1 00:00:12,000 --> 00:00:19,000 So today's lecture talks about the cell cycle, the control of the cell 2 00:00:19,000 --> 00:00:27,000 cycle, and also cell death. So obviously, cell division is 3 00:00:27,000 --> 00:00:34,000 extremely important in multi-cellular organisms. 4 00:00:34,000 --> 00:00:42,000 We've talked a fair bit about control of the cell cycle, 5 00:00:42,000 --> 00:00:50,000 in terms of mitosis and meiosis, in earlier lectures. 6 00:00:50,000 --> 00:00:54,000 Obviously, cell division is necessary in a variety of 7 00:00:54,000 --> 00:00:59,000 circumstances, probably the most obvious is 8 00:00:59,000 --> 00:01:03,000 development. You go from one cell to ten to the thirteenth to ten to 9 00:01:03,000 --> 00:01:08,000 the fourteenth cells. That's accomplished by a tremendous 10 00:01:08,000 --> 00:01:13,000 amount of cell division that goes on over your gestational period. 11 00:01:13,000 --> 00:01:17,000 I've also pointed out, in fact we talked about it last time, 12 00:01:17,000 --> 00:01:22,000 that cell, that wound healing is, in part, a process of cell division. 13 00:01:22,000 --> 00:01:27,000 Fibroblasts for example, 14 00:01:27,000 --> 00:01:31,000 and other epithelial cells, get recruited to divide, in order to 15 00:01:31,000 --> 00:01:36,000 repair damage to a tissue. 16 00:01:36,000 --> 00:01:41,000 And even without damage, there's a lot of cell turnover, 17 00:01:41,000 --> 00:01:46,000 for many tissues. You may not realize this, but your blood, 18 00:01:46,000 --> 00:01:51,000 for example, turns over about every month, so you have to replace all 19 00:01:51,000 --> 00:01:57,000 your red blood cells, and all your white blood cells, 20 00:01:57,000 --> 00:02:01,000 and so on, periodically. Cells in other tissues, 21 00:02:01,000 --> 00:02:05,000 in the skin for example, your skin cells are born, 22 00:02:05,000 --> 00:02:09,000 they migrate up to the superficial layers of your skin, 23 00:02:09,000 --> 00:02:13,000 and then they get sloughed off, and of course, they have to be 24 00:02:13,000 --> 00:02:16,000 replaced. There's a lot of cell division that's going on naturally, 25 00:02:16,000 --> 00:02:20,000 in the process of what we call homeostasis, and to give you an 26 00:02:20,000 --> 00:02:24,000 example of that, if you consider your intestines in a 27 00:02:24,000 --> 00:02:28,000 human, your intestines undergo ten to the eleventh cell divisions per 28 00:02:28,000 --> 00:02:31,000 day. That's a remarkable number. Ten to the eleventh cells dividing 29 00:02:31,000 --> 00:02:35,000 in your intestines per day. If you imagine that a cell in your 30 00:02:35,000 --> 00:02:39,000 intestine is about ten microns in diameter, if you lined up all the 31 00:02:39,000 --> 00:02:43,000 cells next to each other that were born in a given period of time, 32 00:02:43,000 --> 00:02:46,000 in 38 days you would have enough cells lined up end-to-end to go 33 00:02:46,000 --> 00:02:50,000 around the earth, and in a year you'd have enough 34 00:02:50,000 --> 00:02:54,000 cells to make it from the earth to the moon. So you produce a 35 00:02:54,000 --> 00:02:58,000 tremendous number of cells just naturally, in the process of organ 36 00:02:58,000 --> 00:03:02,000 function and homeostasis. Of course we can have too much cell 37 00:03:02,000 --> 00:03:08,000 division, and this is pathological condition, which we typically 38 00:03:08,000 --> 00:03:14,000 associate with cancer. And indeed, many of the factors 39 00:03:14,000 --> 00:03:19,000 that I'm going to talk to you about today, that relate to the normal 40 00:03:19,000 --> 00:03:25,000 control of cell division, and also cell death, are perturbed 41 00:03:25,000 --> 00:03:31,000 in the development of cancer. Cancer, as you almost certainly 42 00:03:31,000 --> 00:03:37,000 know, is a disease of too many cells, and a major factor in that is 43 00:03:37,000 --> 00:03:41,000 excessive cell division. [UNINTELLIGIBLE PHRASE]. 44 00:03:41,000 --> 00:03:44,000 So this happens to be a human cancer cell line growing in tissue 45 00:03:44,000 --> 00:03:47,000 culture, and one of the hallmark features of cancer cells is that 46 00:03:47,000 --> 00:03:49,000 they have, in a sense, unlimited and unregulated cell 47 00:03:49,000 --> 00:03:52,000 division. So if you were to watch this movie, which actually comes 48 00:03:52,000 --> 00:03:55,000 from your book, supplementary materials from your 49 00:03:55,000 --> 00:03:58,000 book, you could watch these cells dividing, and they would do so in an 50 00:03:58,000 --> 00:04:02,000 unnatural fashion. Without response to proper growth 51 00:04:02,000 --> 00:04:07,000 factors that would stimulate cell division, they would divide on top 52 00:04:07,000 --> 00:04:12,000 of one another, which normal cells don't do, 53 00:04:12,000 --> 00:04:17,000 and other places and times when other cells are kept in check, 54 00:04:17,000 --> 00:04:22,000 cancer cells are not. So the basic process, which again is well 55 00:04:22,000 --> 00:04:27,000 familiar to you, is to take a single cell and turn it 56 00:04:27,000 --> 00:04:36,000 into two. 57 00:04:36,000 --> 00:04:40,000 And we discussed the very last stages of this in earlier lectures 58 00:04:40,000 --> 00:04:44,000 on mitosis, how the chromosomes get divided from the mother to the two 59 00:04:44,000 --> 00:04:48,000 daughter cells. We also have briefly alluded to the 60 00:04:48,000 --> 00:04:52,000 fact that there's a second, critical event, and you talked about 61 00:04:52,000 --> 00:04:56,000 the mechanisms of DNA replication. The DNA needs to get duplicated so 62 00:04:56,000 --> 00:05:00,000 that it can be properly divided, and this, as you know, occurs during 63 00:05:00,000 --> 00:05:04,000 a particular phase of the cell cycle, known as S-phase, for 64 00:05:04,000 --> 00:05:08,000 synthesis phase. And then there's chromosome 65 00:05:08,000 --> 00:05:13,000 segregation, and this occurs in a distinct phase of the cell cycle 66 00:05:13,000 --> 00:05:19,000 called M-phase, or mitosis phase. 67 00:05:19,000 --> 00:05:24,000 And the development of cells over the course of, 68 00:05:24,000 --> 00:05:29,000 say development, or in wound healing, 69 00:05:29,000 --> 00:05:35,000 can be thought of as a successive iteration of DNA duplication, 70 00:05:35,000 --> 00:05:40,000 DNA replication, and chromosome segregation. So in a sense, 71 00:05:40,000 --> 00:05:46,000 you go from mitosis to S-phase to mitosis to S-phase. 72 00:05:46,000 --> 00:05:50,000 These segments are separated by periods of time when cells are 73 00:05:50,000 --> 00:05:55,000 accumulating the biomaterials that they need, basically to function, 74 00:05:55,000 --> 00:06:00,000 and also to carry out the next phases of the cell cycle, 75 00:06:00,000 --> 00:06:04,000 and these are refereed to as gap phases, G1 for the one that 76 00:06:04,000 --> 00:06:09,000 separates mitosis from S-phase, and G2, the one that separates the 77 00:06:09,000 --> 00:06:14,000 S-phase from mitosis. Now, this is a cyclic process, 78 00:06:14,000 --> 00:06:19,000 one cell gives rise to two, that then undergoes this process again, 79 00:06:19,000 --> 00:06:24,000 and so we think of these things as cycles from M-to-S, 80 00:06:24,000 --> 00:06:29,000 connected by these gap phases, G1 and G2. Now there's another 81 00:06:29,000 --> 00:06:34,000 phase that we haven't told you about, which many of your cells are 82 00:06:34,000 --> 00:06:40,000 actually in right now, and that's a phase called G0. 