1 00:00:12 --> 00:00:19 So today's lecture talks about the cell cycle, the control of the cell 2 00:00:19 --> 00:00:27 cycle, and also cell death. So obviously, cell division is 3 00:00:27 --> 00:00:34 extremely important in multi-cellular organisms. 4 00:00:34 --> 00:00:42 We've talked a fair bit about control of the cell cycle, 5 00:00:42 --> 00:00:50 in terms of mitosis and meiosis, in earlier lectures. 6 00:00:50 --> 00:00:54 Obviously, cell division is necessary in a variety of 7 00:00:54 --> 00:00:59 circumstances, probably the most obvious is 8 00:00:59 --> 00:01:03 development. You go from one cell to ten to the thirteenth to ten to 9 00:01:03 --> 00:01:08 the fourteenth cells. That's accomplished by a tremendous 10 00:01:08 --> 00:01:13 amount of cell division that goes on over your gestational period. 11 00:01:13 --> 00:01:17 I've also pointed out, in fact we talked about it last time, 12 00:01:17 --> 00:01:22 that cell, that wound healing is, in part, a process of cell division. 13 00:01:22 --> 00:01:27 Fibroblasts for example, 14 00:01:27 --> 00:01:31 and other epithelial cells, get recruited to divide, in order to 15 00:01:31 --> 00:01:36 repair damage to a tissue. 16 00:01:36 --> 00:01:41 And even without damage, there's a lot of cell turnover, 17 00:01:41 --> 00:01:46 for many tissues. You may not realize this, but your blood, 18 00:01:46 --> 00:01:51 for example, turns over about every month, so you have to replace all 19 00:01:51 --> 00:01:57 your red blood cells, and all your white blood cells, 20 00:01:57 --> 00:02:01 and so on, periodically. Cells in other tissues, 21 00:02:01 --> 00:02:05 in the skin for example, your skin cells are born, 22 00:02:05 --> 00:02:09 they migrate up to the superficial layers of your skin, 23 00:02:09 --> 00:02:13 and then they get sloughed off, and of course, they have to be 24 00:02:13 --> 00:02:16 replaced. There's a lot of cell division that's going on naturally, 25 00:02:16 --> 00:02:20 in the process of what we call homeostasis, and to give you an 26 00:02:20 --> 00:02:24 example of that, if you consider your intestines in a 27 00:02:24 --> 00:02:28 human, your intestines undergo ten to the eleventh cell divisions per 28 00:02:28 --> 00:02:31 day. That's a remarkable number. Ten to the eleventh cells dividing 29 00:02:31 --> 00:02:35 in your intestines per day. If you imagine that a cell in your 30 00:02:35 --> 00:02:39 intestine is about ten microns in diameter, if you lined up all the 31 00:02:39 --> 00:02:43 cells next to each other that were born in a given period of time, 32 00:02:43 --> 00:02:46 in 38 days you would have enough cells lined up end-to-end to go 33 00:02:46 --> 00:02:50 around the earth, and in a year you'd have enough 34 00:02:50 --> 00:02:54 cells to make it from the earth to the moon. So you produce a 35 00:02:54 --> 00:02:58 tremendous number of cells just naturally, in the process of organ 36 00:02:58 --> 00:03:02 function and homeostasis. Of course we can have too much cell 37 00:03:02 --> 00:03:08 division, and this is pathological condition, which we typically 38 00:03:08 --> 00:03:14 associate with cancer. And indeed, many of the factors 39 00:03:14 --> 00:03:19 that I'm going to talk to you about today, that relate to the normal 40 00:03:19 --> 00:03:25 control of cell division, and also cell death, are perturbed 41 00:03:25 --> 00:03:31 in the development of cancer. Cancer, as you almost certainly 42 00:03:31 --> 00:03:37 know, is a disease of too many cells, and a major factor in that is 43 00:03:37 --> 00:03:41 excessive cell division. [UNINTELLIGIBLE PHRASE]. 44 00:03:41 --> 00:03:44 So this happens to be a human cancer cell line growing in tissue 45 00:03:44 --> 00:03:47 culture, and one of the hallmark features of cancer cells is that 46 00:03:47 --> 00:03:49 they have, in a sense, unlimited and unregulated cell 47 00:03:49 --> 00:03:52 division. So if you were to watch this movie, which actually comes 48 00:03:52 --> 00:03:55 from your book, supplementary materials from your 49 00:03:55 --> 00:03:58 book, you could watch these cells dividing, and they would do so in an 50 00:03:58 --> 00:04:02 unnatural fashion. Without response to proper growth 51 00:04:02 --> 00:04:07 factors that would stimulate cell division, they would divide on top 52 00:04:07 --> 00:04:12 of one another, which normal cells don't do, 53 00:04:12 --> 00:04:17 and other places and times when other cells are kept in check, 54 00:04:17 --> 00:04:22 cancer cells are not. So the basic process, which again is well 55 00:04:22 --> 00:04:27 familiar to you, is to take a single cell and turn it 56 00:04:27 --> 00:04:36 into two. 57 00:04:36 --> 00:04:40 And we discussed the very last stages of this in earlier lectures 58 00:04:40 --> 00:04:44 on mitosis, how the chromosomes get divided from the mother to the two 59 00:04:44 --> 00:04:48 daughter cells. We also have briefly alluded to the 60 00:04:48 --> 00:04:52 fact that there's a second, critical event, and you talked about 61 00:04:52 --> 00:04:56 the mechanisms of DNA replication. The DNA needs to get duplicated so 62 00:04:56 --> 00:05:00 that it can be properly divided, and this, as you know, occurs during 63 00:05:00 --> 00:05:04 a particular phase of the cell cycle, known as S-phase, for 64 00:05:04 --> 00:05:08 synthesis phase. And then there's chromosome 65 00:05:08 --> 00:05:13 segregation, and this occurs in a distinct phase of the cell cycle 66 00:05:13 --> 00:05:19 called M-phase, or mitosis phase. 67 00:05:19 --> 00:05:24 And the development of cells over the course of, 68 00:05:24 --> 00:05:29 say development, or in wound healing, 69 00:05:29 --> 00:05:35 can be thought of as a successive iteration of DNA duplication, 70 00:05:35 --> 00:05:40 DNA replication, and chromosome segregation. So in a sense, 71 00:05:40 --> 00:05:46 you go from mitosis to S-phase to mitosis to S-phase. 72 00:05:46 --> 00:05:50 These segments are separated by periods of time when cells are 73 00:05:50 --> 00:05:55 accumulating the biomaterials that they need, basically to function, 74 00:05:55 --> 00:06:00 and also to carry out the next phases of the cell cycle, 75 00:06:00 --> 00:06:04 and these are refereed to as gap phases, G1 for the one that 76 00:06:04 --> 00:06:09 separates mitosis from S-phase, and G2, the one that separates the 77 00:06:09 --> 00:06:14 S-phase from mitosis. Now, this is a cyclic process, 78 00:06:14 --> 00:06:19 one cell gives rise to two, that then undergoes this process again, 79 00:06:19 --> 00:06:24 and so we think of these things as cycles from M-to-S, 80 00:06:24 --> 00:06:29 connected by these gap phases, G1 and G2. Now there's another 81 00:06:29 --> 00:06:34 phase that we haven't told you about, which many of your cells are 82 00:06:34 --> 00:06:40 actually in right now, and that's a phase called G0. 