27:46,532 SO THESE TWO COME TOGETHER, AND 661 00:27:46,532 --> 00:27:48,301 THESE PORES ALSO COME TOGETHER, 662 00:27:48,301 --> 00:27:55,508 SO IT CONSTRICTS. 663 00:27:55,508 --> 00:27:56,475 OKAY. 664 00:27:56,475 --> 00:27:59,145 IF WE BLOCK DIE NAM MINIMUM WITH 665 00:27:59,145 --> 00:28:04,250 THESE TWO -- DYNAMI234 WITH 666 00:28:04,250 --> 00:28:06,018 THESE TWO RESULTS, WE PROPOSE 667 00:28:06,018 --> 00:28:11,591 THAT ACTIN 1K3 DYNAMIN IS 668 00:28:11,591 --> 00:28:15,428 INVOLVED MR. IN CONSTRICTION TO 669 00:28:15,428 --> 00:28:17,830 PULL THE VESICLES WITH. 670 00:28:17,830 --> 00:28:20,566 SO ONLY TWO PORES SUFFICIENT TO 671 00:28:20,566 --> 00:28:22,535 GENERATED VESICLE FROM FLAT 672 00:28:22,535 --> 00:28:26,906 MEMBRANE AND OUR COOPERATOR DID 673 00:28:26,906 --> 00:28:28,641 THE MATHEMATICAL MODELING TO 674 00:28:28,641 --> 00:28:31,611 SHOW THAT ONLY TWO FOLDS ARE 675 00:28:31,611 --> 00:28:34,680 SUFFICIENT TO GENERATE VESICLE, 676 00:28:34,680 --> 00:28:39,051 SO THIS HERE, THE VIDEO SHOWING 677 00:28:39,051 --> 00:28:41,687 A PULLING FORCE HERE, AND LAMBDA 678 00:28:41,687 --> 00:28:43,356 CONSTRICTION FORCE WILL GENERATE 679 00:28:43,356 --> 00:28:49,395 OMEGA SHAPE, SO A VIDEO. 680 00:28:49,395 --> 00:28:51,264 SO NOW IT'S PULLING AND THEN 681 00:28:51,264 --> 00:28:53,099 THEY WILL APPLY CONSTRICTION AND 682 00:28:53,099 --> 00:28:58,371 THEN IT BECOMES OMEGA SHAPE. 683 00:28:58,371 --> 00:29:02,108 SO THIS ACTIN IS INVOLVED IN 684 00:29:02,108 --> 00:29:04,277 CONVERTING THE FAT MEMBRANE INTO 685 00:29:04,277 --> 00:29:05,911 A ROUND -- THE FLAT MEMBRANE 686 00:29:05,911 --> 00:29:07,813 INTO A ROUND VESICLE. 687 00:29:07,813 --> 00:29:10,650 I'VE SHOWN YOU THAT THE 688 00:29:10,650 --> 00:29:12,285 PREFORMED OMEGA CAN BE CLOSED 689 00:29:12,285 --> 00:29:13,586 AFTER DEPOLARIZATION, THIS 690 00:29:13,586 --> 00:29:14,920 ACTUALLY PROVIDES US WITH A 691 00:29:14,920 --> 00:29:19,625 GREAT OPPORTUNITY TO SCREEN THE 692 00:29:19,625 --> 00:29:21,360 FISHER MECHANIC -- THE MISSION 693 00:29:21,360 --> 00:29:22,695 MECHANISMS BECAUSE WE DON'T NEED 694 00:29:22,695 --> 00:29:24,997 TO STUDY HOW THE OMEGA PORE IS 695 00:29:24,997 --> 00:29:27,300 FORMED, THIS IS BYPASSING THAT 696 00:29:27,300 --> 00:29:27,533 PROCESS. 697 00:29:27,533 --> 00:29:28,801 THIS SHOWS YOU SOME OF AGAIN 698 00:29:28,801 --> 00:29:30,870 THESE PREFORMED OMEGA PORE, 699 00:29:30,870 --> 00:29:32,338 WAITING FOR THE DEPOLARIZATION 700 00:29:32,338 --> 00:29:34,373 TO CLOSE THE PORES. 701 00:29:34,373 --> 00:29:35,474 WE SCREEN THE PROTEINS AND WHAT 702 00:29:35,474 --> 00:29:41,080 WE FOUND IS REALLY SURPRISING, 703 00:29:41,080 --> 00:29:44,216 INVOLVED IN CLOSING THE PORE, 704 00:29:44,216 --> 00:29:46,552 VERY WELL KNOWN TO COAT THE 705 00:29:46,552 --> 00:29:51,490 VESICLES, HERE IS AN EXAMPLE 10E 706 00:29:51,490 --> 00:29:53,693 VESICLES, COATED ON THE BODY 707 00:29:53,693 --> 00:29:56,429 HERE. 708 00:29:56,429 --> 00:29:56,696 CLATHRIN. 709 00:29:56,696 --> 00:29:59,565 WHAT IS NOT KNOWN IS IF IT 710 00:29:59,565 --> 00:30:00,466 MEDIATES FISSION. 711 00:30:00,466 --> 00:30:08,808 WE KNOCK DOWN CLATHRIN, AND WITH 712 00:30:08,808 --> 00:30:11,644 THE POLYMERIZATION, WE SEE THE 713 00:30:11,644 --> 00:30:13,946 DRAMATIC DECREASE OF THE OMEGA 714 00:30:13,946 --> 00:30:15,348 PREFORM CLOSURE. 715 00:30:15,348 --> 00:30:19,085 SO CARBON IS -- SO CLATHRIN IS 716 00:30:19,085 --> 00:30:21,821 INVOLVED 2349 FITIONZ PROCESS. 717 00:30:21,821 --> 00:30:26,859 -- SO CLATHRIN IS INVOLVED THIS 718 00:30:26,859 --> 00:30:30,930 IN THE FISSION PROCESS. 719 00:30:30,930 --> 00:30:35,735 SO CLATHRIN IS INVOLVED IN THE 720 00:30:35,735 --> 00:30:36,936 FUSION PORE CLOSURE. 721 00:30:36,936 --> 00:30:41,774 THIS MEANS THAT CLATHRIN COULD 722 00:30:41,774 --> 00:30:43,309 ANTAGONIZE THE FUSION PORE 723 00:30:43,309 --> 00:30:43,576 EXPANSION. 724 00:30:43,576 --> 00:30:45,177 IF THAT'S THE CASE, THEN WHEN 725 00:30:45,177 --> 00:30:48,481 YOU BLOCK CLATHRIN, THE FUSION 726 00:30:48,481 --> 00:30:50,116 PORE EXPANSION WILL BE FASTER, 727 00:30:50,116 --> 00:30:53,819 AND WE DO SEE THAT THE KNOCKDOWN 728 00:30:53,819 --> 00:30:57,089 OF CLATHRIN WILL GENERATE A 729 00:30:57,089 --> 00:30:58,524 FASTER RELEASE. 730 00:30:58,524 --> 00:31:02,561 HERE WE LABEL THE VESICLES WITH 731 00:31:02,561 --> 00:31:03,763 NEUROTRANSMITTER INSIDE VERY 732 00:31:03,763 --> 00:31:09,034 WELL KEHL WITH THIS 511 -- THE 733 00:31:09,034 --> 00:31:10,936 FFN 511 AND AFTER DEPOLARIZATION 734 00:31:10,936 --> 00:31:12,338 CAN BE RELEASED. 735 00:31:12,338 --> 00:31:15,040 SO HERE IS AN EXAMPLE I'LL SHOW 736 00:31:15,040 --> 00:31:21,280 YOU AND KNOCKDOWNS OF CLATHRIN, 737 00:31:21,280 --> 00:31:25,117 IT SPEEDS UP THE VESICLE AND 738 00:31:25,117 --> 00:31:26,118 RELEASE OF TRANSMITTEDDER. 