83 00:06:40,000 --> 00:06:44,000 This is a resting phase, and this can be either permanent or 84 00:06:44,000 --> 00:06:49,000 temporary. Many of your cells, when they're born, will stop 85 00:06:49,000 --> 00:06:54,000 dividing forever. Cells in your brain, 86 00:06:54,000 --> 00:06:59,000 for example, or most cells in your brain, cells of your cardiac muscle, 87 00:06:59,000 --> 00:07:04,000 and there are many other examples. Once they get born, 88 00:07:04,000 --> 00:07:09,000 they undergo mitosis, they go into this resting phase, 89 00:07:09,000 --> 00:07:14,000 and they'll never come back out again. 90 00:07:14,000 --> 00:07:17,000 And that's why it's difficult, for example, to deal with brain 91 00:07:17,000 --> 00:07:21,000 injuries, or spinal cord injuries, because those cells cannot be 92 00:07:21,000 --> 00:07:24,000 recruited back into the cell cycle, they can't make more of them. But 93 00:07:24,000 --> 00:07:28,000 there are other cells in which the resting phase is temporary, 94 00:07:28,000 --> 00:07:32,000 and these cells can then reenter the cell cycle, going back from G0 to G1 95 00:07:32,000 --> 00:07:35,000 and through the process again. And a lot of the progenitor cells 96 00:07:35,000 --> 00:07:39,000 that I referred to up there, in terms of the skin and the blood, 97 00:07:39,000 --> 00:07:43,000 and the intestines, are in that situation. 98 00:07:43,000 --> 00:07:47,000 They're resting, and then they be recruited back into 99 00:07:47,000 --> 00:07:51,000 the cell cycle, in order to make more derivative 100 00:07:51,000 --> 00:07:56,000 cells. It's also useful to consider what the chromosomes are doing in 101 00:07:56,000 --> 00:08:00,000 these different phases of the cell cycle. So in a G1 cell, 102 00:08:00,000 --> 00:08:05,000 if we think about a single chromosome, and we're representing 103 00:08:05,000 --> 00:08:09,000 it by a single-line, although this is of course, 104 00:08:09,000 --> 00:08:14,000 a double-helix. In the G1 phase there's a single, 105 00:08:14,000 --> 00:08:18,000 double-helical chromosome, in S-phase that gets duplicated in 106 00:08:18,000 --> 00:08:23,000 the process of DNA replication, initiation shown here, and it would 107 00:08:23,000 --> 00:08:27,000 be completed so that the single-chromosome would be 108 00:08:27,000 --> 00:08:34,000 ultimately duplicated into two. 109 00:08:34,000 --> 00:08:37,000 And then in M-phase, those two chromosomes get separated 110 00:08:37,000 --> 00:08:41,000 from one another, so these are now joined, 111 00:08:41,000 --> 00:08:45,000 separated from one another, eventually into two daughter cells, 112 00:08:45,000 --> 00:08:48,000 which can then go through this process again. 113 00:08:48,000 --> 00:08:52,000 OK? And much of what we think about, when we think about how the 114 00:08:52,000 --> 00:08:56,000 cell cycle is controlled is to deal with the initiation of DNA 115 00:08:56,000 --> 00:09:00,000 replication, and then the initiation of mitosis. 116 00:09:00,000 --> 00:09:03,000 OK, so this is a cyclical process, a highly ordered process, this is a 117 00:09:03,000 --> 00:09:07,000 representation of the cell cycle, as shown in your book, just so you 118 00:09:07,000 --> 00:09:11,000 know it's in there. Exactly as I described to you, 119 00:09:11,000 --> 00:09:15,000 mitosis and S-phase, where the action is, these gap phases, 120 00:09:15,000 --> 00:09:18,000 G1 and G2, and this little arrow represents G0, 121 00:09:18,000 --> 00:09:22,000 and I've also used this term before, interphase, that represents all the 122 00:09:22,000 --> 00:09:26,000 phases where the DNA is not evident under the light microscope. 123 00:09:26,000 --> 00:09:30,000 It's only evident in mitosis because of DNA condensation, 124 00:09:30,000 --> 00:09:34,000 so the rest of the cycle, G1, S, and G2, are called interphase. 125 00:09:34,000 --> 00:09:37,000 So this is a highly ordered process, 126 00:09:37,000 --> 00:09:41,000 each step preceded by a particular other step, leading to a particular 127 00:09:41,000 --> 00:09:44,000 outcome, events leading to a particular outcome, 128 00:09:44,000 --> 00:09:48,000 and in a topical sense, you can think about it as the NCAA 129 00:09:48,000 --> 00:09:51,000 pools, starting tomorrow there'll be individual events, 130 00:09:51,000 --> 00:09:55,000 games, which will lead to next events in the next round, 131 00:09:55,000 --> 00:09:58,000 and next events in the next round, to an ultimate conclusion, in this 132 00:09:58,000 --> 00:10:02,000 case, the NCAA champion, which can only occur if the proper 133 00:10:02,000 --> 00:10:06,000 events have happened, prior to it. 134 00:10:06,000 --> 00:10:12,000 And you'll see another specific example of how the order is assessed 135 00:10:12,000 --> 00:10:18,000 in the development of the stages of the cell cycle. 136 00:10:18,000 --> 00:10:24,000 OK, so we know that the cell cycle is important. The question is, 137 00:10:24,000 --> 00:10:30,000 how is it controlled? How do you accomplish this orderly process of 138 00:10:30,000 --> 00:10:36,000 cell cycle progression? What are the genes -- 139 00:10:36,000 --> 00:10:46,000 -- that regulate this process? 140 00:10:46,000 --> 00:10:50,000 What do the genes encode, what do the proteins, and what do the 141 00:10:50,000 --> 00:10:54,000 proteins do? What are the pathways that they regulate to ensure the 142 00:10:54,000 --> 00:10:58,000 stages of the cell cycle? This was a huge question for 143 00:10:58,000 --> 00:11:02,000 decades, and there was rather little progress in trying to understand how 144 00:11:02,000 --> 00:11:06,000 it happened, especially in us. Our cells are sufficiently complex, 145 00:11:06,000 --> 00:11:10,000 and there's relatively imprecise, or particularly was in the past, 146 00:11:10,000 --> 00:11:14,000 imprecise methods for dissecting complex processes in 147 00:11:14,000 --> 00:11:18,000 mammalian cells. And so it took experiments performed 148 00:11:18,000 --> 00:11:22,000 in yeast, a single-cell, eukaryotic organism, budding yeast 149 00:11:22,000 --> 00:11:27,000 and also fission yeast, to allow us to understand what the 150 00:11:27,000 --> 00:11:31,000 details of the cell cycle are. Budding yeast, as shown here in a 151 00:11:31,000 --> 00:11:36,000 picture from your book, is a single-cell organism. 152 00:11:36,000 --> 00:11:39,000 It's also haploid, or it can be at least, 153 00:11:39,000 --> 00:11:43,000 haploid, which means that it has a single set of chromosomes, 154 00:11:43,000 --> 00:11:46,000 it doesn't have two sets, single set of genes, which makes it amenable to 155 00:11:46,000 --> 00:11:50,000 genetic analysis. You can make mutants more easily, 156 00:11:50,000 --> 00:11:53,000 if you only have one copy of each gene to worry about. 