83 00:06:40 --> 00:06:44 This is a resting phase, and this can be either permanent or 84 00:06:44 --> 00:06:49 temporary. Many of your cells, when they're born, will stop 85 00:06:49 --> 00:06:54 dividing forever. Cells in your brain, 86 00:06:54 --> 00:06:59 for example, or most cells in your brain, cells of your cardiac muscle, 87 00:06:59 --> 00:07:04 and there are many other examples. Once they get born, 88 00:07:04 --> 00:07:09 they undergo mitosis, they go into this resting phase, 89 00:07:09 --> 00:07:14 and they'll never come back out again. 90 00:07:14 --> 00:07:17 And that's why it's difficult, for example, to deal with brain 91 00:07:17 --> 00:07:21 injuries, or spinal cord injuries, because those cells cannot be 92 00:07:21 --> 00:07:24 recruited back into the cell cycle, they can't make more of them. But 93 00:07:24 --> 00:07:28 there are other cells in which the resting phase is temporary, 94 00:07:28 --> 00:07:32 and these cells can then reenter the cell cycle, going back from G0 to G1 95 00:07:32 --> 00:07:35 and through the process again. And a lot of the progenitor cells 96 00:07:35 --> 00:07:39 that I referred to up there, in terms of the skin and the blood, 97 00:07:39 --> 00:07:43 and the intestines, are in that situation. 98 00:07:43 --> 00:07:47 They're resting, and then they be recruited back into 99 00:07:47 --> 00:07:51 the cell cycle, in order to make more derivative 100 00:07:51 --> 00:07:56 cells. It's also useful to consider what the chromosomes are doing in 101 00:07:56 --> 00:08:00 these different phases of the cell cycle. So in a G1 cell, 102 00:08:00 --> 00:08:05 if we think about a single chromosome, and we're representing 103 00:08:05 --> 00:08:09 it by a single-line, although this is of course, 104 00:08:09 --> 00:08:14 a double-helix. In the G1 phase there's a single, 105 00:08:14 --> 00:08:18 double-helical chromosome, in S-phase that gets duplicated in 106 00:08:18 --> 00:08:23 the process of DNA replication, initiation shown here, and it would 107 00:08:23 --> 00:08:27 be completed so that the single-chromosome would be 108 00:08:27 --> 00:08:34 ultimately duplicated into two. 109 00:08:34 --> 00:08:37 And then in M-phase, those two chromosomes get separated 110 00:08:37 --> 00:08:41 from one another, so these are now joined, 111 00:08:41 --> 00:08:45 separated from one another, eventually into two daughter cells, 112 00:08:45 --> 00:08:48 which can then go through this process again. 113 00:08:48 --> 00:08:52 OK? And much of what we think about, when we think about how the 114 00:08:52 --> 00:08:56 cell cycle is controlled is to deal with the initiation of DNA 115 00:08:56 --> 00:09:00 replication, and then the initiation of mitosis. 116 00:09:00 --> 00:09:03 OK, so this is a cyclical process, a highly ordered process, this is a 117 00:09:03 --> 00:09:07 representation of the cell cycle, as shown in your book, just so you 118 00:09:07 --> 00:09:11 know it's in there. Exactly as I described to you, 119 00:09:11 --> 00:09:15 mitosis and S-phase, where the action is, these gap phases, 120 00:09:15 --> 00:09:18 G1 and G2, and this little arrow represents G0, 121 00:09:18 --> 00:09:22 and I've also used this term before, interphase, that represents all the 122 00:09:22 --> 00:09:26 phases where the DNA is not evident under the light microscope. 123 00:09:26 --> 00:09:30 It's only evident in mitosis because of DNA condensation, 124 00:09:30 --> 00:09:34 so the rest of the cycle, G1, S, and G2, are called interphase. 125 00:09:34 --> 00:09:37 So this is a highly ordered process, 126 00:09:37 --> 00:09:41 each step preceded by a particular other step, leading to a particular 127 00:09:41 --> 00:09:44 outcome, events leading to a particular outcome, 128 00:09:44 --> 00:09:48 and in a topical sense, you can think about it as the NCAA 129 00:09:48 --> 00:09:51 pools, starting tomorrow there'll be individual events, 130 00:09:51 --> 00:09:55 games, which will lead to next events in the next round, 131 00:09:55 --> 00:09:58 and next events in the next round, to an ultimate conclusion, in this 132 00:09:58 --> 00:10:02 case, the NCAA champion, which can only occur if the proper 133 00:10:02 --> 00:10:06 events have happened, prior to it. 134 00:10:06 --> 00:10:12 And you'll see another specific example of how the order is assessed 135 00:10:12 --> 00:10:18 in the development of the stages of the cell cycle. 136 00:10:18 --> 00:10:24 OK, so we know that the cell cycle is important. The question is, 137 00:10:24 --> 00:10:30 how is it controlled? How do you accomplish this orderly process of 138 00:10:30 --> 00:10:36 cell cycle progression? What are the genes -- 139 00:10:36 --> 00:10:46 -- that regulate this process? 140 00:10:46 --> 00:10:50 What do the genes encode, what do the proteins, and what do the 141 00:10:50 --> 00:10:54 proteins do? What are the pathways that they regulate to ensure the 142 00:10:54 --> 00:10:58 stages of the cell cycle? This was a huge question for 143 00:10:58 --> 00:11:02 decades, and there was rather little progress in trying to understand how 144 00:11:02 --> 00:11:06 it happened, especially in us. Our cells are sufficiently complex, 145 00:11:06 --> 00:11:10 and there's relatively imprecise, or particularly was in the past, 146 00:11:10 --> 00:11:14 imprecise methods for dissecting complex processes in 147 00:11:14 --> 00:11:18 mammalian cells. And so it took experiments performed 148 00:11:18 --> 00:11:22 in yeast, a single-cell, eukaryotic organism, budding yeast 149 00:11:22 --> 00:11:27 and also fission yeast, to allow us to understand what the 150 00:11:27 --> 00:11:31 details of the cell cycle are. Budding yeast, as shown here in a 151 00:11:31 --> 00:11:36 picture from your book, is a single-cell organism. 152 00:11:36 --> 00:11:39 It's also haploid, or it can be at least, 153 00:11:39 --> 00:11:43 haploid, which means that it has a single set of chromosomes, 154 00:11:43 --> 00:11:46 it doesn't have two sets, single set of genes, which makes it amenable to 155 00:11:46 --> 00:11:50 genetic analysis. You can make mutants more easily, 156 00:11:50 --> 00:11:53 if you only have one copy of each gene to worry about. 