739 00:31:26,118 --> 00:31:29,155 THE QUESTION IS HOW CLATHRIN 740 00:31:29,155 --> 00:31:30,723 CLOSE THE PORE. 741 00:31:30,723 --> 00:31:31,457 SURPRISING RESULTS. 742 00:31:31,457 --> 00:31:33,325 WE LABEL WITH COLORS HERE AND 743 00:31:33,325 --> 00:31:35,961 YOU CAN SEE THE CLATHRIN 744 00:31:35,961 --> 00:31:37,696 SURROUNDS THE BASE OF THE OMEGA 745 00:31:37,696 --> 00:31:40,332 SHAPED PROFILES AND HERE IS A 3D 746 00:31:40,332 --> 00:31:43,602 IMAGING OF CLATHRIN IN MAGENTA 747 00:31:43,602 --> 00:31:46,939 TO SHOW YOU CLATHRIN SURROUNDS 748 00:31:46,939 --> 00:31:49,141 THE BASE OF THE OMEGA PROFILES. 749 00:31:49,141 --> 00:31:53,479 HERE IS THE XY IMAGES ACROSS THE 750 00:31:53,479 --> 00:31:59,118 PORE REGION, MORE EXAMPLES OF 751 00:31:59,118 --> 00:32:00,052 CLATHRIN SURROUNDING THE GREEN 752 00:32:00,052 --> 00:32:00,352 PORE. 753 00:32:00,352 --> 00:32:01,687 SOMETIMES THE GREEN PORE IS TOO 754 00:32:01,687 --> 00:32:03,222 SMALL TO BE RESOLVED BUT WE 755 00:32:03,222 --> 00:32:07,026 STILL SEE THE CLATHRINS. 756 00:32:07,026 --> 00:32:11,197 THIS SHOWS RECENT IMAGING TO SEE 757 00:32:11,197 --> 00:32:13,432 THE INDIVIDUAL CLATHRIN 758 00:32:13,432 --> 00:32:15,034 MOLECULES HERE SURROUNDING THE 759 00:32:15,034 --> 00:32:17,536 REGIONS HERE AS GREEN CONFOCAL 760 00:32:17,536 --> 00:32:18,904 IMAGES, YOU CAN SEE SOMETIMES 761 00:32:18,904 --> 00:32:20,506 THEY ARE VERY CLOSE TO EACH 762 00:32:20,506 --> 00:32:24,477 OTHER, AS IF CLATHRIN IS 763 00:32:24,477 --> 00:32:28,714 POSITIVELY LIMB RIETZED IN 764 00:32:28,714 --> 00:32:30,883 THE --S POLYMERIZED IN THE PORE 765 00:32:30,883 --> 00:32:32,084 REGIONS HERE. 766 00:32:32,084 --> 00:32:37,790 AND WE SEE, THE POLYMERIZED 767 00:32:37,790 --> 00:32:44,730 CLATHRIN AT THE BASE HERE, AND 768 00:32:44,730 --> 00:32:47,766 IT APPEARS TO BE IN THE PORE 769 00:32:47,766 --> 00:32:48,434 REGIONS. 770 00:32:48,434 --> 00:32:51,837 SO IT COULD BE THAT PRELIMINARY 771 00:32:51,837 --> 00:32:56,642 ZIKS OF CLAT -- POLYMERIZATION 772 00:32:56,642 --> 00:33:01,013 OF CLATHRIN. 773 00:33:01,013 --> 00:33:02,548 HERE POLYMERIZATION IS BROUGHT 774 00:33:02,548 --> 00:33:05,384 HERE, AS SHOWN HERE AND UNDER 775 00:33:05,384 --> 00:33:08,888 THESE CONDITIONS, THE FUSION 776 00:33:08,888 --> 00:33:11,557 CLOSURE AND THE PREFORMED OMEGA 777 00:33:11,557 --> 00:33:15,027 PORE CLOSURE IS DECREASED. 778 00:33:15,027 --> 00:33:17,363 SO IT MEANS THAT CLATHRIN 779 00:33:17,363 --> 00:33:19,865 POLYMERIZATION IS REQUIRED TO 780 00:33:19,865 --> 00:33:30,309 MEDIATE THE PORE CLOSURE. 781 00:33:31,644 --> 00:33:34,780 THE MATHEMATICAL MODEL, IT WAS 782 00:33:34,780 --> 00:33:36,181 PRELIMINARY ZIEZED AROUND THE 783 00:33:36,181 --> 00:33:38,684 BASE REGION AND FOUND THE 784 00:33:38,684 --> 00:33:42,855 PROCESS CAN JN RATE CURVE OF 785 00:33:42,855 --> 00:33:44,723 FORCE TO CONSTRICT THE OH 786 00:33:44,723 --> 00:33:48,761 MECHANIC GA PORES, AND PORE IS 787 00:33:48,761 --> 00:33:49,628 UNDERGOING FISSION. 788 00:33:49,628 --> 00:33:52,064 SO WITH THIS WE CONCLUDE THAT 789 00:33:52,064 --> 00:33:55,768 CLATHRIN IS A NEW CLASS OF THE 790 00:33:55,768 --> 00:33:57,736 FISSION ARE, BOLD HYPOTHESIS, 791 00:33:57,736 --> 00:33:59,638 CAN CLOSE THE FISSION PORE AS 792 00:33:59,638 --> 00:34:01,674 WELL AS THE FUSION PORE. 793 00:34:01,674 --> 00:34:02,408 OKAY. 794 00:34:02,408 --> 00:34:05,411 SPEAKING OF THE FUSION PORE, WE 795 00:34:05,411 --> 00:34:07,146 WOULD LIKE TO KNOW THE 796 00:34:07,146 --> 00:34:08,581 MECHANISMS UNDERLYING THE FUSION 797 00:34:08,581 --> 00:34:10,082 PORE DYNAMICS AND BECAUSE OF THE 798 00:34:10,082 --> 00:34:11,850 LIMIT OF TIME, I WILL NOT BE 799 00:34:11,850 --> 00:34:13,385 ABLE TO GO THROUGH ALL THESE 800 00:34:13,385 --> 00:34:14,820 DETAILS, BUT THE BOTTOM LINE 801 00:34:14,820 --> 00:34:18,424 HERE IS THAT THE FUSION PORE 802 00:34:18,424 --> 00:34:19,725 DYNAMICS IS DETERMINED BETWEEN 803 00:34:19,725 --> 00:34:21,026 THE COMPETITION BETWEEN TWO 804 00:34:21,026 --> 00:34:22,595 FORCES, SOMEONE ON THE 805 00:34:22,595 --> 00:34:28,801 RIGHT-HAND SIDE, FUSION PORE 806 00:34:28,801 --> 00:34:33,739 EXTENSIONS, SNARE, ACTIN, 807 00:34:33,739 --> 00:34:36,942 PROMOTES PORE OPEN. 808 00:34:36,942 --> 00:34:40,446 CONSTRICTION, CLATHRIN AND 809 00:34:40,446 --> 00:34:43,082 DYNAMIN WE UNDERSTAND HERE. 810 00:34:43,082 --> 00:34:49,221 AND WHETHER IT STOPS HERE OR 811 00:34:49,221 --> 00:34:50,556 FUSION PORE CLOSURE. 