157 00:11:53,000 --> 00:11:57,000 So that's another reason that yeast was attractive. 158 00:11:57,000 --> 00:12:01,000 And another reason, which is kind of seen here, 159 00:12:01,000 --> 00:12:04,000 not really obvious here, but when yeast cells divide, 160 00:12:04,000 --> 00:12:08,000 they go through a very characteristic, morphological 161 00:12:08,000 --> 00:12:11,000 change. They start off as spheres, 162 00:12:11,000 --> 00:12:15,000 like this one, and as they go through the cell cycle, 163 00:12:15,000 --> 00:12:19,000 they develop a small bud. That bud then gets bigger, 164 00:12:19,000 --> 00:12:22,000 and eventually the two, the joint between the mother cell and the bud, 165 00:12:22,000 --> 00:12:26,000 get severed to form two cells. And you can actually figure out where 166 00:12:26,000 --> 00:12:30,000 you are in the cell cycle, by examining exactly what the 167 00:12:30,000 --> 00:12:34,000 morphology of the yeast cell is, and I'll show you that on the next 168 00:12:34,000 --> 00:12:38,000 slide. This is the yeast cell cycle, 169 00:12:38,000 --> 00:12:42,000 with overlay of the diagram of what the cells look like in the different 170 00:12:42,000 --> 00:12:46,000 phases, so a yeast cell that's in G0, let's say, looks like this, 171 00:12:46,000 --> 00:12:50,000 fairly round, nondescript, as it enters G1 it creates a small bud, 172 00:12:50,000 --> 00:12:54,000 that bud then gets bigger as the cells are now duplicating their DNA, 173 00:12:54,000 --> 00:12:58,000 the bud actually gets bigger. It gets bigger still, 174 00:12:58,000 --> 00:13:02,000 during G2 phase, and then at M-phase you can actually see a little 175 00:13:02,000 --> 00:13:06,000 junction between the two cells, and the mother and the daughter cell 176 00:13:06,000 --> 00:13:10,000 are more-or-less the same size, and then they separate from one 177 00:13:10,000 --> 00:13:14,000 another, and they can go through the cycle again. 178 00:13:14,000 --> 00:13:17,000 And this is useful because you can tell if you have a mutant, 179 00:13:17,000 --> 00:13:21,000 as you'll see in a moment, if you have a mutant in a gene that affects 180 00:13:21,000 --> 00:13:25,000 one of these processes, you can actually figure out where 181 00:13:25,000 --> 00:13:28,000 the mutant gene acts within the cell cycle, based on what the yeast cell 182 00:13:28,000 --> 00:13:32,000 looks like. And these individuals here, won the Nobel prize for their 183 00:13:32,000 --> 00:13:36,000 efforts to understand the cell cycle, they won it about five 184 00:13:36,000 --> 00:13:39,000 years ago, or so. This is Lee Hartwell, 185 00:13:39,000 --> 00:13:43,000 he's an American, happens to run a cancer center out at the University 186 00:13:43,000 --> 00:13:46,000 of Washington, very suave and sophisticated guy. 187 00:13:46,000 --> 00:13:49,000 These two goofballs are Brits, very nice guys actually, 188 00:13:49,000 --> 00:13:53,000 Paul Nurse, who's now the president of Rockefeller University in New 189 00:13:53,000 --> 00:13:56,000 York, and Tim Hunt, actually Sir Tim Hunt, 190 00:13:56,000 --> 00:14:00,000 because he's knighted after this Nobel Prize. And I'm going to 191 00:14:00,000 --> 00:14:03,000 explain the experiments that all three of these guys did today, 192 00:14:03,000 --> 00:14:07,000 which won them the Nobel prize, and gave us tremendous insight into 193 00:14:07,000 --> 00:14:10,000 the control of the cell cycle in yeast, which turned out to tell us 194 00:14:10,000 --> 00:14:14,000 how the cell cycle is controlled, also, in us. 195 00:14:14,000 --> 00:14:17,000 OK, so the first thing we need to do is to create mutants. 196 00:14:17,000 --> 00:14:21,000 That's a general theme in biology. If you want to understand a process, 197 00:14:21,000 --> 00:14:25,000 find a mutant that can't do it, and understand the genes that get 198 00:14:25,000 --> 00:14:29,000 mutated in that process, and so that's what happened in the 199 00:14:29,000 --> 00:14:33,000 hands of Lee Hartwell, the guy in the upper-right, 200 00:14:33,000 --> 00:14:37,000 he took this same yeast, the budding yeast, 201 00:14:37,000 --> 00:14:41,000 which is also Brewer's yeast, the yeast that makes beer. 202 00:14:41,000 --> 00:14:46,000 He took a population of these yeast 203 00:14:46,000 --> 00:14:51,000 cells, and he mutagenized them. 204 00:14:51,000 --> 00:14:54,000 I told you about this before, you can add chemical mutagens that 205 00:14:54,000 --> 00:14:57,000 soak into the cells, bind to the DNA, cause mutations. 206 00:14:57,000 --> 00:15:01,000 Each cell might have one, or a few nucleotides changed, 207 00:15:01,000 --> 00:15:04,000 and at some frequency those mutations will affect genes, 208 00:15:04,000 --> 00:15:08,000 and at some frequency the genes affected will influence the process 209 00:15:08,000 --> 00:15:11,000 that you're trying to study. So he was trying to study growth 210 00:15:11,000 --> 00:15:15,000 control, cell division, so he asked about the ability of the 211 00:15:15,000 --> 00:15:18,000 cells to grow, and particularly, 212 00:15:18,000 --> 00:15:21,000 he did so at two different temperatures. He tested the ability 213 00:15:21,000 --> 00:15:25,000 of the cells to grow at their normal temperature, which is about 25 214 00:15:25,000 --> 00:15:28,000 degrees centigrade, and he found that many of these 215 00:15:28,000 --> 00:15:32,000 yeast cells would grow, following mutagenesis, at 25 degrees. 216 00:15:32,000 --> 00:15:36,000 Probably some of them wouldn't grow 217 00:15:36,000 --> 00:15:40,000 because they would've inactivated some critical gene, 218 00:15:40,000 --> 00:15:44,000 and even at 25 degrees, they couldn't grow. However, 219 00:15:44,000 --> 00:15:48,000 he then took these plates, and he used a technique, 220 00:15:48,000 --> 00:15:53,000 which I referred to in a previous lecture, called replica plating. 221 00:15:53,000 --> 00:15:57,000 He took a stamp, basically, of these colonies, 222 00:15:57,000 --> 00:16:01,000 transferred them onto a fresh plate, and examined how the cells grew at 223 00:16:01,000 --> 00:16:05,000 30 degrees centigrade. And what he found was that, 224 00:16:05,000 --> 00:16:09,000 whereas many of the cells that were able to grow at 25 degrees continued 225 00:16:09,000 --> 00:16:12,000 to grow at 30 degrees, so they produced colonies in exactly 226 00:16:12,000 --> 00:16:16,000 the pattern that he had seen previously. Some of 227 00:16:16,000 --> 00:16:20,000 the cells didn't. This colony here, 228 00:16:20,000 --> 00:16:24,000 while it was successful, the cells were successful in growing 229 00:16:24,000 --> 00:16:29,000 at 25 degrees, failed to grow at 30 degrees. 