157 00:11:53 --> 00:11:57 So that's another reason that yeast was attractive. 158 00:11:57 --> 00:12:01 And another reason, which is kind of seen here, 159 00:12:01 --> 00:12:04 not really obvious here, but when yeast cells divide, 160 00:12:04 --> 00:12:08 they go through a very characteristic, morphological 161 00:12:08 --> 00:12:11 change. They start off as spheres, 162 00:12:11 --> 00:12:15 like this one, and as they go through the cell cycle, 163 00:12:15 --> 00:12:19 they develop a small bud. That bud then gets bigger, 164 00:12:19 --> 00:12:22 and eventually the two, the joint between the mother cell and the bud, 165 00:12:22 --> 00:12:26 get severed to form two cells. And you can actually figure out where 166 00:12:26 --> 00:12:30 you are in the cell cycle, by examining exactly what the 167 00:12:30 --> 00:12:34 morphology of the yeast cell is, and I'll show you that on the next 168 00:12:34 --> 00:12:38 slide. This is the yeast cell cycle, 169 00:12:38 --> 00:12:42 with overlay of the diagram of what the cells look like in the different 170 00:12:42 --> 00:12:46 phases, so a yeast cell that's in G0, let's say, looks like this, 171 00:12:46 --> 00:12:50 fairly round, nondescript, as it enters G1 it creates a small bud, 172 00:12:50 --> 00:12:54 that bud then gets bigger as the cells are now duplicating their DNA, 173 00:12:54 --> 00:12:58 the bud actually gets bigger. It gets bigger still, 174 00:12:58 --> 00:13:02 during G2 phase, and then at M-phase you can actually see a little 175 00:13:02 --> 00:13:06 junction between the two cells, and the mother and the daughter cell 176 00:13:06 --> 00:13:10 are more-or-less the same size, and then they separate from one 177 00:13:10 --> 00:13:14 another, and they can go through the cycle again. 178 00:13:14 --> 00:13:17 And this is useful because you can tell if you have a mutant, 179 00:13:17 --> 00:13:21 as you'll see in a moment, if you have a mutant in a gene that affects 180 00:13:21 --> 00:13:25 one of these processes, you can actually figure out where 181 00:13:25 --> 00:13:28 the mutant gene acts within the cell cycle, based on what the yeast cell 182 00:13:28 --> 00:13:32 looks like. And these individuals here, won the Nobel prize for their 183 00:13:32 --> 00:13:36 efforts to understand the cell cycle, they won it about five 184 00:13:36 --> 00:13:39 years ago, or so. This is Lee Hartwell, 185 00:13:39 --> 00:13:43 he's an American, happens to run a cancer center out at the University 186 00:13:43 --> 00:13:46 of Washington, very suave and sophisticated guy. 187 00:13:46 --> 00:13:49 These two goofballs are Brits, very nice guys actually, 188 00:13:49 --> 00:13:53 Paul Nurse, who's now the president of Rockefeller University in New 189 00:13:53 --> 00:13:56 York, and Tim Hunt, actually Sir Tim Hunt, 190 00:13:56 --> 00:14:00 because he's knighted after this Nobel Prize. And I'm going to 191 00:14:00 --> 00:14:03 explain the experiments that all three of these guys did today, 192 00:14:03 --> 00:14:07 which won them the Nobel prize, and gave us tremendous insight into 193 00:14:07 --> 00:14:10 the control of the cell cycle in yeast, which turned out to tell us 194 00:14:10 --> 00:14:14 how the cell cycle is controlled, also, in us. 195 00:14:14 --> 00:14:17 OK, so the first thing we need to do is to create mutants. 196 00:14:17 --> 00:14:21 That's a general theme in biology. If you want to understand a process, 197 00:14:21 --> 00:14:25 find a mutant that can't do it, and understand the genes that get 198 00:14:25 --> 00:14:29 mutated in that process, and so that's what happened in the 199 00:14:29 --> 00:14:33 hands of Lee Hartwell, the guy in the upper-right, 200 00:14:33 --> 00:14:37 he took this same yeast, the budding yeast, 201 00:14:37 --> 00:14:41 which is also Brewer's yeast, the yeast that makes beer. 202 00:14:41 --> 00:14:46 He took a population of these yeast 203 00:14:46 --> 00:14:51 cells, and he mutagenized them. 204 00:14:51 --> 00:14:54 I told you about this before, you can add chemical mutagens that 205 00:14:54 --> 00:14:57 soak into the cells, bind to the DNA, cause mutations. 206 00:14:57 --> 00:15:01 Each cell might have one, or a few nucleotides changed, 207 00:15:01 --> 00:15:04 and at some frequency those mutations will affect genes, 208 00:15:04 --> 00:15:08 and at some frequency the genes affected will influence the process 209 00:15:08 --> 00:15:11 that you're trying to study. So he was trying to study growth 210 00:15:11 --> 00:15:15 control, cell division, so he asked about the ability of the 211 00:15:15 --> 00:15:18 cells to grow, and particularly, 212 00:15:18 --> 00:15:21 he did so at two different temperatures. He tested the ability 213 00:15:21 --> 00:15:25 of the cells to grow at their normal temperature, which is about 25 214 00:15:25 --> 00:15:28 degrees centigrade, and he found that many of these 215 00:15:28 --> 00:15:32 yeast cells would grow, following mutagenesis, at 25 degrees. 216 00:15:32 --> 00:15:36 Probably some of them wouldn't grow 217 00:15:36 --> 00:15:40 because they would've inactivated some critical gene, 218 00:15:40 --> 00:15:44 and even at 25 degrees, they couldn't grow. However, 219 00:15:44 --> 00:15:48 he then took these plates, and he used a technique, 220 00:15:48 --> 00:15:53 which I referred to in a previous lecture, called replica plating. 221 00:15:53 --> 00:15:57 He took a stamp, basically, of these colonies, 222 00:15:57 --> 00:16:01 transferred them onto a fresh plate, and examined how the cells grew at 223 00:16:01 --> 00:16:05 30 degrees centigrade. And what he found was that, 224 00:16:05 --> 00:16:09 whereas many of the cells that were able to grow at 25 degrees continued 225 00:16:09 --> 00:16:12 to grow at 30 degrees, so they produced colonies in exactly 226 00:16:12 --> 00:16:16 the pattern that he had seen previously. Some of 227 00:16:16 --> 00:16:20 the cells didn't. This colony here, 228 00:16:20 --> 00:16:24 while it was successful, the cells were successful in growing 229 00:16:24 --> 00:16:29 at 25 degrees, failed to grow at 30 degrees. 230 00:16:29 --> 00:16:33 And this type of mutant is referred to as a temperature-sensitive 231 00:16:33 --> 00:16:48 mutant, -- [PAUSE} 232 00:16:48 --> 00:16:52 -- abbreviated TS. We imagine that this mutation 233 00:16:52 --> 00:16:56 affects the amino acid sequence of the protein, at low temperatures the 234 00:16:56 --> 00:17:00 protein is still able to function, but at higher temperatures, maybe it 235 00:17:00 --> 00:17:04 becomes a little unstable, and now it can't function, and 236 00:17:04 --> 00:17:08 that's why you see no growth here. So we had a TS-mutant that affected 237 00:17:08 --> 00:17:11 the growth of the cells, OK? So my question to you is, 238 00:17:11 --> 00:17:15 is that TS-mutant interesting? Is it interesting? 