812 00:34:50,556 --> 00:34:56,095 NEXT WE WOULD LIKE TO KNOW WHY. 813 00:34:56,095 --> 00:35:05,971 WE OBSERVE AND COLLABORATED WITH 814 00:35:05,971 --> 00:35:07,706 COMPUTER SIMULATION AND FOWFNED 815 00:35:07,706 --> 00:35:09,241 POSITIVE DIFFERENCE BETWEEN THE 816 00:35:09,241 --> 00:35:14,046 INTRACELLULAR AND EXTRACELLULAR, 817 00:35:14,046 --> 00:35:16,682 SO THIS PRESSURE DIFFERENCE CAN 818 00:35:16,682 --> 00:35:18,851 SQUEEZE THE MEMBRANE TO MAKE THE 819 00:35:18,851 --> 00:35:21,153 VESICLE MEMBRANE CRUMBLE AND 820 00:35:21,153 --> 00:35:22,454 RELEASE THE TENSION. 821 00:35:22,454 --> 00:35:25,958 HIGHER TENSION AT THE PLASMA 822 00:35:25,958 --> 00:35:27,960 MEMBRANE CAN RELIEVE THE LOAD 823 00:35:27,960 --> 00:35:29,928 TENSION OF THE VESICLE MEMBRANE, 824 00:35:29,928 --> 00:35:31,764 CAUSING THE VESICLES TO SHRINK 825 00:35:31,764 --> 00:35:34,500 AND THIS IS THE SIMULATION 826 00:35:34,500 --> 00:35:36,135 RESULT THAT THEY'RE SHRINKING 827 00:35:36,135 --> 00:35:40,973 INSTEAD OF COLLAPSE INTO THE 828 00:35:40,973 --> 00:35:41,740 MEMBRANE. 829 00:35:41,740 --> 00:35:43,242 SO THE MIDDLE PREDICTED THAT IF 830 00:35:43,242 --> 00:35:45,344 YOU DECREASE THE DRIVING SOURCE 831 00:35:45,344 --> 00:35:48,280 BY APPLYING HYPERTONIC SOLUTION, 832 00:35:48,280 --> 00:35:52,017 THEN YOU SEE REDUCTION OF THE 833 00:35:52,017 --> 00:35:56,388 FUSION, AND WE SEE 834 00:35:56,388 --> 00:35:58,223 EXPERIMENTALLY A DRAMATIC 835 00:35:58,223 --> 00:36:00,092 DECREASE AFTER WE APPLY THE 836 00:36:00,092 --> 00:36:10,069 HYPERTONIC SOLUTION. 837 00:36:10,069 --> 00:36:15,774 SO WHEN WE APPLY F-ACTIN, IT 838 00:36:15,774 --> 00:36:17,076 REDUCES THE TENSION HERE AND 839 00:36:17,076 --> 00:36:18,744 BLOCKS THE FUSION AS WE SEE 840 00:36:18,744 --> 00:36:19,378 HERE. 841 00:36:19,378 --> 00:36:22,648 SO THAT'S AGAIN CONSISTENT WITH 842 00:36:22,648 --> 00:36:26,085 THE MODEL'S PREDICTION, AND WE 843 00:36:26,085 --> 00:36:28,353 INCREASE THE DRIVING FORCE BY 844 00:36:28,353 --> 00:36:30,656 APPLYING HYPOTONIC, LAYER 845 00:36:30,656 --> 00:36:32,491 SOLUTION, THEN IT WILL RESCUE 846 00:36:32,491 --> 00:36:35,327 POSSIBLY AT LEAST THE INHIBITION 847 00:36:35,327 --> 00:36:38,764 OF THE FUSION AS SHOWN HERE IN 848 00:36:38,764 --> 00:36:40,065 THIS BLUE BAR HERE. 849 00:36:40,065 --> 00:36:44,870 SO WE CONCLUDE THE TENSION AND 850 00:36:44,870 --> 00:36:46,538 ABILITYIN ARE INVOLVED IN 851 00:36:46,538 --> 00:36:49,608 SHRINKING THE VESICLE. 852 00:36:49,608 --> 00:36:53,312 SO THIS IS THE MECHANISMS 853 00:36:53,312 --> 00:36:54,947 BIOMECHANICS THAT I'VE STUDIED 854 00:36:54,947 --> 00:36:57,750 SO FAR, SHOWN HERE. 855 00:36:57,750 --> 00:36:59,218 IN THE NEXT FEW MINUTES I WOULD 856 00:36:59,218 --> 00:37:01,386 LIKE TO TALK ABOUT SOME 857 00:37:01,386 --> 00:37:04,256 FUNCTIONS OF THESE 858 00:37:04,256 --> 00:37:05,457 TRANSFORMATIONS THAT WE HAVE NOT 859 00:37:05,457 --> 00:37:08,060 BEEN ABLE TO RECOGNIZE BEFORE. 860 00:37:08,060 --> 00:37:12,030 THE FIRST ONE IS THE HEMIFUSION 861 00:37:12,030 --> 00:37:18,604 WHICH CAN ACTUALLY GENERATE -- 862 00:37:18,604 --> 00:37:25,177 IT CAN CONTROL RELEASE ASSEMBLY. 863 00:37:25,177 --> 00:37:28,347 ARE SO IT COULD CAUSE RELEASE 864 00:37:28,347 --> 00:37:29,248 FAILURE. 865 00:37:29,248 --> 00:37:31,416 HEMIFUSION TO FULL FUSION IS 866 00:37:31,416 --> 00:37:32,951 DELAYED AS SHOWN HERE, IT 867 00:37:32,951 --> 00:37:34,286 GENERATES ASYNCHRONOUS RELEASE 868 00:37:34,286 --> 00:37:36,555 WHICH HAS NOT BEEN RECOGNIZED 869 00:37:36,555 --> 00:37:37,656 BEFORE AS MECHANISM. 870 00:37:37,656 --> 00:37:41,693 THE OTHER WAY TO GENERATE 871 00:37:41,693 --> 00:37:43,896 ASYNCHRONOUS RELEASE IS BY THE 872 00:37:43,896 --> 00:37:45,631 SEQUENTIAL COMPOUND FUSION, AND 873 00:37:45,631 --> 00:37:47,833 HERE WE'LL SHOW YOU AN EXAMPLE 874 00:37:47,833 --> 00:37:57,676 OF THIS FUSION, VESICLES WITH 875 00:37:57,676 --> 00:38:00,179 THE PHOSPHONEUROTRANSMITTER, 876 00:38:00,179 --> 00:38:02,581 FOLLOWED BY THIS VESICLE RELEASE 877 00:38:02,581 --> 00:38:07,519 AND THIS WILL FORM ASYNCHRONOUS 878 00:38:07,519 --> 00:38:08,720 VESICLES, THIS IS THE GREEN 879 00:38:08,720 --> 00:38:10,355 PROBE THAT WE'LL LABEL THE 880 00:38:10,355 --> 00:38:12,457 MEMBRANES, I'LL SHOW YOU SEVERAL 881 00:38:12,457 --> 00:38:13,859 TIMES HERE. 882 00:38:13,859 --> 00:38:15,060 VERY FAST. 883 00:38:15,060 --> 00:38:16,829 RELEASE AND REPEAT AGAIN. 884 00:38:16,829 --> 00:38:25,571 NO, IT DOES NOT GO. 885 00:38:25,571 --> 00:38:26,138 ONE MORE TIME. 886 00:38:26,138 --> 00:38:28,340 SO NOW IT REPEATS. 887 00:38:28,340 --> 00:38:31,076 OKAY. 888 00:38:31,076 --> 00:38:33,712 SO THIS IS A I SEQUENTIAL COMPOD 889 00:38:33,712 --> 00:38:35,647 FUSION THAT CAN GENERATE SAING 890 00:38:35,647 --> 00:38:36,381 NUS RELEASE. 