230 00:16:29,000 --> 00:16:33,000 And this type of mutant is referred to as a temperature-sensitive 231 00:16:33,000 --> 00:16:48,000 mutant, -- [PAUSE} 232 00:16:48,000 --> 00:16:52,000 -- abbreviated TS. We imagine that this mutation 233 00:16:52,000 --> 00:16:56,000 affects the amino acid sequence of the protein, at low temperatures the 234 00:16:56,000 --> 00:17:00,000 protein is still able to function, but at higher temperatures, maybe it 235 00:17:00,000 --> 00:17:04,000 becomes a little unstable, and now it can't function, and 236 00:17:04,000 --> 00:17:08,000 that's why you see no growth here. So we had a TS-mutant that affected 237 00:17:08,000 --> 00:17:11,000 the growth of the cells, OK? So my question to you is, 238 00:17:11,000 --> 00:17:15,000 is that TS-mutant interesting? Is it interesting? 239 00:17:15,000 --> 00:17:22,000 Well we can't actually know whether 240 00:17:22,000 --> 00:17:26,000 it's interesting yet, because there are two general 241 00:17:26,000 --> 00:17:30,000 classes of mutations that would give you this same phenotype. 242 00:17:30,000 --> 00:17:35,000 One of them is interesting, does that cement always show, 243 00:17:35,000 --> 00:17:39,000 or do we have a problem here? It's always there? Never noticed it, 244 00:17:39,000 --> 00:17:43,000 in all these years. You can distinguish between these two 245 00:17:43,000 --> 00:17:48,000 classes of boring and interesting mutants, based on the morphology of 246 00:17:48,000 --> 00:17:52,000 the yeast cells when you shift the temperature. If you start with a 247 00:17:52,000 --> 00:17:57,000 population of yeast cells growing just randomly at 25 degrees 248 00:17:57,000 --> 00:18:01,000 centigrade, including these mutants, if you were to look under the 249 00:18:01,000 --> 00:18:05,000 microscope, you would find mutants, cells that were at various stages of 250 00:18:05,000 --> 00:18:10,000 the cell cycle. OK? Now, let's imagine that our mutation 251 00:18:10,000 --> 00:18:14,000 affects a general enzyme that's required for cell viability, 252 00:18:14,000 --> 00:18:18,000 DNA polymerase or maybe, better still, some ribosomal protein that 253 00:18:18,000 --> 00:18:22,000 all cells need all the time, in order to function. If I were to 254 00:18:22,000 --> 00:18:26,000 take these cells which were growing at 25 degrees centigrade, 255 00:18:26,000 --> 00:18:30,000 and I were to shift them to 30 degrees centigrade, 256 00:18:30,000 --> 00:18:34,000 and look under the microscope, what would I see? 257 00:18:34,000 --> 00:18:41,000 Well, if it's a ribosomal protein, 258 00:18:41,000 --> 00:18:45,000 and now translation just stops, these cells are going nowhere. 259 00:18:45,000 --> 00:18:49,000 Regardless of where they were in the cell cycle, 260 00:18:49,000 --> 00:18:52,000 they don't go any further because you need protein synthesis to do 261 00:18:52,000 --> 00:18:56,000 anything, so if you look under the microscope at these cells, 262 00:18:56,000 --> 00:19:00,000 following a temperature shift, you would still find a distribution 263 00:19:00,000 --> 00:19:04,000 of cells in the different phases of the cell cycle. 264 00:19:04,000 --> 00:19:08,000 Lee Hartwell was not interested in this class of mutants. 265 00:19:08,000 --> 00:19:13,000 They could be anything, he didn't care. However, 266 00:19:13,000 --> 00:19:17,000 he reasoned that if he were dealing with a mutant that affected 267 00:19:17,000 --> 00:19:22,000 specifically a stage in the cell cycle, he might find that the cells, 268 00:19:22,000 --> 00:19:26,000 upon temperature shift from 25 degrees to 30 degrees, 269 00:19:26,000 --> 00:19:31,000 got to a particular point in the cell cycle and couldn't go any 270 00:19:31,000 --> 00:19:35,000 further, that this gene affected one of these important transitions, 271 00:19:35,000 --> 00:19:40,000 and so for example -- -- if he were to look under the 272 00:19:40,000 --> 00:19:45,000 microscope, all of the cells would have arrested with a small bud, 273 00:19:45,000 --> 00:19:50,000 would have arrested in G1, or maybe they would've arrested looking like 274 00:19:50,000 --> 00:19:55,000 this, just prior to mitosis, or like this, somewhere in G2. 275 00:19:55,000 --> 00:20:00,000 But importantly, they would arrest with a specific morphology that 276 00:20:00,000 --> 00:20:05,000 would indicate that they had a specific cell cycle block. 277 00:20:05,000 --> 00:20:10,000 And so he did this, and he found many, many such mutants. 278 00:20:10,000 --> 00:20:15,000 He called them cell division cycle mutants, or CDC mutants. 279 00:20:15,000 --> 00:20:20,000 And it was his methodology, which really broke this field open, 280 00:20:20,000 --> 00:20:26,000 and won him the Nobel prize. Now one example of a CDC mutant that he 281 00:20:26,000 --> 00:20:31,000 discovered, called CDC2, was particularly important. 282 00:20:31,000 --> 00:20:37,000 It's been renamed CDK1, or cyclin-dependent kinase one, 283 00:20:37,000 --> 00:20:44,000 for reasons that I'll come to. Cyclin, sorry, cyclin-dependent, 284 00:20:44,000 --> 00:20:54,000 kinase one. 285 00:20:54,000 --> 00:21:00,000 And this one acts at a particular phase in the cell cycle, 286 00:21:00,000 --> 00:21:07,000 early on in the transition from G1 to S. So if you imagine cells in G0, 287 00:21:07,000 --> 00:21:13,000 going to G1, and then progressing from G1 into S-phase, 288 00:21:13,000 --> 00:21:20,000 into G2, and finally, in mitosis. This particular mutant blocked 289 00:21:20,000 --> 00:21:27,000 right here, and so it said that this gene, CDC2 or CDK1, 290 00:21:27,000 --> 00:21:34,000 was required for this transition. 291 00:21:34,000 --> 00:21:38,000 And just to emphasize the TS-temperature shift and building up 292 00:21:38,000 --> 00:21:42,000 of the cells in the cell cycle concept, imagine a cell that looked 293 00:21:42,000 --> 00:21:46,000 like this, before the temperature shift. Ok? If you now do the 294 00:21:46,000 --> 00:21:50,000 temperature shift, this cell will progress to this 295 00:21:50,000 --> 00:21:54,000 point, but it won't go any further because CDC2 is required. 296 00:21:54,000 --> 00:21:58,000 Maybe I'll draw this over here, to make it even more obvious. 297 00:21:58,000 --> 00:22:01,000 CDC2 is required to go beyond this point, so that cell will arrest 298 00:22:01,000 --> 00:22:05,000 right here. If there were a cell that looked like this, 299 00:22:05,000 --> 00:22:09,000 in the population of 25 degrees, and now you shifted the temperature, 300 00:22:09,000 --> 00:22:13,000 it would complete this phase because CDC2 is not required. 301 00:22:13,000 --> 00:22:16,000 It would make it all the way to here, it would keep going, 302 00:22:16,000 --> 00:22:19,000 and then it would get stuck here again. And that's why you get cells 303 00:22:19,000 --> 00:22:23,000 building up with a particular morphology. OK? 304 00:22:23,000 --> 00:22:26,000 So this was successful, and as I said, he isolated a large 305 00:22:26,000 --> 00:22:29,000 number of such CDC mutants, which have taught us not about just 306 00:22:29,000 --> 00:22:33,000 that transition, but many other transitions in the 307 00:22:33,000 --> 00:22:37,000 cell. Now what Paul Nurse did, 308 00:22:37,000 --> 00:22:43,000 this guy here, he actually was performing very similar experiments 309 00:22:43,000 --> 00:22:48,000 in a related yeast species, but the critical experiment that he 310 00:22:48,000 --> 00:22:54,000 did was to clone the gene that is responsible for CDC2 function. 