239 00:17:15 --> 00:17:22 Well we can't actually know whether 240 00:17:22 --> 00:17:26 it's interesting yet, because there are two general 241 00:17:26 --> 00:17:30 classes of mutations that would give you this same phenotype. 242 00:17:30 --> 00:17:35 One of them is interesting, does that cement always show, 243 00:17:35 --> 00:17:39 or do we have a problem here? It's always there? Never noticed it, 244 00:17:39 --> 00:17:43 in all these years. You can distinguish between these two 245 00:17:43 --> 00:17:48 classes of boring and interesting mutants, based on the morphology of 246 00:17:48 --> 00:17:52 the yeast cells when you shift the temperature. If you start with a 247 00:17:52 --> 00:17:57 population of yeast cells growing just randomly at 25 degrees 248 00:17:57 --> 00:18:01 centigrade, including these mutants, if you were to look under the 249 00:18:01 --> 00:18:05 microscope, you would find mutants, cells that were at various stages of 250 00:18:05 --> 00:18:10 the cell cycle. OK? Now, let's imagine that our mutation 251 00:18:10 --> 00:18:14 affects a general enzyme that's required for cell viability, 252 00:18:14 --> 00:18:18 DNA polymerase or maybe, better still, some ribosomal protein that 253 00:18:18 --> 00:18:22 all cells need all the time, in order to function. If I were to 254 00:18:22 --> 00:18:26 take these cells which were growing at 25 degrees centigrade, 255 00:18:26 --> 00:18:30 and I were to shift them to 30 degrees centigrade, 256 00:18:30 --> 00:18:34 and look under the microscope, what would I see? 257 00:18:34 --> 00:18:41 Well, if it's a ribosomal protein, 258 00:18:41 --> 00:18:45 and now translation just stops, these cells are going nowhere. 259 00:18:45 --> 00:18:49 Regardless of where they were in the cell cycle, 260 00:18:49 --> 00:18:52 they don't go any further because you need protein synthesis to do 261 00:18:52 --> 00:18:56 anything, so if you look under the microscope at these cells, 262 00:18:56 --> 00:19:00 following a temperature shift, you would still find a distribution 263 00:19:00 --> 00:19:04 of cells in the different phases of the cell cycle. 264 00:19:04 --> 00:19:08 Lee Hartwell was not interested in this class of mutants. 265 00:19:08 --> 00:19:13 They could be anything, he didn't care. However, 266 00:19:13 --> 00:19:17 he reasoned that if he were dealing with a mutant that affected 267 00:19:17 --> 00:19:22 specifically a stage in the cell cycle, he might find that the cells, 268 00:19:22 --> 00:19:26 upon temperature shift from 25 degrees to 30 degrees, 269 00:19:26 --> 00:19:31 got to a particular point in the cell cycle and couldn't go any 270 00:19:31 --> 00:19:35 further, that this gene affected one of these important transitions, 271 00:19:35 --> 00:19:40 and so for example -- -- if he were to look under the 272 00:19:40 --> 00:19:45 microscope, all of the cells would have arrested with a small bud, 273 00:19:45 --> 00:19:50 would have arrested in G1, or maybe they would've arrested looking like 274 00:19:50 --> 00:19:55 this, just prior to mitosis, or like this, somewhere in G2. 275 00:19:55 --> 00:20:00 But importantly, they would arrest with a specific morphology that 276 00:20:00 --> 00:20:05 would indicate that they had a specific cell cycle block. 277 00:20:05 --> 00:20:10 And so he did this, and he found many, many such mutants. 278 00:20:10 --> 00:20:15 He called them cell division cycle mutants, or CDC mutants. 279 00:20:15 --> 00:20:20 And it was his methodology, which really broke this field open, 280 00:20:20 --> 00:20:26 and won him the Nobel prize. Now one example of a CDC mutant that he 281 00:20:26 --> 00:20:31 discovered, called CDC2, was particularly important. 282 00:20:31 --> 00:20:37 It's been renamed CDK1, or cyclin-dependent kinase one, 283 00:20:37 --> 00:20:44 for reasons that I'll come to. Cyclin, sorry, cyclin-dependent, 284 00:20:44 --> 00:20:54 kinase one. 285 00:20:54 --> 00:21:00 And this one acts at a particular phase in the cell cycle, 286 00:21:00 --> 00:21:07 early on in the transition from G1 to S. So if you imagine cells in G0, 287 00:21:07 --> 00:21:13 going to G1, and then progressing from G1 into S-phase, 288 00:21:13 --> 00:21:20 into G2, and finally, in mitosis. This particular mutant blocked 289 00:21:20 --> 00:21:27 right here, and so it said that this gene, CDC2 or CDK1, 290 00:21:27 --> 00:21:34 was required for this transition. 291 00:21:34 --> 00:21:38 And just to emphasize the TS-temperature shift and building up 292 00:21:38 --> 00:21:42 of the cells in the cell cycle concept, imagine a cell that looked 293 00:21:42 --> 00:21:46 like this, before the temperature shift. Ok? If you now do the 294 00:21:46 --> 00:21:50 temperature shift, this cell will progress to this 295 00:21:50 --> 00:21:54 point, but it won't go any further because CDC2 is required. 296 00:21:54 --> 00:21:58 Maybe I'll draw this over here, to make it even more obvious. 297 00:21:58 --> 00:22:01 CDC2 is required to go beyond this point, so that cell will arrest 298 00:22:01 --> 00:22:05 right here. If there were a cell that looked like this, 299 00:22:05 --> 00:22:09 in the population of 25 degrees, and now you shifted the temperature, 300 00:22:09 --> 00:22:13 it would complete this phase because CDC2 is not required. 301 00:22:13 --> 00:22:16 It would make it all the way to here, it would keep going, 302 00:22:16 --> 00:22:19 and then it would get stuck here again. And that's why you get cells 303 00:22:19 --> 00:22:23 building up with a particular morphology. OK? 304 00:22:23 --> 00:22:26 So this was successful, and as I said, he isolated a large 305 00:22:26 --> 00:22:29 number of such CDC mutants, which have taught us not about just 306 00:22:29 --> 00:22:33 that transition, but many other transitions in the 307 00:22:33 --> 00:22:37 cell. Now what Paul Nurse did, 308 00:22:37 --> 00:22:43 this guy here, he actually was performing very similar experiments 309 00:22:43 --> 00:22:48 in a related yeast species, but the critical experiment that he 310 00:22:48 --> 00:22:54 did was to clone the gene that is responsible for CDC2 function. 