The Dynamic Mitotic Spindle
Transcript of Part 1: The Dynamic Mitotic Spindle
00:00:17;01 My name is Shinya Inoue. 00:00:19;29 I'm going to start off by showing you a movie that I took 00:00:23;01 and then explain what the movie means as we go along. 00:00:26;12 Here we're seeing a dividing pollen mother cell of an Easter lily. 00:00:32;08 And, the movie is showing structures that people had not seen inside living cells before 00:00:41;05 because scientists had been fixing cells in order to visualize those structures, 00:00:48;17 if they could see them. Here, we see the fibers pulling chromosomes apart, 00:00:54;01 and then new filaments appearing between the separating sets of chromosomes 00:01:01;05 and laying down the cell plate. 00:01:03;12 And, the significance of all this is why looking at the living cell, 00:01:12;04 we can not only tell where the fibers are 00:01:16;14 and what they're doing, but tell what the molecules are doing inside them. 00:01:22;00 Especially what I'd like to talk about is about how cells divide, 00:01:27;15 and what we found out about how molecules come together or fall apart, 00:01:33;14 and how this plays a very important role in terms of how cells divide. 00:01:38;16 So, how did this whole thing happen? 00:01:42;06 Well, it happened because I met Professor Katsuma Dan. 00:01:47;12 He was a very unusual teacher 00:01:51;03 because he had taken his PhD at the University of Pennsylvania 00:01:55;15 and had come back to Japan in 1937 with his American wife, 00:02:01;26 and then I was in the first class that he ever taught in Japan. 00:02:07;18 And, what he did was so different from the other Japanese high schools that I attended. 00:02:16;13 He even let us not do experiments that he had prescribed for us, 00:02:25;06 but let us try things that were of interest to us in the lab. 00:02:30;18 And so what I did was to try Lilly's iron wire model; of nerve conduction. 00:02:39;20 And this was such an interesting project that this really took me into biology. 00:02:44;14 And then a few years later, when I met KD at his Injean Dans; home, 00:02:51;03 he showed me this book which is on the right-hand side. 00:02:54;26 This was by W J Schmidt in Essen, Germany, who had a picture that's shown in the middle. 00:03:01;02 Those were of sea urchin eggs in which you see these bright or dark structures. 00:03:07;23 Schmidt thought that those were chromosomes when he wrote this book, 00:03:12;02 but in 1939, he revised his view and said those were actually mitotic spindles. 00:03:19;25 So, this is of interest to us, especially to Katsuma Dan, 00:03:24;18 because Dan thought that the spindle elongating 00:03:28;13 was what was responsible for what divided cells. 00:03:32;10 So, he said, "Let's try and repeat this experiment of Schmidt's." 00:03:36;26 And we tried once in the dark, but it didn't work. 00:03:40;22 After the war, we got back together in Misaki at the Marine Biological Laboratory, 00:03:46;23 after he recovered it from the occupation army. 00:03:51;03 And I built this microscope, which is a polarizing microscope, 00:03:56;14 somewhat different from what is used in mineralogy. 00:03:59;21 And this has a very good extinction polarizer and analyzer, 00:04:04;26 and a compensator, and so on. 00:04:06;27 Very bright light source, which makes it easier to see the various things inside living cells. 00:04:13;13 And so, using this, we were able to see images somewhat better than what W J Schmidt saw 00:04:20;28 and were able to follow what was going on inside dividing cells. 00:04:25;27 So, this is what started me on the whole quest for following living cells 00:04:31;16 and asking with the polarizing microscope, "What are molecules doing inside yourself?" 00:04:38;21 The microscope that is shown here is what I built when I came to Princeton in 1948 00:04:48;18 as a graduate student. 00:04:50;24 And with the microscope, I was able to show what part of the cell was birefringent. 00:05:02;12 Birefringence is a word I'll keep on using that means 00:05:05;27 it has different optical properties than the rest, 00:05:08;24 which you can see by using a polarized light microscope. 00:05:12;19 And by using polarized light, not only can you visualize a structure that is birefringent, 00:05:20;27 but birefringence tells us how molecules are lined up, how they change, and so on. 00:05:27;08 So, this is the special kind of microscope that I built for that purpose. 