311 00:22:54,000 --> 00:23:00,000 So he took CDC2 mutant cells, which at 30 degrees -- 312 00:23:00,000 --> 00:23:08,000 -- will not grow. 313 00:23:08,000 --> 00:23:14,000 And he added, through a cDNA library, CDC2 cDNA, 314 00:23:14,000 --> 00:23:20,000 of yeast origin. So he made a cDNA library from yeast, 315 00:23:20,000 --> 00:23:26,000 he introduced it into these mutant cells, and then he asked whether the 316 00:23:26,000 --> 00:23:32,000 cells could now grow at 30 degrees -- 317 00:23:32,000 --> 00:23:40,000 -- and the answer was yes. 318 00:23:40,000 --> 00:23:44,000 That is, if the cells now carried an extra copy of CDC2, 319 00:23:44,000 --> 00:23:49,000 in the form of this cDNA, they now could grow at 30 degrees. 320 00:23:49,000 --> 00:23:53,000 He complemented the mutation through the addition of a normal 321 00:23:53,000 --> 00:23:57,000 copy of the CDC2 cDNA. Quite surprisingly, he did the same 322 00:23:57,000 --> 00:24:05,000 experiment -- 323 00:24:05,000 --> 00:24:09,000 -- using not a yeast gene, but a human gene. Frankly, 324 00:24:09,000 --> 00:24:13,000 there was great skepticism in the field about whether what these guys 325 00:24:13,000 --> 00:24:17,000 were doing in yeast had anything to do with cell cycle control in humans. 326 00:24:17,000 --> 00:24:21,000 Most people actually assumed that it probably had nothing to do with 327 00:24:21,000 --> 00:24:25,000 it, and so therefore, when Paul Nurse proposed to try to 328 00:24:25,000 --> 00:24:29,000 complement his yeast mutation with a human gene, people 329 00:24:29,000 --> 00:24:36,000 were skeptical. 330 00:24:36,000 --> 00:24:40,000 But in fact, it worked. And this told him, and the field, 331 00:24:40,000 --> 00:24:45,000 that the machinery that controls the cell cycle in this simple, 332 00:24:45,000 --> 00:24:49,000 single-celled eukaryote, is highly conserved, all the way through to 333 00:24:49,000 --> 00:24:54,000 humans, and that we could therefore understand cell cycle control in us, 334 00:24:54,000 --> 00:24:59,000 by doing experiments like this in yeast. So this really broke the 335 00:24:59,000 --> 00:25:03,000 field open, and that's why Nurse won the Nobel prize at that same time. 336 00:25:03,000 --> 00:25:08,000 However, there was a problem. They did in fact clone the gene, 337 00:25:08,000 --> 00:25:12,000 they were able to sequence the gene, and they found that the sequence of 338 00:25:12,000 --> 00:25:17,000 the gene, which I'm going to refer to now by the other name, 339 00:25:17,000 --> 00:25:22,000 CDK1, looked like a kinase. It had amino acid sequence, 340 00:25:22,000 --> 00:25:26,000 which made it resemble known kinases, so it was assumed to be a protein 341 00:25:26,000 --> 00:25:31,000 kinase. However, it didn't have any kinase activity. 342 00:25:43,000 --> 00:25:46,000 If you mixed it with various substrate molecules in the presence 343 00:25:46,000 --> 00:25:49,000 of ATP, those substrates were not changed, they were not 344 00:25:49,000 --> 00:25:52,000 phosphorylated. So the purified CDK enzyme had no 345 00:25:52,000 --> 00:25:56,000 kinase activity, and therefore, it wasn't entirely 346 00:25:56,000 --> 00:25:59,000 clear what its function really was, and the field sort of got stuck 347 00:25:59,000 --> 00:26:02,000 there for a while, trying to understand the biochemical 348 00:26:02,000 --> 00:26:06,000 function of CDK1, and other cell cycle regulators. 349 00:26:06,000 --> 00:26:11,000 And that then led to the experiments done by Tim Hunt, 350 00:26:11,000 --> 00:26:17,000 this guy here. And Tim Hunt, actually doing experiments at Woods 351 00:26:17,000 --> 00:26:22,000 Hole, down at the Cape, in a summer course, with summer 352 00:26:22,000 --> 00:26:28,000 students, did a famous experiment using sea urchins. 353 00:26:28,000 --> 00:26:33,000 Sea urchins, when they're fertilized, 354 00:26:33,000 --> 00:26:37,000 undergo very rapid, and very synchronous, 355 00:26:37,000 --> 00:26:42,000 cell divisions. In the first few hours after you fertilize sea urchin 356 00:26:42,000 --> 00:26:46,000 eggs, they will divide and divide again. And importantly, 357 00:26:46,000 --> 00:26:51,000 they will do so in a very synchronous manner, 358 00:26:51,000 --> 00:26:55,000 so all the cells will produce two cells at around the same time, 359 00:26:55,000 --> 00:27:00,000 and those will all produce four cells at around the same time. 360 00:27:00,000 --> 00:27:04,000 And this is important if one wants to do biochemical experiments, 361 00:27:04,000 --> 00:27:09,000 to understand what is changing within these cells as they're going 362 00:27:09,000 --> 00:27:14,000 through these various cell cycles. And so, what Tim and his students 363 00:27:14,000 --> 00:27:18,000 did was to label the proteins in these dividing sea urchin cells, 364 00:27:18,000 --> 00:27:23,000 with a radioactive amino acid, S35 methionine. And then they simply 365 00:27:23,000 --> 00:27:28,000 made extracts of these cells at different time points thereafter, 366 00:27:28,000 --> 00:27:32,000 and asked whether anything was changing in an interesting pattern, 367 00:27:32,000 --> 00:27:37,000 at different times that correspond to different stages 368 00:27:37,000 --> 00:27:43,000 of the cell cycle. So they ran protein gels, 369 00:27:43,000 --> 00:27:50,000 they separated the proteins and then exposed the gels to x-ray film, 370 00:27:50,000 --> 00:27:57,000 to visual what the protein concentrations were at different 371 00:27:57,000 --> 00:28:03,000 time points. They took cells at time zero, they took cells after 30 372 00:28:03,000 --> 00:28:10,000 minutes, after 60 minutes, after 90 minutes, 120 minutes, 373 00:28:10,000 --> 00:28:16,000 150 minutes, 180 minutes. And they knew already, 374 00:28:16,000 --> 00:28:21,000 from their earlier analysis, that this corresponded to the first 375 00:28:21,000 --> 00:28:27,000 cell cycle, and this corresponded to the second cell cycle. 376 00:28:27,000 --> 00:28:32,000 OK? Now some proteins, when they visualize them that way, 377 00:28:32,000 --> 00:28:37,000 didn't change. At the different time points they saw roughly equal 378 00:28:37,000 --> 00:28:43,000 concentrations of that protein throughout, but interestingly, 379 00:28:43,000 --> 00:28:48,000 other proteins changed in abundance, and did so according to a pattern. 380 00:29:02,000 --> 00:29:07,000 They seemed to oscillate, they seemed to cycle, in a pattern 381 00:29:07,000 --> 00:29:12,000 that corresponded with the cell cycle. And so he called these 382 00:29:12,000 --> 00:29:17,000 cyclins, and he suggested that they might have something to do with the 383 00:29:17,000 --> 00:29:22,000 control of the cell division cycle. Well, meanwhile, Nurse and Hartwell 384 00:29:22,000 --> 00:29:27,000 were doing their thing on CDKs, and so they came up with the idea 385 00:29:27,000 --> 00:29:32,000 that maybe these two things have something to do with one another. 386 00:29:32,000 --> 00:29:37,000 And particularly, maybe the failure of CDK to function 387 00:29:37,000 --> 00:29:42,000 as a kinase was due to the fact that it didn't have an accessory protein 388 00:29:42,000 --> 00:29:47,000 that it needed, mainly the cyclin. 