891 00:38:36,381 --> 00:38:37,850 OF COURSE WHEN THE FUSION PORE 892 00:38:37,850 --> 00:38:40,452 IS NOT RESERVABLE, IT WILL VERY 893 00:38:40,452 --> 00:38:42,554 OFTEN ACCOMPANIED BY THE SLOW 894 00:38:42,554 --> 00:38:43,989 RELEASE SHOWN HERE AND WE CAN 895 00:38:43,989 --> 00:38:47,259 SEE THE FUSION PORE, THEN 896 00:38:47,259 --> 00:38:49,461 RELEASE OF THIS FORCED 897 00:38:49,461 --> 00:38:50,429 NEUROTRANSMITTER WOULD BE MUCH 898 00:38:50,429 --> 00:38:50,896 FASTER. 899 00:38:50,896 --> 00:38:53,432 SO THE FUSION PORE CAN SHOW THE 900 00:38:53,432 --> 00:38:55,901 RATE OF THE RELEASE. 901 00:38:55,901 --> 00:39:00,072 AND ALSO OUR RESULT DEFINED KISS 902 00:39:00,072 --> 00:39:00,739 AND RUN. 903 00:39:00,739 --> 00:39:02,140 KISS AND RUN IS ALWAYS DEFINED 904 00:39:02,140 --> 00:39:03,575 AS VERY FAST FUSION FOR OPENING 905 00:39:03,575 --> 00:39:05,210 AND CLOSING WITHIN THE PORE AND 906 00:39:05,210 --> 00:39:07,512 NOW IT CAN BE ACTUALLY FAST OR 907 00:39:07,512 --> 00:39:09,348 SLOW, IT CAN BE NARROW OR LARGE, 908 00:39:09,348 --> 00:39:12,317 IT ALSO CAN FORM LARGE VESICLES 909 00:39:12,317 --> 00:39:14,119 OR SMALL VESICLES. 910 00:39:14,119 --> 00:39:15,554 IF THE VESICLE ARE LARGE, THEY 911 00:39:15,554 --> 00:39:17,990 CAN CAUSE THE PORE TO FORM LARGE 912 00:39:17,990 --> 00:39:18,323 VESICLES. 913 00:39:18,323 --> 00:39:20,292 IF SHRINK RS CAN CAUSE THE PORE 914 00:39:20,292 --> 00:39:21,927 TO FORM SMALL VESICLES AND 915 00:39:21,927 --> 00:39:27,299 THEREFORE TO CONTROL THE VESICLE 916 00:39:27,299 --> 00:39:29,801 SIZE. 917 00:39:29,801 --> 00:39:31,703 OBSERVATION ALSO SHOWS THE FOCAL 918 00:39:31,703 --> 00:39:35,307 FUSION AS THE DOMINANT 919 00:39:35,307 --> 00:39:38,176 MECHANISMS IN THE FUSION. 920 00:39:38,176 --> 00:39:41,113 ALSO, I WOULD LIKE TO TALK ABOUT 921 00:39:41,113 --> 00:39:42,781 THE ENDOCYTOSIS. 922 00:39:42,781 --> 00:39:45,851 THE ENDOCYTOSIS WE SEE HERE FROM 923 00:39:45,851 --> 00:39:47,452 WHOLE CELL POINT OF VIEW, MOST 924 00:39:47,452 --> 00:39:49,755 PEOPLE WOULD INTERPRET THAT AS 925 00:39:49,755 --> 00:39:56,228 THE FLAT TO O TRANSITIONS AS WE 926 00:39:56,228 --> 00:39:56,895 SEE HERE. 927 00:39:56,895 --> 00:39:59,531 WHAT WE SEE HERE AGAIN IS REALLY 928 00:39:59,531 --> 00:40:03,135 A SURPRISE TOWSES, THAT THE FLAT 929 00:40:03,135 --> 00:40:07,205 TO O TRANSITION IS MINIMAL FROM 930 00:40:07,205 --> 00:40:09,141 AFTER THE DEPOLARIZATION SH 931 00:40:09,141 --> 00:40:12,444 THESE TWO FORMS OF MECHANISMS 932 00:40:12,444 --> 00:40:14,947 ARE THE DOMINANT ONE TO FORM 933 00:40:14,947 --> 00:40:16,148 VESICLES RATHER THAN THE FLAT TO 934 00:40:16,148 --> 00:40:20,953 O TRANSITIONS. 935 00:40:20,953 --> 00:40:22,487 BECAUSE THIS IS A SURPRISE, I 936 00:40:22,487 --> 00:40:26,124 WOULD LIKE TO RECONFIRM THIS BY 937 00:40:26,124 --> 00:40:29,294 RECONSTRUCTING THE POPULATION 938 00:40:29,294 --> 00:40:32,030 EXO AND DOANCYTOSIS FROM 939 00:40:32,030 --> 00:40:33,131 INDIVIDUAL ENDOCYTOSIS HERE. 940 00:40:33,131 --> 00:40:35,200 THIS SHOWS YOU THE CELLS 941 00:40:35,200 --> 00:40:36,835 RECORDING, WHOLE CELL RECORDINGS 942 00:40:36,835 --> 00:40:41,206 HERE OF THE CAPACITANCE DECAYS, 943 00:40:41,206 --> 00:40:46,244 FAST DECAYS, AND EXO HYPHEN 944 00:40:46,244 --> 00:40:49,648 DOUGH FROM EACH INDIVIDUAL -- 945 00:40:49,648 --> 00:40:51,984 EXO AND ENDO, EACH OF THE CELLS 946 00:40:51,984 --> 00:40:53,618 WE SEE THE FUSION EVENTS. 947 00:40:53,618 --> 00:40:55,287 IF THERE'S A FUSION EVENT, WE 948 00:40:55,287 --> 00:40:58,457 GIVE UP STEPS. 949 00:40:58,457 --> 00:41:00,559 IF FUSION CLOSURE, WE GIVE DOWN 950 00:41:00,559 --> 00:41:01,760 STEP. 951 00:41:01,760 --> 00:41:06,031 IF NO FUSION CLOASH -- WE SEE 952 00:41:06,031 --> 00:41:08,433 TONS OF THEM IN ONE CELL AND WE 953 00:41:08,433 --> 00:41:09,935 ADD THEM HERE, THIS SHOWS THE 954 00:41:09,935 --> 00:41:11,403 NUMBER OF EVENTS, FUSION OPENING 955 00:41:11,403 --> 00:41:12,404 AND CLOSURES. 956 00:41:12,404 --> 00:41:14,673 SO WE CAN MIMIC THE CAPACITANCE 957 00:41:14,673 --> 00:41:19,378 INCREASE HERE. 958 00:41:19,378 --> 00:41:23,315 WE GIVE A DOWN STEP HERE AND WE 959 00:41:23,315 --> 00:41:24,983 ADD ALL OF THEM TOGETHER, WE SEE 960 00:41:24,983 --> 00:41:27,486 SOME FIVE TO TEN EVENTS HERE, 961 00:41:27,486 --> 00:41:29,888 REFLECTING THE PREFORMED OMEGA 962 00:41:29,888 --> 00:41:31,089 PROFILE CLOSING EVENTS. 963 00:41:31,089 --> 00:41:32,724 THEN WE ADD ALL THIS TOGETHER 964 00:41:32,724 --> 00:41:39,064 AND WE GET THE RED THAT SHOWS 965 00:41:39,064 --> 00:41:43,435 RESEMBLES CAPACITANCE RECORDED, 966 00:41:43,435 --> 00:41:46,538 AND WE SEE THE FUSION CLOSURE 967 00:41:46,538 --> 00:41:50,575 AND PREFORMED OMEGA CLOSURE IS 968 00:41:50,575 --> 00:41:53,412 RESPONSIBLE FOR THE SMALL 969 00:41:53,412 --> 00:41:55,280 ENDOCYTOSIS, FAST AND 970 00:41:55,280 --> 00:41:58,450 COMPENSATORY ENDOCYTOSIS. 971 00:41:58,450 --> 00:42:02,721 RATHER THAN THE FLAT TO ROUND. 