00:05:32;27 Now, with that, what I was able to do was to, first of all, show that in dividing cells, 00:05:43;23 there are actually fibers that are pulling chromosomes apart during cell division. 00:05:49;12 And this was important because when I first came, it was argued back and forth, 00:05:55;13 even though many people had studied cells after fixation, 00:06:00;27 that the cells may not have been quite happy or alive. 00:06:06;07 And so we needed to find a condition where the cells were alive 00:06:11;18 and still see the fibers that pulled the chromosomes, 00:06:15;10 and those were not visible except by using polarized light, 00:06:19;14 and the polarizing microscopes that were available 00:06:22;14 were not good enough to show the details, 00:06:26;25 so this is why I built the microscope. 00:06:28;10 Now, what can we see here? The bright structures are the birefringent spindle fibers 00:06:35;15 which had been thought to be present, but not necessarily proven. 00:06:40;08 And those are pulling the chromosomes apart, 00:06:43;11 and after the chromosomes are pulled apart, then you see some filaments, 00:06:47;08 which in the case of plant cells, lay down the cell plate, 00:06:51;23 and that divides the cell into two. 00:06:54;08 So, after seeing this, even the skeptics could not argue anymore 00:06:59;03 that these were artifacts of fixation, 00:07:02;01 but were really present inside the living cells. 00:07:05;04 That was fun enough as it was, but what was really interesting to me 00:07:12;09 was to find out that these birefringent structures 00:07:16;01 were not just there to move chromosomes and so on, 00:07:20;00 but had some very intriguing properties. They would come and go. 00:07:24;07 Here's a dividing sea urchin egg, which is about to divide, 00:07:28;29 and you see the birefringent spindle in the middle. 00:07:32;08 But now I'm going to drop the temperature, and raise it again, drop it, and raise it, 00:07:38;00 and as you see, this is a time-lapse movie, each time I drop the temperature, 00:07:42;18 then the birefringence just disappears. 00:07:46;04 What it means is that the molecules are not bound there together tightly, 00:07:51;23 but can fall apart very easily. 00:07:54;07 Again, dropping the temperature, and this doesn't affect what the cell does. 00:07:58;24 It's perfectly happy and keeps on going. Drop the temperature, raise again. 00:08:03;26 So, we can keep on doing this experiment over and over again. 00:08:07;28 This tells us that the molecules that make up these fibers are in a dynamic equilibrium 00:08:14;25 with something... they can either fall apart or be put together again, 00:08:19;03 and we'll see more of this in the next slide. 00:08:22;15 Here, what we see is the effect of colchicine. 00:08:27;16 It's a well-known drug, known from Egyptian tombs, even, 00:08:32;27 which was used for treating gout. 00:08:36;26 But more recently, it's been used for collecting chromosomes, 00:08:41;28 because one can collect metaphase chromosomes and use this for diagnosing 00:08:49;07 how chromosomes... whether they are normal or not. 00:08:54;03 Now, what happened with colchicine is, when I applied colchicine to living cells... 00:09:01;09 In this case, it's a oocyte -- a cell that forms an egg -- of a marine worm, Chaetopterus. 00:09:13;27 These are my favorite material because the cell stays in metaphase 00:09:19;08 unless you stimulate it to go further. 00:09:21;15 So, it has a metaphase spindle built in. 00:09:25;28 But, then when you apply colchicine, then as you see in the lower row, 00:09:32;01 the birefringence gradually disappears in a few minutes. 00:09:36;19 And then, if you use a lower concentration, then as you see in the upper row, 00:09:42;09 then not only does the birefringence disappear, 00:09:45;06 but the spindle gets shorter and shorter and shorter, 00:09:48;19 and at the same time, it pulls the chromosomes to the cell surface. 00:09:53;15 So, from this, I concluded that colchicine, just like cold, 00:10:02;06 is one of the agents that makes the spindle material fall apart. 00:10:07;24 But what's interesting is that, as the molecules are falling apart, 00:10:12;18 they can generate force for pulling. This is a very strange concept 00:10:17;15 that you can generate pulling force that will pull chromosomes 00:10:24;00 and the inner spindle pole to the cell surface. 