389 00:29:47,000 --> 00:29:53,000 And so, whereas CDK2, sorry, CDK1, was an inactive protein 390 00:29:53,000 --> 00:30:04,000 kinase. 391 00:30:04,000 --> 00:30:09,000 Now in a test tube, in a biochemical experiment, 392 00:30:09,000 --> 00:30:14,000 if they mixed CDK1 with one of these cyclins, they observed kinase 393 00:30:14,000 --> 00:30:20,000 activity. And thus the name, cyclin-dependent kinase. It's not a 394 00:30:20,000 --> 00:30:25,000 kinase, it's not an active kinase in the absence of cyclin, 395 00:30:25,000 --> 00:30:31,000 it only becomes active in the presence of cyclin. 396 00:30:31,000 --> 00:30:36,000 So let me give you two quick examples of proteins that are then 397 00:30:36,000 --> 00:30:41,000 phosphorylated by this active kinase, to give you a sense of how this 398 00:30:41,000 --> 00:30:47,000 kinase regulates cell cycle transitions. There's a class of 399 00:30:47,000 --> 00:30:52,000 proteins that are involved in the regulation of replication initiation. 400 00:30:52,000 --> 00:30:58,000 We'll just call them replication initiation factors. 401 00:30:58,000 --> 00:31:03,000 In the absence of phosphorylation, they're inactive, and that's one of 402 00:31:03,000 --> 00:31:09,000 the reasons that replication is not initiated at those times 403 00:31:09,000 --> 00:31:15,000 in the cell cycle. However, in the presence of CDK 404 00:31:15,000 --> 00:31:22,000 cyclin, now the protein becomes phosphorylated, 405 00:31:22,000 --> 00:31:29,000 and becomes active. OK? So one of the ways you trigger 406 00:31:29,000 --> 00:31:36,000 the transition from G1 into S-phase, is to turn on this enzyme which 407 00:31:36,000 --> 00:31:43,000 phosphorylated this target protein, and stimulates S-phase entry. 408 00:31:43,000 --> 00:31:51,000 A second example is a protein called 409 00:31:51,000 --> 00:31:56,000 pRB. In its un-phosphorylated state, it is active, different from here 410 00:31:56,000 --> 00:32:01,000 where, in its un-phosphorylated state, it was inactive, 411 00:32:01,000 --> 00:32:06,000 and the function of the RB protein, in its active state, is to block 412 00:32:06,000 --> 00:32:11,000 again, the transition from S-phase to G1. This is actually an 413 00:32:11,000 --> 00:32:16,000 important cancer gene, it's mutated in a large number of 414 00:32:16,000 --> 00:32:21,000 cancers, and so we'll talk about its function, exactly how it blocks the 415 00:32:21,000 --> 00:32:26,000 transition, in later lectures, but suffice it to say, it does that. 416 00:32:26,000 --> 00:32:35,000 And when it is phosphorylated by CDK 417 00:32:35,000 --> 00:32:42,000 cyclins, it becomes inactive, thereby allowing cells to progress 418 00:32:42,000 --> 00:32:50,000 from G1 into S-phase. OK? So those are two examples of 419 00:32:50,000 --> 00:33:16,000 how CDK cyclins operate. 420 00:33:16,000 --> 00:33:23,000 Now, importantly, cyclin kinase activity is determined 421 00:33:23,000 --> 00:33:31,000 by the level of the cyclin. As I told you, cyclin levels 422 00:33:31,000 --> 00:33:38,000 oscillate, where this is cycle one, cycle two, cycle three. So these 423 00:33:38,000 --> 00:33:46,000 are cyclin concentrations inside the cell. CDK levels do 424 00:33:46,000 --> 00:34:00,000 not oscillate. 425 00:34:00,000 --> 00:34:04,000 Concentration of the CDKs inside the cells is rather constant. 426 00:34:04,000 --> 00:34:09,000 However, at this point in the cell cycle, when there's not enough 427 00:34:09,000 --> 00:34:13,000 cyclin, you don't have kinase activity. Only when you go past the 428 00:34:13,000 --> 00:34:18,000 threshold, do you get kinase activity. 429 00:34:18,000 --> 00:34:24,000 When it drops again, 430 00:34:24,000 --> 00:34:28,000 you lose kinase activity. But in the next cell cycle, 431 00:34:28,000 --> 00:34:33,000 the cyclin levels increase again, and you get kinase activity, and so 432 00:34:33,000 --> 00:34:38,000 on. So the oscillation of cyclins determines the oscillation of kinase 433 00:34:38,000 --> 00:34:42,000 activity, which determines the periodicity of the cell cycle. 434 00:34:42,000 --> 00:34:47,000 Now, for the truth in advertising, the situation is actually more 435 00:34:47,000 --> 00:34:52,000 complex, there are actually multiple CDKs and multiple cyclins. 436 00:34:52,000 --> 00:35:02,000 So in our cells, for example, if we imagine G1, 437 00:35:02,000 --> 00:35:13,000 S, M, G2, G1, S, M, G2, there's a cyclin called cyclin-D, 438 00:35:13,000 --> 00:35:24,000 which comes up in G1, goes down, comes up in the next G1, goes down. 439 00:35:24,000 --> 00:35:32,000 There's another cyclin called 440 00:35:32,000 --> 00:35:38,000 cyclin-E, which comes up a little bit later, in S-phase goes down, 441 00:35:38,000 --> 00:35:43,000 stays down, comes up in the next S-phase. And there's finally 442 00:35:43,000 --> 00:35:49,000 another one called cyclin-B, which comes up in G2, and comes down, 443 00:35:49,000 --> 00:36:14,000 goes up in the next G2. 444 00:36:14,000 --> 00:36:20,000 Anyway, the point is that there are different cyclins that get induced 445 00:36:20,000 --> 00:36:27,000 in different phases of the cell cycle, and actually control, 446 00:36:27,000 --> 00:36:33,000 through binding to different CDKs, different transitions in the 447 00:36:33,000 --> 00:36:40,000 different phases of the cell cycle. OK. Let's see. 448 00:36:40,000 --> 00:36:44,000 So, another concept: the transitions from the cell cycle don't occur, 449 00:36:44,000 --> 00:36:49,000 from one cell cycle position to the next don't occur, 450 00:36:49,000 --> 00:36:53,000 unless the previous cell cycle event has been completed. 451 00:36:53,000 --> 00:36:58,000 And that makes sense because you don't want to, 452 00:36:58,000 --> 00:37:02,000 for example, try to divide your DNA in mitosis if you haven't fully 453 00:37:02,000 --> 00:37:07,000 replicated your DNA in S-phase. So there are processes that are 454 00:37:07,000 --> 00:37:12,000 overlaid on top of cell cycle control, which ensure the completion 455 00:37:12,000 --> 00:37:17,000 of one phase before the next phase is initiated. And I draw, 456 00:37:17,000 --> 00:37:22,000 as an analogy, your washing machine, which likewise, has checkpoints 457 00:37:22,000 --> 00:37:27,000 which will determine whether or not the previous phase of the wash cycle 458 00:37:27,000 --> 00:37:33,000 has been completed. For example, you don't spin your 459 00:37:33,000 --> 00:37:39,000 wash until all the water has been rinsed out. There's a sensor that 460 00:37:39,000 --> 00:37:45,000 will determine whether that's not true, and if that sensor is tripped, 461 00:37:45,000 --> 00:37:51,000 it blocks the wash cycle at that point. Your cells have very similar 462 00:37:51,000 --> 00:37:57,000 checkpoints that will monitor and regulate cell cycle transitions, 463 00:37:57,000 --> 00:38:08,000 and they're called checkpoints. 