972 00:42:02,721 --> 00:42:05,323 SO FLAT TO ROUND IS NOT THE 973 00:42:05,323 --> 00:42:07,459 PRIMARY MECHANISM, RATHER IT'S 974 00:42:07,459 --> 00:42:13,999 THE TWO HERE, FUSION CLOSURES 975 00:42:13,999 --> 00:42:14,699 AND OMEGA. 976 00:42:14,699 --> 00:42:17,869 SO I'VE SHOWN YOU VERY QUICKLY 977 00:42:17,869 --> 00:42:20,072 20 YEARS LAB IN VISUALIZING THE 978 00:42:20,072 --> 00:42:21,706 FUSION AND BUDDING 979 00:42:21,706 --> 00:42:22,908 TRANSFORMATIONS MECHANISMS AND 980 00:42:22,908 --> 00:42:24,209 FUNCTIONS WE HAVE STUDIED SO 981 00:42:24,209 --> 00:42:26,044 FAR, AND THIS IS QUITE DIFFERENT 982 00:42:26,044 --> 00:42:29,047 FROM THE CLASSICAL MODEL THAT WE 983 00:42:29,047 --> 00:42:30,449 STARTED WITH. 984 00:42:30,449 --> 00:42:31,783 FINALLY, I WOULD LIKE TO THANK 985 00:42:31,783 --> 00:42:36,588 MANY, MANY PEOPLE INVOLVED IN 986 00:42:36,588 --> 00:42:38,657 THIS WORK. 987 00:42:38,657 --> 00:42:39,558 [READING NAIMEDZ] 988 00:42:39,558 --> 00:42:41,526 INVOLVED IN ESTABLISHING THE 989 00:42:41,526 --> 00:42:43,495 WHOLE CELL AND CELL, MANY PEOPLE 990 00:42:43,495 --> 00:42:45,097 HERE, VERY IMPORTANT 991 00:42:45,097 --> 00:42:47,099 CONTRIBUTIONS TO OUR MANY YEARS 992 00:42:47,099 --> 00:42:54,106 OF STUDIES HERE. 993 00:42:54,106 --> 00:42:55,540 [READING NAMES] 994 00:42:55,540 --> 00:43:04,716 I WANT TO SINCERELY THANK. 995 00:43:04,716 --> 00:43:08,553 AND MANY OTHERS ARE INVOLVED IN 996 00:43:08,553 --> 00:43:11,923 THE IMAGING EXPERIMENTS. 997 00:43:11,923 --> 00:43:13,992 PROVIDED MANY OF THE VIDEOS, AND 998 00:43:13,992 --> 00:43:16,361 THESE ARE MY LAB MEMBERS. 999 00:43:16,361 --> 00:43:20,031 I WOULD ALSO LIKE TO THANK 1000 00:43:20,031 --> 00:43:22,667 CAROLYN SMITH FOR HER SUPPORT 1001 00:43:22,667 --> 00:43:25,070 AND MANY OF THE COLLABORATIONS I 1002 00:43:25,070 --> 00:43:26,571 MENTIONED IN MY TALK. 1003 00:43:26,571 --> 00:43:31,409 ALSO HUGE THANKS TO NINDS FOR 1004 00:43:31,409 --> 00:43:32,377 REALLY STRONG SUPPORT. 1005 00:43:32,377 --> 00:43:33,578 I ALMOST CANNOT IMAGINE ANY 1006 00:43:33,578 --> 00:43:37,182 OTHER PLACE THAT WILL ALLOW ME 1007 00:43:37,182 --> 00:43:38,850 TO TRANSFORM ELECTROPHYSIOLOGY 1008 00:43:38,850 --> 00:43:42,454 INTO AN IMAGING IN THE FRONT. 1009 00:43:42,454 --> 00:43:44,956 SO IMPORTANT, HUGE THINGS TO 1010 00:43:44,956 --> 00:43:46,291 NINDS, AND ALSO FINALLY THANK 1011 00:43:46,291 --> 00:43:48,126 YOU VERY MUCH FOR YOUR ALL 1012 00:43:48,126 --> 00:43:48,493 ATTENTION. 1013 00:43:48,493 --> 00:43:58,670 [APPLAUSE] 1014 00:44:04,876 --> 00:44:05,510 >> HI. 1015 00:44:05,510 --> 00:44:06,545 CAN YOU HEAR ME? 1016 00:44:06,545 --> 00:44:08,280 >> YES. 1017 00:44:08,280 --> 00:44:10,348 >> I HAVE PERHAPS A NAIVE 1018 00:44:10,348 --> 00:44:11,449 QUESTION, BECAUSE I KNOW NOTHING 1019 00:44:11,449 --> 00:44:13,618 ABOUT VESICLE FUSION, BUT WHAT 1020 00:44:13,618 --> 00:44:15,820 IS THE MECHANISM FOR THE 1021 00:44:15,820 --> 00:44:17,889 COMPOUND VESICLE FUSION? 1022 00:44:17,889 --> 00:44:18,990 BECAUSE I CAN UNDERSTAND LIKE 1023 00:44:18,990 --> 00:44:21,860 THE NORMAL VESICLE FUSION 1024 00:44:21,860 --> 00:44:24,029 INVOLVES, THE FORCE REQUIRED BY 1025 00:44:24,029 --> 00:44:25,263 THE SNARE COMPLEX AND CALCIUM. 1026 00:44:25,263 --> 00:44:27,199 >> YEAH. 1027 00:44:27,199 --> 00:44:29,301 THAT'S A VERY GOOD QUESTION. 1028 00:44:29,301 --> 00:44:30,502 I ACTUALLY DO NOT KNOW. 1029 00:44:30,502 --> 00:44:34,873 BUT WHAT WE KNOW IS THAT WHEN WE 1030 00:44:34,873 --> 00:44:41,246 KNOCK OUT TECH 1 -- SNARE 2 I'M 1031 00:44:41,246 --> 00:44:42,881 SORRY, WE SEE A DRAMATIC 1032 00:44:42,881 --> 00:44:44,082 REDUCTION OF THE COMPOUND 1033 00:44:44,082 --> 00:44:44,583 FUSIONS. 1034 00:44:44,583 --> 00:44:47,652 SO IT MEANS THAT IT WASN'T 1035 00:44:47,652 --> 00:44:49,721 REQUIRED CALCIUM SENSORS, AND 1036 00:44:49,721 --> 00:44:51,289 YOU'RE ASKING WHETHER THE 1037 00:44:51,289 --> 00:44:53,858 SNARES, I BELIEVE IT'S INVOLVED, 1038 00:44:53,858 --> 00:44:57,529 AND VESICLE HAS NOT BEEN KNOWN 1039 00:44:57,529 --> 00:45:00,232 TO CONTAIN SYNTACTIC SNARES BUT 1040 00:45:00,232 --> 00:45:03,902 I THINK SOME OF THEM SHOULD BE 1041 00:45:03,902 --> 00:45:06,972 THERE, OTHERWISE -- I CANNOT 1042 00:45:06,972 --> 00:45:08,039 IMAGINE WITHOUT SNARE THEY WOULD 1043 00:45:08,039 --> 00:45:08,240 FUSE. 1044 00:45:08,240 --> 00:45:11,676 >> A RELATED QUESTION ABOUT THE 1045 00:45:11,676 --> 00:45:14,946 PULLING FROM THE LAMBDA TO OMEGA 1046 00:45:14,946 --> 00:45:18,016 TRANSITIONS, I'M ASSUMING THE 1047 00:45:18,016 --> 00:45:19,985 ACTIN FILAMENT WOULD HAVE TO LET 1048 00:45:19,985 --> 00:45:21,953 GO OF THE VESICLE, OR DOES THAT 1049 00:45:21,953 --> 00:45:25,123 STAY ATTACHED AND THE PULLING 1050 00:45:25,123 --> 00:45:27,659 FORCE JUST CONTINUES? 