311 00:22:54 --> 00:23:00 So he took CDC2 mutant cells, which at 30 degrees -- 312 00:23:00 --> 00:23:08 -- will not grow. 313 00:23:08 --> 00:23:14 And he added, through a cDNA library, CDC2 cDNA, 314 00:23:14 --> 00:23:20 of yeast origin. So he made a cDNA library from yeast, 315 00:23:20 --> 00:23:26 he introduced it into these mutant cells, and then he asked whether the 316 00:23:26 --> 00:23:32 cells could now grow at 30 degrees -- 317 00:23:32 --> 00:23:40 -- and the answer was yes. 318 00:23:40 --> 00:23:44 That is, if the cells now carried an extra copy of CDC2, 319 00:23:44 --> 00:23:49 in the form of this cDNA, they now could grow at 30 degrees. 320 00:23:49 --> 00:23:53 He complemented the mutation through the addition of a normal 321 00:23:53 --> 00:23:57 copy of the CDC2 cDNA. Quite surprisingly, he did the same 322 00:23:57 --> 00:24:05 experiment -- 323 00:24:05 --> 00:24:09 -- using not a yeast gene, but a human gene. Frankly, 324 00:24:09 --> 00:24:13 there was great skepticism in the field about whether what these guys 325 00:24:13 --> 00:24:17 were doing in yeast had anything to do with cell cycle control in humans. 326 00:24:17 --> 00:24:21 Most people actually assumed that it probably had nothing to do with 327 00:24:21 --> 00:24:25 it, and so therefore, when Paul Nurse proposed to try to 328 00:24:25 --> 00:24:29 complement his yeast mutation with a human gene, people 329 00:24:29 --> 00:24:36 were skeptical. 330 00:24:36 --> 00:24:40 But in fact, it worked. And this told him, and the field, 331 00:24:40 --> 00:24:45 that the machinery that controls the cell cycle in this simple, 332 00:24:45 --> 00:24:49 single-celled eukaryote, is highly conserved, all the way through to 333 00:24:49 --> 00:24:54 humans, and that we could therefore understand cell cycle control in us, 334 00:24:54 --> 00:24:59 by doing experiments like this in yeast. So this really broke the 335 00:24:59 --> 00:25:03 field open, and that's why Nurse won the Nobel prize at that same time. 336 00:25:03 --> 00:25:08 However, there was a problem. They did in fact clone the gene, 337 00:25:08 --> 00:25:12 they were able to sequence the gene, and they found that the sequence of 338 00:25:12 --> 00:25:17 the gene, which I'm going to refer to now by the other name, 339 00:25:17 --> 00:25:22 CDK1, looked like a kinase. It had amino acid sequence, 340 00:25:22 --> 00:25:26 which made it resemble known kinases, so it was assumed to be a protein 341 00:25:26 --> 00:25:31 kinase. However, it didn't have any kinase activity. 342 00:25:31 --> 00:25:43 343 00:25:43 --> 00:25:46 If you mixed it with various substrate molecules in the presence 344 00:25:46 --> 00:25:49 of ATP, those substrates were not changed, they were not 345 00:25:49 --> 00:25:52 phosphorylated. So the purified CDK enzyme had no 346 00:25:52 --> 00:25:56 kinase activity, and therefore, it wasn't entirely 347 00:25:56 --> 00:25:59 clear what its function really was, and the field sort of got stuck 348 00:25:59 --> 00:26:02 there for a while, trying to understand the biochemical 349 00:26:02 --> 00:26:06 function of CDK1, and other cell cycle regulators. 350 00:26:06 --> 00:26:11 And that then led to the experiments done by Tim Hunt, 351 00:26:11 --> 00:26:17 this guy here. And Tim Hunt, actually doing experiments at Woods 352 00:26:17 --> 00:26:22 Hole, down at the Cape, in a summer course, with summer 353 00:26:22 --> 00:26:28 students, did a famous experiment using sea urchins. 354 00:26:28 --> 00:26:33 Sea urchins, when they're fertilized, 355 00:26:33 --> 00:26:37 undergo very rapid, and very synchronous, 356 00:26:37 --> 00:26:42 cell divisions. In the first few hours after you fertilize sea urchin 357 00:26:42 --> 00:26:46 eggs, they will divide and divide again. And importantly, 358 00:26:46 --> 00:26:51 they will do so in a very synchronous manner, 359 00:26:51 --> 00:26:55 so all the cells will produce two cells at around the same time, 360 00:26:55 --> 00:27:00 and those will all produce four cells at around the same time. 361 00:27:00 --> 00:27:04 And this is important if one wants to do biochemical experiments, 362 00:27:04 --> 00:27:09 to understand what is changing within these cells as they're going 363 00:27:09 --> 00:27:14 through these various cell cycles. And so, what Tim and his students 364 00:27:14 --> 00:27:18 did was to label the proteins in these dividing sea urchin cells, 365 00:27:18 --> 00:27:23 with a radioactive amino acid, S35 methionine. And then they simply 366 00:27:23 --> 00:27:28 made extracts of these cells at different time points thereafter, 367 00:27:28 --> 00:27:32 and asked whether anything was changing in an interesting pattern, 368 00:27:32 --> 00:27:37 at different times that correspond to different stages 369 00:27:37 --> 00:27:43 of the cell cycle. So they ran protein gels, 370 00:27:43 --> 00:27:50 they separated the proteins and then exposed the gels to x-ray film, 371 00:27:50 --> 00:27:57 to visual what the protein concentrations were at different 372 00:27:57 --> 00:28:03 time points. They took cells at time zero, they took cells after 30 373 00:28:03 --> 00:28:10 minutes, after 60 minutes, after 90 minutes, 120 minutes, 374 00:28:10 --> 00:28:16 150 minutes, 180 minutes. And they knew already, 375 00:28:16 --> 00:28:21 from their earlier analysis, that this corresponded to the first 376 00:28:21 --> 00:28:27 cell cycle, and this corresponded to the second cell cycle. 377 00:28:27 --> 00:28:32 OK? Now some proteins, when they visualize them that way, 378 00:28:32 --> 00:28:37 didn't change. At the different time points they saw roughly equal 379 00:28:37 --> 00:28:43 concentrations of that protein throughout, but interestingly, 380 00:28:43 --> 00:28:48 other proteins changed in abundance, and did so according to a pattern. 381 00:28:48 --> 00:29:02 382 00:29:02 --> 00:29:07 They seemed to oscillate, they seemed to cycle, in a pattern 383 00:29:07 --> 00:29:12 that corresponded with the cell cycle. And so he called these 384 00:29:12 --> 00:29:17 cyclins, and he suggested that they might have something to do with the 385 00:29:17 --> 00:29:22 control of the cell division cycle. Well, meanwhile, Nurse and Hartwell 386 00:29:22 --> 00:29:27 were doing their thing on CDKs, and so they came up with the idea 387 00:29:27 --> 00:29:32 that maybe these two things have something to do with one another. 