464 00:38:08,000 --> 00:38:12,000 There are checkpoints, actually, that operate at different 465 00:38:12,000 --> 00:38:16,000 phases of the cell cycle. I'll only give you one example, 466 00:38:16,000 --> 00:38:20,000 it's actually the one that's best known. It occurs at the transition 467 00:38:20,000 --> 00:38:30,000 in mitosis, from metaphase -- 468 00:38:30,000 --> 00:38:36,000 -- where, if you'll recall, the duplicated chromosomes line up 469 00:38:36,000 --> 00:38:42,000 on the metaphase plate. In the process of anaphase, 470 00:38:42,000 --> 00:38:48,000 where the chromosomes separate from one another, the chromatids I should 471 00:38:48,000 --> 00:38:54,000 say, separate from one another. There's a cell cycle checkpoint 472 00:38:54,000 --> 00:39:00,000 that makes sure that this happens before that can happen. 473 00:39:00,000 --> 00:39:04,000 And in particular, if you have a chromosome which is 474 00:39:04,000 --> 00:39:08,000 only attached to one side of the mitotic spindle, 475 00:39:08,000 --> 00:39:12,000 so if you recall, these are centered in the middle 476 00:39:12,000 --> 00:39:16,000 through attachment to microtubules that are emanating from the two 477 00:39:16,000 --> 00:39:20,000 poles. If you have a chromosome that is only attached to one of the 478 00:39:20,000 --> 00:39:24,000 two spindles, it's called monopolar attachment, whereas perhaps other 479 00:39:24,000 --> 00:39:28,000 chromosomes, maybe all of the other chromosomes, are properly attached 480 00:39:28,000 --> 00:39:32,000 at the metaphase plate. This one, unattached chromosome will 481 00:39:32,000 --> 00:39:37,000 literally send a signal, a biochemical signal, which is the 482 00:39:37,000 --> 00:39:42,000 equivalent of "wait for me". And that signal will inhibit cell 483 00:39:42,000 --> 00:39:47,000 cycle progression. It'll specifically inhibit the 484 00:39:47,000 --> 00:39:52,000 transition from metaphase to anaphase. While that signal is 485 00:39:52,000 --> 00:39:57,000 being sent, the cells will just sit there, waiting for another 486 00:39:57,000 --> 00:40:02,000 microtubule to bind to this end of the chromosome, 487 00:40:02,000 --> 00:40:07,000 thereby extinguishing the signal, relieving this inhibition, and 488 00:40:07,000 --> 00:40:12,000 allowing anaphase to progress. OK, so these checkpoints are 489 00:40:12,000 --> 00:40:16,000 critical in insuring that these things happen in a timely and 490 00:40:16,000 --> 00:40:20,000 ordered fashion. OK. That's it for the cell cycle, 491 00:40:20,000 --> 00:40:24,000 so for the final ten minutes, and I always give short shrift to the next 492 00:40:24,000 --> 00:40:29,000 topic, which is cell death, which is another fascinating topic, 493 00:40:29,000 --> 00:40:33,000 fortunately there's not a lot of board work to show you. 494 00:40:33,000 --> 00:40:37,000 Cell death. So cells are born, divide, as we've just been talking 495 00:40:37,000 --> 00:40:42,000 about, but remarkable numbers of cells in your body die. 496 00:40:42,000 --> 00:40:46,000 They will die, too, because of cellular injury, 497 00:40:46,000 --> 00:40:50,000 but they'll also die because they're programmed to die, 498 00:40:50,000 --> 00:40:54,000 or they'll decide to commit suicide, or they'll be murdered by other 499 00:40:54,000 --> 00:40:59,000 cells. This happens in development, for example, your brains, as 500 00:40:59,000 --> 00:41:03,000 developing fetuses, have ten times the number of cells 501 00:41:03,000 --> 00:41:07,000 than you end up with as a young infant, because 90%, 502 00:41:07,000 --> 00:41:11,000 for some of you more than 90% of the cells will actually be killed 503 00:41:11,000 --> 00:41:16,000 through this process of programmed cell death. 504 00:41:16,000 --> 00:41:20,000 Other cells, in your immune system, for example, are eliminated by this 505 00:41:20,000 --> 00:41:25,000 process, so as to avoid them attacking your own body. 506 00:41:25,000 --> 00:41:29,000 And there's many other examples of relevant and important programmed 507 00:41:29,000 --> 00:41:34,000 cell death. So we want to understand this process, 508 00:41:34,000 --> 00:41:39,000 partly because it's critical in normal development, 509 00:41:39,000 --> 00:41:43,000 and also because deregulation of this process is critical 510 00:41:43,000 --> 00:41:48,000 for many diseases. Too little cell death can give you 511 00:41:48,000 --> 00:41:53,000 proliferate diseases like cancer or autoimmune disease, 512 00:41:53,000 --> 00:41:58,000 too much cell death can give you diseases like neurodegenerative 513 00:41:58,000 --> 00:42:04,000 diseases, too many cells in your brain dying. So the regulation of 514 00:42:04,000 --> 00:42:09,000 cell death is quite important. Here are two other famous examples 515 00:42:09,000 --> 00:42:14,000 of programmed cell death. This is the loss of the tadpole's 516 00:42:14,000 --> 00:42:18,000 tail that occurs through an orderly, cellular suicide program, in which 517 00:42:18,000 --> 00:42:23,000 all of these cells die, giving rise to a frog with no tail. 518 00:42:23,000 --> 00:42:27,000 And likewise, in development, your hands are formed in such a way that 519 00:42:27,000 --> 00:42:32,000 the digits are actually connected by other cells, but through this 520 00:42:32,000 --> 00:42:37,000 process of programmed cell death, the cells in the middle, the 521 00:42:37,000 --> 00:42:41,000 interdigital cells, are eliminated, thereby sculpting 522 00:42:41,000 --> 00:42:46,000 the formation of your fingers. And this can be regulated, 523 00:42:46,000 --> 00:42:50,000 and has been regulated, in evolution, in some species, 524 00:42:50,000 --> 00:42:54,000 like ducks, the loss of the interdigital cells doesn't take 525 00:42:54,000 --> 00:42:59,000 place, so they have webbing. In other species of birds, like in 526 00:42:59,000 --> 00:43:03,000 us, the interdigital cells are removed, so they have sculpted toes. 527 00:43:03,000 --> 00:43:07,000 This is what programmed cell death looks like. It is a very rapid, 528 00:43:07,000 --> 00:43:12,000 and as I said, a very orderly process. 529 00:43:12,000 --> 00:43:15,000 Cells get signals to die, they can get signals because they 530 00:43:15,000 --> 00:43:19,000 get damaged, or they can get signals from their neighbors. 531 00:43:19,000 --> 00:43:23,000 When they get those signals, they undergo a series of biochemical 532 00:43:23,000 --> 00:43:26,000 changes. Their nucleus gets condensed, the DNA within the 533 00:43:26,000 --> 00:43:30,000 nucleus breaks up, and then the cells themselves break 534 00:43:30,000 --> 00:43:34,000 up into small fragments called apoptotic bodies, 535 00:43:34,000 --> 00:43:38,000 and then interestingly, the cells that surround those cells, 536 00:43:38,000 --> 00:43:41,000 eat the damage. It's like disposing of the body 537 00:43:41,000 --> 00:43:45,000 after a crime, the cells are eliminated through a 538 00:43:45,000 --> 00:43:49,000 process of phagocytosis, this is a very clean process then, 539 00:43:49,000 --> 00:43:53,000 there's very little junk, there's very little cellular debris that 540 00:43:53,000 --> 00:43:56,000 remains when this process of programmed cell death is completed. 