1051 00:45:27,659 --> 00:45:29,060 12K3W4R50 IT'S A MODEL BECAUSE 1052 00:45:29,060 --> 00:45:32,664 WE SEE THE ACTIN FILAMENTS 1053 00:45:32,664 --> 00:45:34,532 THERE, AND IT APPEARS TO PULL 1054 00:45:34,532 --> 00:45:35,500 MEMBRANES, BUT WE DON'T KNOW 1055 00:45:35,500 --> 00:45:37,369 WHERE THE ACTIN HOLDS. 1056 00:45:37,369 --> 00:45:39,104 MAIB THE MICROTUBULE OR 1057 00:45:39,104 --> 00:45:42,307 SOMETHING AND HOW THE ACTIN 1058 00:45:42,307 --> 00:45:43,508 FILAMENT WOULD PULL THE 1059 00:45:43,508 --> 00:45:45,043 MEMBRANE, WE DO NOT KNOW AT THIS 1060 00:45:45,043 --> 00:45:45,343 POINT. 1061 00:45:45,343 --> 00:45:47,279 SO IF ANYONE HAS GOOD IDEAS, I 1062 00:45:47,279 --> 00:45:48,480 WOULD LOVE TO TAKE IT. 1063 00:45:48,480 --> 00:45:51,916 >> THANK YOU. 1064 00:45:51,916 --> 00:46:00,191 >> THANK YOU. 1065 00:46:00,191 --> 00:46:01,393 >> AGAIN. 1066 00:46:01,393 --> 00:46:03,495 THANKS FOR THAT. 1067 00:46:03,495 --> 00:46:05,463 THAT'S AN AMAZING ARC OF 1068 00:46:05,463 --> 00:46:09,167 ACHIEVEMENT OVER A LONG TIME. 1069 00:46:09,167 --> 00:46:11,036 IT'S JUST AMAZING TO SEE. 1070 00:46:11,036 --> 00:46:14,239 I THINK WE TAKE FOR GRANTED 1071 00:46:14,239 --> 00:46:16,074 PRETTY IMAGING PICTURES THESE 1072 00:46:16,074 --> 00:46:18,276 DAYS, BUT THE ACHIEVEMENT IS 1073 00:46:18,276 --> 00:46:18,510 AMAZING. 1074 00:46:18,510 --> 00:46:19,144 >> THANK YOU. 1075 00:46:19,144 --> 00:46:22,647 >> AS YOU ALLUDED TO, THE 1076 00:46:22,647 --> 00:46:25,517 ELECTROPHYSIOLOGY WASN'T SO EASY 1077 00:46:25,517 --> 00:46:25,750 EITHER. 1078 00:46:25,750 --> 00:46:28,320 IT'S VERY CLEVER HOW YOU TOOK 1079 00:46:28,320 --> 00:46:31,022 ADVANTAGE OF THE BLEACHING OF 1080 00:46:31,022 --> 00:46:34,359 THE RED DYE, AND TO SHOW THAT 1081 00:46:34,359 --> 00:46:36,127 THE VESICLE HAD CLOSED OFF. 1082 00:46:36,127 --> 00:46:38,296 IT GOT ME THINKING ABOUT THE 1083 00:46:38,296 --> 00:46:40,498 OTHER IMAGING THAT YOU WERE 1084 00:46:40,498 --> 00:46:40,799 DOING. 1085 00:46:40,799 --> 00:46:43,368 IS THERE ANY OTHER BLEACHING 1086 00:46:43,368 --> 00:46:44,102 GOING ON? 1087 00:46:44,102 --> 00:46:48,273 I MEAN, YOU HAVE THE DYNAMIN 1088 00:46:48,273 --> 00:46:49,708 WHICH IS IN THE SAME SORT OF 1089 00:46:49,708 --> 00:46:51,776 AREA, THE SPECTRUM, PRESUMABLY 1090 00:46:51,776 --> 00:46:53,311 FORMING A STRUCTURE AROUND, BUT 1091 00:46:53,311 --> 00:46:56,348 THAT DOESN'T BLEACH? 1092 00:46:56,348 --> 00:47:02,187 >> WE HAVE TO USE A VERY DIM 1093 00:47:02,187 --> 00:47:03,388 LIGHT SO THERE'S A BALANCE. 1094 00:47:03,388 --> 00:47:05,757 IF IT'S A STRONG LIGHT, THEN 1095 00:47:05,757 --> 00:47:07,525 VISION IS HIGHER BUT THEN YOU 1096 00:47:07,525 --> 00:47:10,362 BLEACH BEFORE YOU CAN SEE IT. 1097 00:47:10,362 --> 00:47:12,464 SO WE USE VERY STRONG INCREASE 1098 00:47:12,464 --> 00:47:16,534 OUR POWER TO 80% TO BLEACH THEM 1099 00:47:16,534 --> 00:47:18,169 VERY QUICKLY. 1100 00:47:18,169 --> 00:47:21,639 OTHERWISE, WE HAVE TO -- THE 1101 00:47:21,639 --> 00:47:23,208 DYNAMIN, IT'S MUCH LESS 1102 00:47:23,208 --> 00:47:24,075 BLEACHING, SO THEY ARE STILL 1103 00:47:24,075 --> 00:47:25,477 THERE AND SHOULD BE FINE. 1104 00:47:25,477 --> 00:47:25,677 YEAH. 1105 00:47:25,677 --> 00:47:27,912 >> SO JUST RELATED TO THAT, AND 1106 00:47:27,912 --> 00:47:30,749 I'M SORRY, YOUR TRICK THAT WAS 1107 00:47:30,749 --> 00:47:32,250 MAKING ME THINK ABOUT THIS. 1108 00:47:32,250 --> 00:47:33,585 BUT WHEN YOU SHOWED THAT THE 1109 00:47:33,585 --> 00:47:37,489 VESICLE KIND OF DISAPPEARS, IS 1110 00:47:37,489 --> 00:47:40,058 IT POSSIBLE THAT YOU COULD BE 1111 00:47:40,058 --> 00:47:41,259 RESTRICTING THE FLOW OF THE 1112 00:47:41,259 --> 00:47:43,161 LABEL OF IP3 OR WHATEVER IT IS 1113 00:47:43,161 --> 00:47:46,398 TO THAT AREA AND IT'S JUST 1114 00:47:46,398 --> 00:47:47,699 BLEACHING, OR -- 1115 00:47:47,699 --> 00:47:50,135 >> IS IT POSSIBLE? 1116 00:47:50,135 --> 00:47:53,405 SO WHEN THE DYE IS BLEACHING, WE 1117 00:47:53,405 --> 00:47:59,244 THINK IT'S BECAUSE IT'S CLOTHED 1118 00:47:59,244 --> 00:48:00,645 SO WE COULD ALSO -- SO YOUR 1119 00:48:00,645 --> 00:48:02,680 QUESTION IS, IS IT POSSIBLE THA- 1120 00:48:02,680 --> 00:48:04,849 >> I WAS WAITING TO SEE A 1121 00:48:04,849 --> 00:48:06,251 VESICLE THAT HAD BEEN FULLY 1122 00:48:06,251 --> 00:48:07,786 PINCHED OFF AND I JUST DON'T 1123 00:48:07,786 --> 00:48:08,420 REMEMBER SEEING THAT. 1124 00:48:08,420 --> 00:48:09,654 I WAS WONDERING WHETHER THAT 1125 00:48:09,654 --> 00:48:11,055 WOULD BLEACH VERY QUICKLY, AND 1126 00:48:11,055 --> 00:48:13,925 IF SO, COULD A SIMILAR THING 1127 00:48:13,925 --> 00:48:17,796 EXPLAIN YOUR DISAPPEARING -- 1128 00:48:17,796 --> 00:48:19,397 >> YEAH, VERY GOOD POINT. 