388 00:29:32 --> 00:29:37 And particularly, maybe the failure of CDK to function 389 00:29:37 --> 00:29:42 as a kinase was due to the fact that it didn't have an accessory protein 390 00:29:42 --> 00:29:47 that it needed, mainly the cyclin. 391 00:29:47 --> 00:29:53 And so, whereas CDK2, sorry, CDK1, was an inactive protein 392 00:29:53 --> 00:30:04 kinase. 393 00:30:04 --> 00:30:09 Now in a test tube, in a biochemical experiment, 394 00:30:09 --> 00:30:14 if they mixed CDK1 with one of these cyclins, they observed kinase 395 00:30:14 --> 00:30:20 activity. And thus the name, cyclin-dependent kinase. It's not a 396 00:30:20 --> 00:30:25 kinase, it's not an active kinase in the absence of cyclin, 397 00:30:25 --> 00:30:31 it only becomes active in the presence of cyclin. 398 00:30:31 --> 00:30:36 So let me give you two quick examples of proteins that are then 399 00:30:36 --> 00:30:41 phosphorylated by this active kinase, to give you a sense of how this 400 00:30:41 --> 00:30:47 kinase regulates cell cycle transitions. There's a class of 401 00:30:47 --> 00:30:52 proteins that are involved in the regulation of replication initiation. 402 00:30:52 --> 00:30:58 We'll just call them replication initiation factors. 403 00:30:58 --> 00:31:03 In the absence of phosphorylation, they're inactive, and that's one of 404 00:31:03 --> 00:31:09 the reasons that replication is not initiated at those times 405 00:31:09 --> 00:31:15 in the cell cycle. However, in the presence of CDK 406 00:31:15 --> 00:31:22 cyclin, now the protein becomes phosphorylated, 407 00:31:22 --> 00:31:29 and becomes active. OK? So one of the ways you trigger 408 00:31:29 --> 00:31:36 the transition from G1 into S-phase, is to turn on this enzyme which 409 00:31:36 --> 00:31:43 phosphorylated this target protein, and stimulates S-phase entry. 410 00:31:43 --> 00:31:51 A second example is a protein called 411 00:31:51 --> 00:31:56 pRB. In its un-phosphorylated state, it is active, different from here 412 00:31:56 --> 00:32:01 where, in its un-phosphorylated state, it was inactive, 413 00:32:01 --> 00:32:06 and the function of the RB protein, in its active state, is to block 414 00:32:06 --> 00:32:11 again, the transition from S-phase to G1. This is actually an 415 00:32:11 --> 00:32:16 important cancer gene, it's mutated in a large number of 416 00:32:16 --> 00:32:21 cancers, and so we'll talk about its function, exactly how it blocks the 417 00:32:21 --> 00:32:26 transition, in later lectures, but suffice it to say, it does that. 418 00:32:26 --> 00:32:35 And when it is phosphorylated by CDK 419 00:32:35 --> 00:32:42 cyclins, it becomes inactive, thereby allowing cells to progress 420 00:32:42 --> 00:32:50 from G1 into S-phase. OK? So those are two examples of 421 00:32:50 --> 00:33:16 how CDK cyclins operate. 422 00:33:16 --> 00:33:23 Now, importantly, cyclin kinase activity is determined 423 00:33:23 --> 00:33:31 by the level of the cyclin. As I told you, cyclin levels 424 00:33:31 --> 00:33:38 oscillate, where this is cycle one, cycle two, cycle three. So these 425 00:33:38 --> 00:33:46 are cyclin concentrations inside the cell. CDK levels do 426 00:33:46 --> 00:34:00 not oscillate. 427 00:34:00 --> 00:34:04 Concentration of the CDKs inside the cells is rather constant. 428 00:34:04 --> 00:34:09 However, at this point in the cell cycle, when there's not enough 429 00:34:09 --> 00:34:13 cyclin, you don't have kinase activity. Only when you go past the 430 00:34:13 --> 00:34:18 threshold, do you get kinase activity. 431 00:34:18 --> 00:34:24 When it drops again, 432 00:34:24 --> 00:34:28 you lose kinase activity. But in the next cell cycle, 433 00:34:28 --> 00:34:33 the cyclin levels increase again, and you get kinase activity, and so 434 00:34:33 --> 00:34:38 on. So the oscillation of cyclins determines the oscillation of kinase 435 00:34:38 --> 00:34:42 activity, which determines the periodicity of the cell cycle. 436 00:34:42 --> 00:34:47 Now, for the truth in advertising, the situation is actually more 437 00:34:47 --> 00:34:52 complex, there are actually multiple CDKs and multiple cyclins. 438 00:34:52 --> 00:35:02 So in our cells, for example, if we imagine G1, 439 00:35:02 --> 00:35:13 S, M, G2, G1, S, M, G2, there's a cyclin called cyclin-D, 440 00:35:13 --> 00:35:24 which comes up in G1, goes down, comes up in the next G1, goes down. 441 00:35:24 --> 00:35:32 There's another cyclin called 442 00:35:32 --> 00:35:38 cyclin-E, which comes up a little bit later, in S-phase goes down, 443 00:35:38 --> 00:35:43 stays down, comes up in the next S-phase. And there's finally 444 00:35:43 --> 00:35:49 another one called cyclin-B, which comes up in G2, and comes down, 445 00:35:49 --> 00:36:14 goes up in the next G2. 446 00:36:14 --> 00:36:20 Anyway, the point is that there are different cyclins that get induced 447 00:36:20 --> 00:36:27 in different phases of the cell cycle, and actually control, 448 00:36:27 --> 00:36:33 through binding to different CDKs, different transitions in the 449 00:36:33 --> 00:36:40 different phases of the cell cycle. OK. Let's see. 450 00:36:40 --> 00:36:44 So, another concept: the transitions from the cell cycle don't occur, 451 00:36:44 --> 00:36:49 from one cell cycle position to the next don't occur, 452 00:36:49 --> 00:36:53 unless the previous cell cycle event has been completed. 453 00:36:53 --> 00:36:58 And that makes sense because you don't want to, 454 00:36:58 --> 00:37:02 for example, try to divide your DNA in mitosis if you haven't fully 455 00:37:02 --> 00:37:07 replicated your DNA in S-phase. So there are processes that are 456 00:37:07 --> 00:37:12 overlaid on top of cell cycle control, which ensure the completion 457 00:37:12 --> 00:37:17 of one phase before the next phase is initiated. And I draw, 458 00:37:17 --> 00:37:22 as an analogy, your washing machine, which likewise, has checkpoints 459 00:37:22 --> 00:37:27 which will determine whether or not the previous phase of the wash cycle 460 00:37:27 --> 00:37:33 has been completed. For example, you don't spin your 461 00:37:33 --> 00:37:39 wash until all the water has been rinsed out. There's a sensor that 462 00:37:39 --> 00:37:45 will determine whether that's not true, and if that sensor is tripped, 463 00:37:45 --> 00:37:51 it blocks the wash cycle at that point. Your cells have very similar 464 00:37:51 --> 00:37:57 checkpoints that will monitor and regulate cell cycle transitions, 465 00:37:57 --> 00:38:08 and they're called checkpoints. 