541 00:43:56,000 --> 00:44:00,000 This is a normal looking lymphocyte, and this is a lymphocyte undergoing 542 00:44:00,000 --> 00:44:04,000 cell death. It's like, you know, this is your brain, 543 00:44:04,000 --> 00:44:08,000 this is your brain on drugs, this is a cell, this is a cell undergoing 544 00:44:08,000 --> 00:44:12,000 apoptosis. It's a fairly violent death, 545 00:44:12,000 --> 00:44:16,000 at least visually. This movie shows you an example of that. 546 00:44:16,000 --> 00:44:20,000 Here are cells undergoing apoptosis, I hope. 547 00:44:20,000 --> 00:44:27,000 Maybe not. Oh well. 548 00:44:27,000 --> 00:44:30,000 It comes from your book, so you can see it yourselves, 549 00:44:30,000 --> 00:44:33,000 in fear that that would happen, I'll show you another set of stills. 550 00:44:33,000 --> 00:44:36,000 Here are normal cells growing in the tissue culture dish, 551 00:44:36,000 --> 00:44:39,000 you add some agent which induces apoptosis, the cells begin to round 552 00:44:39,000 --> 00:44:42,000 up as you can see here, and their cell surfaces actually 553 00:44:42,000 --> 00:44:45,000 become this horrible mess of blebbing membrane, 554 00:44:45,000 --> 00:44:48,000 and they will eventually break up, as you can see starting to happen 555 00:44:48,000 --> 00:44:52,000 here. So apoptosis is critical and 556 00:44:52,000 --> 00:44:56,000 actually very, very interesting. 557 00:44:56,000 --> 00:45:00,000 As I said, too much cell death can give rise to various diseases, 558 00:45:00,000 --> 00:45:04,000 neurodegeneration stroke is complicated because of the effects 559 00:45:04,000 --> 00:45:08,000 of apoptosis, too much apoptosis, even in AIDS, there's a lot of cell 560 00:45:08,000 --> 00:45:12,000 death that takes place in apoptosis, and too little cell death, as I said, 561 00:45:12,000 --> 00:45:16,000 is important in cancer, autoimmune disease, and certain 562 00:45:16,000 --> 00:45:19,000 viral infections. Importantly, we know about the genes 563 00:45:19,000 --> 00:45:23,000 that are regulated in cell death, that regulate cell death, through 564 00:45:23,000 --> 00:45:26,000 genetic experiments. And here, the genetic experiments 565 00:45:26,000 --> 00:45:29,000 were performed, in large part, at MIT, 566 00:45:29,000 --> 00:45:33,000 by a professor in the biology department named Bob Horvitz. 567 00:45:33,000 --> 00:45:36,000 He took comfort in the fact that the process in C. 568 00:45:36,000 --> 00:45:39,000 elegans, a simple worm, has very many similarities to the 569 00:45:39,000 --> 00:45:43,000 process that I've outlined to you, and therefore, he hoped that the 570 00:45:43,000 --> 00:45:46,000 genes that regulate programmed cell death in C. elegans, 571 00:45:46,000 --> 00:45:50,000 were similar to the ones that do so in mammals. 572 00:45:50,000 --> 00:45:54,000 And so he then undertook a study to ask what genes regulate the cell 573 00:45:54,000 --> 00:45:58,000 death process in this simple organism called C. 574 00:45:58,000 --> 00:46:02,000 elegans, which as an adult has only 1,090 cells. And the beauty of this 575 00:46:02,000 --> 00:46:06,000 organism is that you can actually see through it during development, 576 00:46:06,000 --> 00:46:10,000 and you can therefore follow the fate of individual cells over the 577 00:46:10,000 --> 00:46:14,000 course of development. And he and his students and fellows 578 00:46:14,000 --> 00:46:18,000 did that, he observed that in the development of certain cell lineages 579 00:46:18,000 --> 00:46:22,000 of the developing worm, individual cells died. 580 00:46:22,000 --> 00:46:27,000 In fact, about 130 cells underwent this process of apoptosis. 581 00:46:27,000 --> 00:46:32,000 And then he carried out a genetic experiment. He asked, 582 00:46:32,000 --> 00:46:38,000 if I mutagenize worms, can I find ones in which this process doesn't 583 00:46:38,000 --> 00:46:43,000 happen, or perhaps, in which this process happens too 584 00:46:43,000 --> 00:46:49,000 much. What are the genes that regulate apoptosis? 585 00:46:49,000 --> 00:46:54,000 And he found several such genes that turn viable cells into cells 586 00:46:54,000 --> 00:47:00,000 that have undergone programmed cell death, or apoptosis. 587 00:47:00,000 --> 00:47:05,000 In particular, there were two genes that positively 588 00:47:05,000 --> 00:47:10,000 regulated this process. They were called Ced-3 and Ced-4. 589 00:47:10,000 --> 00:47:15,000 They were required for apoptosis to occur. Mutants in Ced-3 and Ced-4 590 00:47:15,000 --> 00:47:21,000 didn't have this cell death process in those developing worms. 591 00:47:21,000 --> 00:47:26,000 And another gene called Ced-9 was required to prevent apoptosis. 592 00:47:26,000 --> 00:47:32,000 In a Ced-9 mutant, there was too much apoptosis going on, OK? 593 00:47:32,000 --> 00:47:38,000 They then cloned these genes, and it turned out they all have 594 00:47:38,000 --> 00:47:44,000 homologs, versions, in our cells, and those genes in our 595 00:47:44,000 --> 00:47:50,000 cells likewise regulate apoptosis in us. And we now know that at least 596 00:47:50,000 --> 00:47:56,000 members of this family of genes are important in diseases, 597 00:47:56,000 --> 00:48:02,000 including cancer. I'll just mention the function of one gene, 598 00:48:02,000 --> 00:48:08,000 right now. Ced-3, it is a protease, which cleaves target proteins -- 599 00:48:08,000 --> 00:48:22,000 -- into fragments. 600 00:48:22,000 --> 00:48:26,000 So, one of the key things that happens in apoptosis is, 601 00:48:26,000 --> 00:48:30,000 you turn on this enzyme, this protease, and it goes around 602 00:48:30,000 --> 00:48:34,000 cutting up various target proteins, which eventually lead to the death 603 00:48:34,000 --> 00:48:38,000 of the cell. There are many target proteins that get cleaved when these 604 00:48:38,000 --> 00:48:43,000 caspases, these proteases are called caspases, when these proteases get 605 00:48:43,000 --> 00:48:47,000 activated, and I just gave you one example of one such target. 606 00:48:47,000 --> 00:48:51,000 There's an enzyme that is normally involved in cleaving your DNA, 607 00:48:51,000 --> 00:48:55,000 this is a DNA gel, of viable cells and cells that have undergone 608 00:48:55,000 --> 00:49:00,000 apoptosis after a certain number of hours. 609 00:49:00,000 --> 00:49:04,000 The DNA of the cells is normally intact, and large, 610 00:49:04,000 --> 00:49:09,000 because this enzyme is kept in check. However, when its inhibitor is 611 00:49:09,000 --> 00:49:13,000 cleaved by the caspsase, it now goes about cleaving the DNA, 612 00:49:13,000 --> 00:49:18,000 and this is one of the reasons why apoptotic cells die, 613 00:49:18,000 --> 00:49:22,000 because their DNA gets cut up into very small fragments. 614 00:49:22,000 --> 00:49:27,000 There are a number of other targets, you can read a little bit more about 615 00:49:27,000 --> 00:49:31,000 this in your book, I apologize that we had to rush 616 00:49:31,000 --> 00:49:36,000 through apoptosis, it is important, and we will in fact 617 00:49:36,000 --> 00:49:39,000 come back to it in a discussion of cancer.