00:10:28;07 And then if you take the colchicine away, 00:10:30;29 the reverse happens -- the molecules come back together, 00:10:34;10 and it pushes the chromosomes and the inner pole towards the middle. 00:10:38;26 So, this gave rise to the whole concept that molecules that are falling apart can actually 00:10:45;28 generate pulling forces. 00:10:48;12 Now, this seems so strange. It took 20 years until, in the test tube, 00:10:55;04 this was proven to be correct. 00:10:57;13 One other graduate student working with us, Howard Fuhr;, did another experiment. 00:11:03;25 He used a small spot of ultraviolet light, as you see in the top, second to the left panel... 00:11:14;09 a bright spot of ultraviolet light. 00:11:17;06 When you shine a bright microbeam of ultraviolet light on the spindle 00:11:22;07 and watch the birefringence, then you see that spot itself loses birefringence, 00:11:28;29 and you develop an area of reduced birefringence -- Arb as you see there. 00:11:34;01 What was really surprising is that this Arb then gradually migrates to the spindle pole 00:11:42;10 and then disappears, which means that there must be some 00:11:47;08 movement of the spindle material 00:11:49;28 from the chromosomes towards the spindle pole. 00:11:53;04 And while this is going on, the part between the chromosomes and the Arb, 00:11:59;20 and between the Arb and the pole, 00:12:02;05 the birefringence doesn't change 00:12:04;01 which meant that there must be a microtubule organizing center 00:12:09;22 both at the chromosomes and at the spindle poles. 00:12:15;01 So, this was quite a revolution... a revelation. 00:12:18;17 And, when we put all of this together, 00:12:22;20 then as Ted and I put together the summary article later on, 00:12:28;04 the spindle has organizing centers both at the spindle poles 00:12:34;29 and right at the chromosome kinetochore. 00:12:38;07 And then those are both responsible for lining up microtubule material, tubulin, 00:12:44;21 but the tubulin is constantly flowing from the chromosomes towards the spindle pole. 00:12:50;21 And as Tim Mitchison and others showed later on, this occurs very, very rapidly. 00:12:56;22 But, in spite of this, then if we apply cold or colchicine, 00:13:02;01 or hydrostatic pressure, that this whole equilibrium is shifted towards depolymerization, 00:13:07;26 so in spite of all this flow and so on taking place, we get a shorter spindle 00:13:14;01 and the microtubules fall apart and form tubulin. Again, this is completely reversible. 00:13:19;20 So, we have a dynamic equilibrium between microtubules 00:13:24;16 which are flowing from the chromosomes towards the pole, 00:13:27;28 and then which can be made to fall apart or come back together, 00:13:35;01 so this forms one of the major current concepts of how spindles work. 00:13:40;03 Of course, there are motor protein molecules, 00:13:46;13 in addition to this, which also play important roles. 00:13:50;01 And, as people have found out recently, even at the kinetochore itself, 00:13:57;05 on the chromosome, there are 40 different protein molecules 00:14:03;13 that are organizing the spindle microtubules. 00:14:07;28 So, the whole story gets more and more complex 00:14:10;25 and is not as simple as I portrayed it initially. 00:14:15;14 But, nevertheless, the whole general scheme seems to hold. 00:14:19;08 And finally, as a summary reflection, polarized light and... 00:14:27;04 I didn't get a chance to talk about video microscopy, 00:14:30;22 but both of these combined together have allowed us to probe the dynamic behavior 00:14:36;24 of cell architecture and the structural molecules which are far smaller 00:14:42;11 than the resolution limit of the light microscope. 00:14:45;07 And we can do so directly and non-destructively in living cells. 00:14:50;15 Of course, there are non-destructive methods these days. 00:14:54;07 But, these are especially powerful approaches, 00:14:58;02 and still I believe that we've only scratched the surface. 00:15:01;24 Now, I look forward to further developments based on key insights 00:15:07;28 into the interaction of polarized light, 00:15:10;00 (which is an electromagnetic wave) with matter, 00:15:13;09 and the broader biological explanation through which distinct living cells 00:15:20;16 which may not be ordinarily used can teach us the finer mechanisms 00:15:25;25 underlying the mysteries of nature and of life itself. 00:15:29;22 So, this is my summary reflection. Thank you very much for listening.