466 00:38:08 --> 00:38:12 There are checkpoints, actually, that operate at different 467 00:38:12 --> 00:38:16 phases of the cell cycle. I'll only give you one example, 468 00:38:16 --> 00:38:20 it's actually the one that's best known. It occurs at the transition 469 00:38:20 --> 00:38:30 in mitosis, from metaphase -- 470 00:38:30 --> 00:38:36 -- where, if you'll recall, the duplicated chromosomes line up 471 00:38:36 --> 00:38:42 on the metaphase plate. In the process of anaphase, 472 00:38:42 --> 00:38:48 where the chromosomes separate from one another, the chromatids I should 473 00:38:48 --> 00:38:54 say, separate from one another. There's a cell cycle checkpoint 474 00:38:54 --> 00:39:00 that makes sure that this happens before that can happen. 475 00:39:00 --> 00:39:04 And in particular, if you have a chromosome which is 476 00:39:04 --> 00:39:08 only attached to one side of the mitotic spindle, 477 00:39:08 --> 00:39:12 so if you recall, these are centered in the middle 478 00:39:12 --> 00:39:16 through attachment to microtubules that are emanating from the two 479 00:39:16 --> 00:39:20 poles. If you have a chromosome that is only attached to one of the 480 00:39:20 --> 00:39:24 two spindles, it's called monopolar attachment, whereas perhaps other 481 00:39:24 --> 00:39:28 chromosomes, maybe all of the other chromosomes, are properly attached 482 00:39:28 --> 00:39:32 at the metaphase plate. This one, unattached chromosome will 483 00:39:32 --> 00:39:37 literally send a signal, a biochemical signal, which is the 484 00:39:37 --> 00:39:42 equivalent of "wait for me". And that signal will inhibit cell 485 00:39:42 --> 00:39:47 cycle progression. It'll specifically inhibit the 486 00:39:47 --> 00:39:52 transition from metaphase to anaphase. While that signal is 487 00:39:52 --> 00:39:57 being sent, the cells will just sit there, waiting for another 488 00:39:57 --> 00:40:02 microtubule to bind to this end of the chromosome, 489 00:40:02 --> 00:40:07 thereby extinguishing the signal, relieving this inhibition, and 490 00:40:07 --> 00:40:12 allowing anaphase to progress. OK, so these checkpoints are 491 00:40:12 --> 00:40:16 critical in insuring that these things happen in a timely and 492 00:40:16 --> 00:40:20 ordered fashion. OK. That's it for the cell cycle, 493 00:40:20 --> 00:40:24 so for the final ten minutes, and I always give short shrift to the next 494 00:40:24 --> 00:40:29 topic, which is cell death, which is another fascinating topic, 495 00:40:29 --> 00:40:33 fortunately there's not a lot of board work to show you. 496 00:40:33 --> 00:40:37 Cell death. So cells are born, divide, as we've just been talking 497 00:40:37 --> 00:40:42 about, but remarkable numbers of cells in your body die. 498 00:40:42 --> 00:40:46 They will die, too, because of cellular injury, 499 00:40:46 --> 00:40:50 but they'll also die because they're programmed to die, 500 00:40:50 --> 00:40:54 or they'll decide to commit suicide, or they'll be murdered by other 501 00:40:54 --> 00:40:59 cells. This happens in development, for example, your brains, as 502 00:40:59 --> 00:41:03 developing fetuses, have ten times the number of cells 503 00:41:03 --> 00:41:07 than you end up with as a young infant, because 90%, 504 00:41:07 --> 00:41:11 for some of you more than 90% of the cells will actually be killed 505 00:41:11 --> 00:41:16 through this process of programmed cell death. 506 00:41:16 --> 00:41:20 Other cells, in your immune system, for example, are eliminated by this 507 00:41:20 --> 00:41:25 process, so as to avoid them attacking your own body. 508 00:41:25 --> 00:41:29 And there's many other examples of relevant and important programmed 509 00:41:29 --> 00:41:34 cell death. So we want to understand this process, 510 00:41:34 --> 00:41:39 partly because it's critical in normal development, 511 00:41:39 --> 00:41:43 and also because deregulation of this process is critical 512 00:41:43 --> 00:41:48 for many diseases. Too little cell death can give you 513 00:41:48 --> 00:41:53 proliferate diseases like cancer or autoimmune disease, 514 00:41:53 --> 00:41:58 too much cell death can give you diseases like neurodegenerative 515 00:41:58 --> 00:42:04 diseases, too many cells in your brain dying. So the regulation of 516 00:42:04 --> 00:42:09 cell death is quite important. Here are two other famous examples 517 00:42:09 --> 00:42:14 of programmed cell death. This is the loss of the tadpole's 518 00:42:14 --> 00:42:18 tail that occurs through an orderly, cellular suicide program, in which 519 00:42:18 --> 00:42:23 all of these cells die, giving rise to a frog with no tail. 520 00:42:23 --> 00:42:27 And likewise, in development, your hands are formed in such a way that 521 00:42:27 --> 00:42:32 the digits are actually connected by other cells, but through this 522 00:42:32 --> 00:42:37 process of programmed cell death, the cells in the middle, the 523 00:42:37 --> 00:42:41 interdigital cells, are eliminated, thereby sculpting 524 00:42:41 --> 00:42:46 the formation of your fingers. And this can be regulated, 525 00:42:46 --> 00:42:50 and has been regulated, in evolution, in some species, 526 00:42:50 --> 00:42:54 like ducks, the loss of the interdigital cells doesn't take 527 00:42:54 --> 00:42:59 place, so they have webbing. In other species of birds, like in 528 00:42:59 --> 00:43:03 us, the interdigital cells are removed, so they have sculpted toes. 529 00:43:03 --> 00:43:07 This is what programmed cell death looks like. It is a very rapid, 530 00:43:07 --> 00:43:12 and as I said, a very orderly process. 531 00:43:12 --> 00:43:15 Cells get signals to die, they can get signals because they 532 00:43:15 --> 00:43:19 get damaged, or they can get signals from their neighbors. 533 00:43:19 --> 00:43:23 When they get those signals, they undergo a series of biochemical 534 00:43:23 --> 00:43:26 changes. Their nucleus gets condensed, the DNA within the 535 00:43:26 --> 00:43:30 nucleus breaks up, and then the cells themselves break 536 00:43:30 --> 00:43:34 up into small fragments called apoptotic bodies, 537 00:43:34 --> 00:43:38 and then interestingly, the cells that surround those cells, 538 00:43:38 --> 00:43:41 eat the damage. It's like disposing of the body 539 00:43:41 --> 00:43:45 after a crime, the cells are eliminated through a 540 00:43:45 --> 00:43:49 process of phagocytosis, this is a very clean process then, 541 00:43:49 --> 00:43:53 there's very little junk, there's very little cellular debris that 542 00:43:53 --> 00:43:56 remains when this process of programmed cell death is completed. 