1129 00:48:19,397 --> 00:48:21,232 SO WHEN WE SEE THE KISS AND 1130 00:48:21,232 --> 00:48:26,404 RUNS, WE ALWAYS ASSUME THAT THE 1131 00:48:26,404 --> 00:48:28,706 VESICLE IS DIGGING AWAY AND WE 1132 00:48:28,706 --> 00:48:30,341 TRY VERY HARD AND WE DON'T SEE 1133 00:48:30,341 --> 00:48:32,644 THEM, THEY ALL STAYED THERE, 1134 00:48:32,644 --> 00:48:34,612 SOME STRUCTURE STILL ATTACHED. 1135 00:48:34,612 --> 00:48:39,117 WHAT WE SEE IS THE DYE IS 1136 00:48:39,117 --> 00:48:39,984 BLEACHING -- THE VESICLE 1137 00:48:39,984 --> 00:48:41,419 MEMBRANE IS STILL THERE AND DYE 1138 00:48:41,419 --> 00:48:41,953 IS BLEACHING. 1139 00:48:41,953 --> 00:48:47,091 SO THE VESICLE STAYS THERE. 1140 00:48:47,091 --> 00:48:49,727 I HAVEN'T SEEN ONE IMAGE TO -- 1141 00:48:49,727 --> 00:48:52,564 SO HERE IS IMAGING, I THINK THIS 1142 00:48:52,564 --> 00:48:58,036 IS THE CLATHRIN HERE AND 1143 00:48:58,036 --> 00:49:01,773 UNDERNEATH HERE, AND AFTER 1144 00:49:01,773 --> 00:49:05,376 DEPOLARIZING, WE DON'T SEE THEM 1145 00:49:05,376 --> 00:49:07,879 FOR THE LARGE VESICLE. 1146 00:49:07,879 --> 00:49:09,547 LARGE VESICLES, THEY STAY THERE 1147 00:49:09,547 --> 00:49:10,748 FOR SOME TIME. 1148 00:49:10,748 --> 00:49:14,152 OCCASIONALLY WE SEE THAT DATA 1149 00:49:14,152 --> 00:49:19,491 BUT VERY FEW, AND I DON'T KNOW 1150 00:49:19,491 --> 00:49:20,625 WHY. 1151 00:49:20,625 --> 00:49:22,126 >> (AWAY FROM MICROPHONE) AROUND 1152 00:49:22,126 --> 00:49:23,761 THE VESICLE? 1153 00:49:23,761 --> 00:49:26,831 >> THAT SUGGESTED CLATHRIN, THIS 1154 00:49:26,831 --> 00:49:30,101 ONE, YEAH, COULD BE CLATHRIN, 1155 00:49:30,101 --> 00:49:32,203 THIS BINDS TO CLATHRIN AS WELL 1156 00:49:32,203 --> 00:49:40,879 AS THE LIPID PITP, AND THEN IT 1157 00:49:40,879 --> 00:49:41,179 DISAPPEARS. 1158 00:49:41,179 --> 00:49:43,948 >> SO MUCH FOR YOUR BEAUTIFUL 1159 00:49:43,948 --> 00:49:44,249 TALK. 1160 00:49:44,249 --> 00:49:45,350 -- THANK YOU SO MUCH FOR YOUR 1161 00:49:45,350 --> 00:49:46,451 BEAUTIFUL TALK. 1162 00:49:46,451 --> 00:49:47,886 I WAS THINKING ABOUT THE 1163 00:49:47,886 --> 00:49:50,088 CLATHRIN YOU SHOWED BENEATH THE 1164 00:49:50,088 --> 00:49:50,321 VESICLE. 1165 00:49:50,321 --> 00:49:52,056 THIS IS A NEUROSCIENCE SEMINAR 1166 00:49:52,056 --> 00:49:53,157 SERIES, SO DO YOU THINK WE'RE 1167 00:49:53,157 --> 00:49:54,792 JUST MISS GOING IN NEURONS AND 1168 00:49:54,792 --> 00:49:56,528 THAT IT'S ALSO PRESENT AT THE 1169 00:49:56,528 --> 00:50:00,598 PORE IN NEURONS, SYNAPTIC AS 1170 00:50:00,598 --> 00:50:00,932 WELL? 1171 00:50:00,932 --> 00:50:01,633 >> VERY GOOD POINTED. 1172 00:50:01,633 --> 00:50:03,134 THE QUESTION IS WHETHER THE 1173 00:50:03,134 --> 00:50:04,569 CLATHRIN FOR THE SMALL VESICLE 1174 00:50:04,569 --> 00:50:08,640 RS THERE ALSO IN THE BASE OR NOT 1175 00:50:08,640 --> 00:50:13,545 1234 I DO NOT KNOW. 1176 00:50:13,545 --> 00:50:16,814 BUT THE SIMULATION A LONG TIME 1177 00:50:16,814 --> 00:50:21,419 AGO IN 2003, 20 YEARS AGO, 1178 00:50:21,419 --> 00:50:23,821 SHOWING THAT CLATHRIN ON THIS 1179 00:50:23,821 --> 00:50:25,590 COATED AREA CAN ALSO CONSTRICT 1180 00:50:25,590 --> 00:50:27,525 THE MEMBRANE, SO THEY DON'T NEED 1181 00:50:27,525 --> 00:50:32,397 TO BE ON THE BASE. 1182 00:50:32,397 --> 00:50:33,898 IF THEY ARE ONLY AT THE HEAD 1183 00:50:33,898 --> 00:50:35,133 THEY CAN ALSO CONSIDER THE PORE, 1184 00:50:35,133 --> 00:50:44,108 SO ALSO IT'S SHOWN HERE THAT TH, 1185 00:50:44,108 --> 00:50:48,046 SHOWING THE PIT CAN ALSO BE -- 1186 00:50:48,046 --> 00:50:50,682 THE CLATHRIN CAN BLOCK THE VERY 1187 00:50:50,682 --> 00:50:52,216 WELL KEHL PINCH SO I THINK IT'S 1188 00:50:52,216 --> 00:50:55,720 ALSO INVOLVED IN THE SMALL 1189 00:50:55,720 --> 00:50:57,188 VESICLES PINCH PROCESS. 1190 00:50:57,188 --> 00:50:59,524 >> HI LING GANG. 1191 00:50:59,524 --> 00:51:00,525 THANKS FOR THE GREAT 1192 00:51:00,525 --> 00:51:00,858 PRESENTATION. 1193 00:51:00,858 --> 00:51:02,393 I THINK YOU BROKE THE WORLD 1194 00:51:02,393 --> 00:51:03,795 RECORD ON AVERAGING. 1195 00:51:03,795 --> 00:51:07,131 I'VE NEVER SEEN A SIGNAL WITH 1196 00:51:07,131 --> 00:51:08,366 300,000 AVERAGING, SO THANK YOU 1197 00:51:08,366 --> 00:51:09,601 SO MUCH FOR THE BEAUTIFUL WORK. 1198 00:51:09,601 --> 00:51:10,835 I HAVE A COUPLE QUESTIONS. 1199 00:51:10,835 --> 00:51:13,905 ONE IS THAT SOME OF THIS 1200 00:51:13,905 --> 00:51:15,740 MOVEMENT -- HOW DO YOU DEAL WITH 1201 00:51:15,740 --> 00:51:19,711 MOVEMENT IN THE SORT OF XY 1202 00:51:19,711 --> 00:51:19,978 DIRECTION? 1203 00:51:19,978 --> 00:51:21,346 YOU KNOW, DO THINGS BASICALLY 1204 00:51:21,346 --> 00:51:22,213 STAY THE SAME? 1205 00:51:22,213 --> 00:51:25,049 1K3 THEN THE SECOND THING IS, I 1206 00:51:25,049 --> 00:51:29,754 THOUGHT THAT THE MODEL WITH THE 1207 00:51:29,754 --> 00:51:30,955 OSMOLARITY WAS 1208 00:51:30,955 --> 00:51:31,356 SUPER-INTERESTING. 