543 00:43:56 --> 00:44:00 This is a normal looking lymphocyte, and this is a lymphocyte undergoing 544 00:44:00 --> 00:44:04 cell death. It's like, you know, this is your brain, 545 00:44:04 --> 00:44:08 this is your brain on drugs, this is a cell, this is a cell undergoing 546 00:44:08 --> 00:44:12 apoptosis. It's a fairly violent death, 547 00:44:12 --> 00:44:16 at least visually. This movie shows you an example of that. 548 00:44:16 --> 00:44:20 Here are cells undergoing apoptosis, I hope. 549 00:44:20 --> 00:44:27 Maybe not. Oh well. 550 00:44:27 --> 00:44:30 It comes from your book, so you can see it yourselves, 551 00:44:30 --> 00:44:33 in fear that that would happen, I'll show you another set of stills. 552 00:44:33 --> 00:44:36 Here are normal cells growing in the tissue culture dish, 553 00:44:36 --> 00:44:39 you add some agent which induces apoptosis, the cells begin to round 554 00:44:39 --> 00:44:42 up as you can see here, and their cell surfaces actually 555 00:44:42 --> 00:44:45 become this horrible mess of blebbing membrane, 556 00:44:45 --> 00:44:48 and they will eventually break up, as you can see starting to happen 557 00:44:48 --> 00:44:52 here. So apoptosis is critical and 558 00:44:52 --> 00:44:56 actually very, very interesting. 559 00:44:56 --> 00:45:00 As I said, too much cell death can give rise to various diseases, 560 00:45:00 --> 00:45:04 neurodegeneration stroke is complicated because of the effects 561 00:45:04 --> 00:45:08 of apoptosis, too much apoptosis, even in AIDS, there's a lot of cell 562 00:45:08 --> 00:45:12 death that takes place in apoptosis, and too little cell death, as I said, 563 00:45:12 --> 00:45:16 is important in cancer, autoimmune disease, and certain 564 00:45:16 --> 00:45:19 viral infections. Importantly, we know about the genes 565 00:45:19 --> 00:45:23 that are regulated in cell death, that regulate cell death, through 566 00:45:23 --> 00:45:26 genetic experiments. And here, the genetic experiments 567 00:45:26 --> 00:45:29 were performed, in large part, at MIT, 568 00:45:29 --> 00:45:33 by a professor in the biology department named Bob Horvitz. 569 00:45:33 --> 00:45:36 He took comfort in the fact that the process in C. 570 00:45:36 --> 00:45:39 elegans, a simple worm, has very many similarities to the 571 00:45:39 --> 00:45:43 process that I've outlined to you, and therefore, he hoped that the 572 00:45:43 --> 00:45:46 genes that regulate programmed cell death in C. elegans, 573 00:45:46 --> 00:45:50 were similar to the ones that do so in mammals. 574 00:45:50 --> 00:45:54 And so he then undertook a study to ask what genes regulate the cell 575 00:45:54 --> 00:45:58 death process in this simple organism called C. 576 00:45:58 --> 00:46:02 elegans, which as an adult has only 1,090 cells. And the beauty of this 577 00:46:02 --> 00:46:06 organism is that you can actually see through it during development, 578 00:46:06 --> 00:46:10 and you can therefore follow the fate of individual cells over the 579 00:46:10 --> 00:46:14 course of development. And he and his students and fellows 580 00:46:14 --> 00:46:18 did that, he observed that in the development of certain cell lineages 581 00:46:18 --> 00:46:22 of the developing worm, individual cells died. 582 00:46:22 --> 00:46:27 In fact, about 130 cells underwent this process of apoptosis. 583 00:46:27 --> 00:46:32 And then he carried out a genetic experiment. He asked, 584 00:46:32 --> 00:46:38 if I mutagenize worms, can I find ones in which this process doesn't 585 00:46:38 --> 00:46:43 happen, or perhaps, in which this process happens too 586 00:46:43 --> 00:46:49 much. What are the genes that regulate apoptosis? 587 00:46:49 --> 00:46:54 And he found several such genes that turn viable cells into cells 588 00:46:54 --> 00:47:00 that have undergone programmed cell death, or apoptosis. 589 00:47:00 --> 00:47:05 In particular, there were two genes that positively 590 00:47:05 --> 00:47:10 regulated this process. They were called Ced-3 and Ced-4. 591 00:47:10 --> 00:47:15 They were required for apoptosis to occur. Mutants in Ced-3 and Ced-4 592 00:47:15 --> 00:47:21 didn't have this cell death process in those developing worms. 593 00:47:21 --> 00:47:26 And another gene called Ced-9 was required to prevent apoptosis. 594 00:47:26 --> 00:47:32 In a Ced-9 mutant, there was too much apoptosis going on, OK? 595 00:47:32 --> 00:47:38 They then cloned these genes, and it turned out they all have 596 00:47:38 --> 00:47:44 homologs, versions, in our cells, and those genes in our 597 00:47:44 --> 00:47:50 cells likewise regulate apoptosis in us. And we now know that at least 598 00:47:50 --> 00:47:56 members of this family of genes are important in diseases, 599 00:47:56 --> 00:48:02 including cancer. I'll just mention the function of one gene, 600 00:48:02 --> 00:48:08 right now. Ced-3, it is a protease, which cleaves target proteins -- 601 00:48:08 --> 00:48:22 -- into fragments. 602 00:48:22 --> 00:48:26 So, one of the key things that happens in apoptosis is, 603 00:48:26 --> 00:48:30 you turn on this enzyme, this protease, and it goes around 604 00:48:30 --> 00:48:34 cutting up various target proteins, which eventually lead to the death 605 00:48:34 --> 00:48:38 of the cell. There are many target proteins that get cleaved when these 606 00:48:38 --> 00:48:43 caspases, these proteases are called caspases, when these proteases get 607 00:48:43 --> 00:48:47 activated, and I just gave you one example of one such target. 608 00:48:47 --> 00:48:51 There's an enzyme that is normally involved in cleaving your DNA, 609 00:48:51 --> 00:48:55 this is a DNA gel, of viable cells and cells that have undergone 610 00:48:55 --> 00:49:00 apoptosis after a certain number of hours. 611 00:49:00 --> 00:49:04 The DNA of the cells is normally intact, and large, 612 00:49:04 --> 00:49:09 because this enzyme is kept in check. However, when its inhibitor is 613 00:49:09 --> 00:49:13 cleaved by the caspsase, it now goes about cleaving the DNA, 614 00:49:13 --> 00:49:18 and this is one of the reasons why apoptotic cells die, 615 00:49:18 --> 00:49:22 because their DNA gets cut up into very small fragments. 616 00:49:22 --> 00:49:27 There are a number of other targets, you can read a little bit more about 617 00:49:27 --> 00:49:31 this in your book, I apologize that we had to rush 618 00:49:31 --> 00:49:36 through apoptosis, it is important, and we will in fact 619 00:49:36 --> 00:49:39 come back to it in a discussion of cancer.