1209 00:51:31,356 --> 00:51:33,124 SO DOES THAT MEAN THAT INSIDE 1210 00:51:33,124 --> 00:51:37,528 THE VESICLE YOU PRODUCE LIKE A 1211 00:51:37,528 --> 00:51:39,297 HYPOTONIC -- HOW IS THAT REALLY 1212 00:51:39,297 --> 00:51:39,731 WORKING? 1213 00:51:39,731 --> 00:51:41,466 SO YOU HAVE SOMETHING THAT'S 1214 00:51:41,466 --> 00:51:42,433 HYPOTONIC INSIDE SO THAT THE 1215 00:51:42,433 --> 00:51:46,971 SOLUTION IS MORE LIKELY TO 1216 00:51:46,971 --> 00:51:47,305 LEAVE? 1217 00:51:47,305 --> 00:51:48,940 THE CONTROL FOR SORT OF MAKING 1218 00:51:48,940 --> 00:51:52,677 THE VESICLE SMALLER USING THE 1219 00:51:52,677 --> 00:51:55,513 OSMOLARITY INSIDE THE VESICLE, 1220 00:51:55,513 --> 00:51:57,582 INSIDE THE CELL, INSIDE THE 1221 00:51:57,582 --> 00:52:01,285 VESICLE, I'M ASSUMING 1234. 1222 00:52:01,285 --> 00:52:03,154 >> IT'S SOLUTIONS. 1223 00:52:03,154 --> 00:52:04,589 SO IT'S THE SECOND QUESTION. 1224 00:52:04,589 --> 00:52:12,363 SO WE APPLY THE DIFFERENT OSMO 1225 00:52:12,363 --> 00:52:18,970 SOLUTION OUTSIDE THE CELL. 1226 00:52:18,970 --> 00:52:22,106 WE HAVE THE IMAGES I DID NOT 1227 00:52:22,106 --> 00:52:25,643 SHOW YOU, BUT THE CELLS CRUMBLE 1228 00:52:25,643 --> 00:52:27,912 AND TENSION IS LOSE, WE ALSO 1229 00:52:27,912 --> 00:52:28,913 MEASURED THE TENSION AFTER THAT 1230 00:52:28,913 --> 00:52:31,849 AND WE SEE THE TENSION REDUCED. 1231 00:52:31,849 --> 00:52:36,020 THEN WE APPLY THE HYPOTONIC, 1232 00:52:36,020 --> 00:52:37,889 WHERE IT GETS INTO THE CELL AND 1233 00:52:37,889 --> 00:52:40,725 WE SEE THE CELL SWELLING AND THE 1234 00:52:40,725 --> 00:52:44,028 TENSION IS HIGHER. 1235 00:52:44,028 --> 00:52:47,331 WE MEASURED THE TENSION WITH 1236 00:52:47,331 --> 00:52:52,570 PIPETTE ASPIRATION AND SO I 1237 00:52:52,570 --> 00:52:53,971 THINK IT SHOULD BE RIGHT, WE 1238 00:52:53,971 --> 00:52:54,706 HAVE EVIDENCE TO SUPPORT. 1239 00:52:54,706 --> 00:52:56,841 >> SO YOU THINK THAT'S A NORMAL 1240 00:52:56,841 --> 00:52:58,476 MECHANISM BY WHICH HOW THE 1241 00:52:58,476 --> 00:52:59,844 VESICLE GETS SMALLER? 1242 00:52:59,844 --> 00:53:02,547 >> MY VESICLE GETS SMALLER. 1243 00:53:02,547 --> 00:53:06,718 IT CHANGE THE TENSIONS. 1244 00:53:06,718 --> 00:53:09,020 SO THE TENSION AT THE MEMBRANE, 1245 00:53:09,020 --> 00:53:10,555 PLASMA MEMBRANE IS HIGH. 1246 00:53:10,555 --> 00:53:13,057 NOW, IF YOU REDUCE THE MEMBRANE 1247 00:53:13,057 --> 00:53:16,994 TENSIONS, THEN THIS LOWER 1248 00:53:16,994 --> 00:53:22,900 MEMBRANE TENSION, THE -- THE 1249 00:53:22,900 --> 00:53:25,470 VESICLE MEMBRANE, TO LOWER THIS 1250 00:53:25,470 --> 00:53:28,706 VESICLE MEMBRANE, YOU NEED 1251 00:53:28,706 --> 00:53:30,308 HIGHER MEMBER TENSION, SO 1252 00:53:30,308 --> 00:53:32,310 THERE'S A DIFFERENT TENSION AT 1253 00:53:32,310 --> 00:53:33,978 THE PLASMA MEMBRANE AND THE 1254 00:53:33,978 --> 00:53:36,781 FUSION VESICLE AND THAT PROVIDES 1255 00:53:36,781 --> 00:53:38,983 THE DRIVING FORCE TO LOWER THE 1256 00:53:38,983 --> 00:53:41,753 MEMBRANE TENSION, LIKE WATER 1257 00:53:41,753 --> 00:53:43,588 FORCING INTO THE FOLD, ACCORDING 1258 00:53:43,588 --> 00:53:44,889 TO THE SIMULATION. 1259 00:53:44,889 --> 00:53:49,293 I'M JUST TRANSLATING MY 1260 00:53:49,293 --> 00:53:49,861 COLLABORATORS. 1261 00:53:49,861 --> 00:53:55,700 AND FOR THE FIRST QUESTION -- 1262 00:53:55,700 --> 00:54:00,304 >> (AWAY FROM MICROPHONE). 1263 00:54:00,304 --> 00:54:02,206 >> MY FIRST TWO PAPERS, I GOT 1264 00:54:02,206 --> 00:54:03,641 THIS QUESTION, AND WE HAD TO 1265 00:54:03,641 --> 00:54:05,510 SPEND HALF A YEAR TO ONE YEAR TO 1266 00:54:05,510 --> 00:54:08,379 ADDRESS THAT PARTICULAR 1267 00:54:08,379 --> 00:54:08,646 QUESTION. 1268 00:54:08,646 --> 00:54:11,649 THE QUESTION IS WHETHER WHAT WE 1269 00:54:11,649 --> 00:54:17,755 SEE IS SIMPLY THE MOVEMENT AWAY. 1270 00:54:17,755 --> 00:54:21,058 IT'S NOT BECAUSE XY AND XZ AND I 1271 00:54:21,058 --> 00:54:22,160 DO NOT HAVE THE DATA HERE TO 1272 00:54:22,160 --> 00:54:25,463 SHOW YOU, BUT WHAT WE'RE TRYING 1273 00:54:25,463 --> 00:54:33,204 TO DO IS DO THE 3D'S, INSTEAD OF 1274 00:54:33,204 --> 00:54:35,606 SINGLE, SO EVEN THOUGH THEY ARE 1275 00:54:35,606 --> 00:54:36,707 MOVING, WE WILL KNOW. 1276 00:54:36,707 --> 00:54:40,411 THE ANSWER IS THEY ARE NOT THE 1277 00:54:40,411 --> 00:54:41,212 MOVEMENTS. 1278 00:54:41,212 --> 00:54:43,381 BUT BECAUSE THEY CAN BE BLOCKED. 1279 00:54:43,381 --> 00:54:45,349 IF THEY ARE MOVEMENT, IT CANNOT 1280 00:54:45,349 --> 00:54:47,952 BE BLOCKED BY ANYTHING. 1281 00:54:47,952 --> 00:54:57,161 AND THEN OTHER 3D MEASUREMENTS. 1282 00:54:57,161 --> 00:54:59,130 THANK YOU VERY MUCH. 1283 00:54:59,130 --> 00:55:09,307 [APPLAUSE]